Masaryk University
Faculty of Medicine
Pathophysiology practicals
for General Medicine and Dental Medicine courses
Kateřina Kaňková et al.
Brno 2008
Editor:
Assoc. Prof. Kateřina Kaňková, MD, PhD
Authors:
Julie Bienertová-Vašků, MD
Prof. Lydie Izakovičová-Hollá, MD, PhD
Michal Jurajda, MD, PhD
Assoc. Prof. Kateřina Kaňková, MD, PhD
Michal Masařík, MA, PhD
Lukáš Pácal, MA
Prof. Anna Vašků, MD, PhD
Reviewer:
Assoc. Prof. Marie Nováková, MD, PhD
Illustrated by: Assoc. Prof. Kateřina Kaňková, MD, PhD
2
Preface
Pathophysiology is a medical discipline that seeks to provide understanding of the body (mal)functioning
upon the disruption of bodily functions by a disease or an abnormal situation that does not qualify to be called disease. In practical medical terms it means study of the aetiology (= cause) and pathogenesis (= course) of disease.
Pathophysiology can be seen as a follow-up of normal physiology that study function of a healthy body under the
normal conditions. Pathophysiology looks at body malfunctioning in any possible way, from the whole body,
through organ and tissue to molecular level applying all available routine as well as experimental methodology.
However, there are several drawbacks in the above mentioned simplified definition of pathophysiology.
First, body functions in a broad sense are never the same in any two individuals. This so-called interindividual variability is a result of genetic variability which always produces a unique phenotype in a given subject. Second, there
is hardly any disease to be found that would be a consequence of a single pathogenic cause. Typically, several or
multiple factors (both endogenous and environmental) operate together at the time of disease origin and during its
course. Thus, ethiopathogenic and temporal plasticity of disease is a great challenge for contemporary pathophysiology.
One of the possible approaches to study human diseases and their complexity is to use non-human experimental model organisms. History of the use of animals for learning and research purposes parallels that of medicine itself and possess many ethical and legal issues. Use of animals in medicine and industry is a subject of intensive debate both in professional and lay communities. Nevertheless, whatever the future trend might be, animals are
recently only used in research – under the strict control and evidence - when the answers to scientific questions
cannot be obtained in any other way and it is necessary to see what happens in the whole living body. Experimental
work with laboratory animals is, for the same reason, an inseparable part of pathophysiology curriculum.
Brno 2008
Kateřina Kaňková
3
Table of contents
1
2
USE OF ANIMALS AS EXPERIMENTAL SUBJECTS ......................................................... 5
1.1
Introduction ....................................................................................................... 5
1.1.1 History .......................................................................................................................................................... 5
1.1.2 Ethical principles, animal protection and legal issues .................................................................................. 6
1.1.3 Commonly used animal species & areas of research .................................................................................... 6
1.1.4 Use of animals in education .......................................................................................................................... 7
1.1.5 Laboratory animal genetics & microbiology ................................................................................................ 7
1.1.6 Alternative and complementary techniques .................................................................................................. 8
1.2
Work with laboratory animals – principles ......................................................... 9
1.2.1 Animal husbandry & handling ..................................................................................................................... 9
1.2.2 Anaesthesia ................................................................................................................................................... 9
1.2.3 Resuscitation .............................................................................................................................................. 10
1.2.4 Euthanasia .................................................................................................................................................. 10
1.3
Reference values of selected physiological parameters in commonly used adult
laboratory animals ............................................................................................ 10
EXPERIMENTAL PRACTICAL MANUAL........................................................................ 11
2.1
Animal experiments .......................................................................................... 11
2.1.1 Explorative laparotomy in laboratory animal ............................................................................................. 11
2.1.2 Experimentally induced acute radiation syndrome in laboratory animal.................................................... 13
2.1.3 Experimentally induced atherosclerosis in laboratory animal .................................................................... 16
2.1.4 ECG monitoring of experimentally induced arrhythmias in laboratory animal – models of
hyperkalemia, hypocalcemia and enhanced adrenergic stimulation ........................................................... 18
2.1.5 Experimentally induced renal ischemia in laboratory animal as a model of secondary
(renovascular) hypertension ....................................................................................................................... 20
2.1.6 Experimentally induced acute renal insufficiency in laboratory animal – measurement of GFR
based on kinetics of renal inulin excretion ................................................................................................. 22
2.1.7 Experimentally induced renal failure - peritoneal dialysis in laboratory animal ........................................ 24
2.1.8 Experimentally induced peptic ulcer in laboratory animal ......................................................................... 26
2.1.9 Quantification of gastric juice secretion after its pharmacological manipulation in laboratory
animal ......................................................................................................................................................... 27
2.1.10 Experimentally induced diabetes mellitus in laboratory animal – principles of diagnostics by
glucose tolerance test .................................................................................................................................. 29
2.1.11 Experimentally induced haemolytic, hepatotoxic and obstruction hyperbilirubinemia/jaundice in
laboratory animal ........................................................................................................................................ 31
2.1.12 Experimentally induced venous thrombosis in laboratory animal .............................................................. 33
2.1.13 Effect of selected pharmacologically active substances on microcirculation in laboratory animal ............ 35
2.1.14 Experimentally induced anaphylactic response in laboratory animal ......................................................... 36
2.2
Experiments using alternative models – humans & in vitro techniques ............ 38
2.2.1 Principles and demonstration of peripheral blood flow examination using Doppler ultrasonography ....... 38
2.2.2 Measurement of resting and ambulatory blood pressure and heart rate, postural changes, effect of
isometric and aerobic exercise .................................................................................................................... 40
2.2.3 Pathophysiology of ventilation disorders – pulmonary function tests: spirometry ..................................... 42
2.2.4 Use of enzymes as diagnostic markers – evaluation of lactate dehydrogenase (LDH) isoenzymes
by agarose gel electrophoresis .................................................................................................................... 44
2.2.5 Molecular biological methods in haematology – diagnostics of familial thrombofilias: detection of
the factor V Leiden mutation by PCR ........................................................................................................ 45
4
1
Use of animals as experimental subjects
1.1
Introduction
There are many approaches to study human diseases and their complexity, one of them being research using non-human experimental model organisms. Annually ten to hundreds of millions of vertebrate animals (from
zebra-fish to non-human primates) are used worldwide accompanied by even larger number of invertebrates, mainly flies (Drosophila melanogaster) and worms (Caenorhabditis elegans). Aside from basic science (such as genetics, behavioural sciences, developmental biology etc.) they are used in applied research incl. biomedical research,
pharmacology and pharmaceutical industry, transplantation medicine, toxicology etc. not mentioning industrial
testing, military and breeding purposes. The numbers are, fortunately, falling annually since alternative methods
(such as various in vitro techniques and computer modelling) are being developed and introduced into research
practise.
It is not worth concealing that the topic of animal use in research and testing is highly controversial.
There are supporters as well as opponents of the practise. Those in favour propose that very little of the progress in
medicine would be made without use of animals and that virtually every medical achievement in 20 th and 21st century involved animals in some way. Each of us, himself, family member or friend, is a consumer of some of the
many outcomes which came from animal research. Moreover, even with the most sophisticated computer algorithms, there are no means available yet to comprehend and predict all possible interactions operating in multilevel
organisms and thus spare animals from science completely. The human body is very complex and is much more
than just a sum of its parts. Opponents argue that animal research is cruel and unjustifiable even when providing
benefit for humans since they can be seen as yet another of many species inhabiting this world with no superior
moral rights. Still, not all but quite a large number of those who criticise the use of animals are happy to enjoy medical benefits obtained by animal research. But even when accepting the justifiable use of animals in research generally, there are stil new and new hot topics being added into already heated discussion such as animal (and human)
cloning, stem cell research, animal-to-human organ transplants, use of monkeys in brain research etc.
The issue of ethical justification of animal research is one of the toughest philosophical questions nowadays. Mere fact that so many people benefit from animal research is not sufficient enough (we could probably benefit the same way from experiments performed by Nazis on humans). Unlike humans animal can’t give an informed
consent so we can only try to perform research as “humanly” as possible to minimize their suffering the way we
would do with each of us. Another important issue - from the perspective of psychology and sociology - is that
humans have obligations to ourselves that they do not have to animals. Although opponents of animal research
often state that all living organisms have the same rights in the nature, the everyday practise, even within human
society, does not indicate such an egalitarian principle. Humans tend to see themselves as a superior life form and
indeed there are many reasons for doing so. Whether one likes it or not, human’s “aristocratic” view of the world is
evident in nearly all ways we deal with the world (environment, resources etc.) including animals. The history of
animal research shows similar moral progress seen within the human society itself (rejection of slavery, feudalism,
sexual, ethnic and religious discrimination etc.). The field of animal research is a subject of very intensive and serious debate and, quite correctly, tight control preventing unnecessary animal exploitation.
On the other hand, put into perspective, animal research is just one of many complicated ways humans interfere with other living creatures. While millions of animals a year used for research purposes may sound like a
large number, compared to other ways we use and treat animals in our society – animals consumed as a food, kept
in captivity or like pets (sometimes mistreated), hunted as a game, purposefully killed as vermin (rodents, pigeons,
seagulls etc.) – animal research suddenly represents just a tiny fraction.
1.1.1
History
Use of animals for medical experimenting dates back to the beginning of the subject itself, originally with
the aim to find out basic principles of body structure and function. The earliest references to animal testing are
found in the writings of the Greeks (Corpus Hippocraticum, ~400 BCE), animals experiments were carried by Aristotle or Erasistratus. In Rome, Galen dissected pigs and goats to study anatomy and physiology. Significant progress in our understanding of medicine using animals was achieved during renaissance period (Anatomica de Motu
Cordis et Sanguinis in Animalibus, Exercitatio, 1628).
While the anatomical and physiological studies were performed on dead animals, discovery of anaesthetics in the first half of the 19th century revolutionised medical research since it allowed performing in vivo experiments. Animals have been used throughout the history of scientific research. Animals stand behind scientific milestones in medicine such as work of L. Pasteur (anthrax in sheep), I. Pavlov (conditioning in dogs), F. Banting &
Ch. Best (insulin from dogs) and numerous others until recently. The crucial importance of animal research was
5
highlighted by French physiologist Claude Bernard (1813-1878) in his book “An Introduction to the Study of Experimental Medicine” (1865). He was a professor of physiology in College de France and, amongst many other
scientific achievements, author of the concept of homeostasis (“Constance of the internal environment is the condition for a free and independent life”). Interestingly, while C. Bernard practiced animal experiments as a routine part
of his professional work since he firmly believed that the advancement of medicine and the relief of human suffering justified the suffering of animals, his wife and daughter were not convinced and the couple were officially separated in 1869 and his wife went on to actively campaign against the practice of vivisection.
This marks another important history. In parallel to the expanding use of animals first initiatives opposing
the practise were appearing initially in Great Britain where the first law protecting animals was enacted by British
Parliament in 1876 (“Cruelty to Animals Act“). The idea of rigorous legislation and strict control over animal experiments was promoted, among others, by Ch. Darwin. Similarly, in the United States the “American Society for
the Prevention of Cruelty to Animals” was founded in 1860 and the campaign culminated in federal legislation in
1966 (“Laboratory Animal Welfare Act”).
1.1.2
Ethical principles, animal protection and legal issues
First of all, people who are involved in animal research - scientists, doctors, vets, animal carers – have,
similar to anyone having a pet or just being normal caring human being, no desire to mistreat animals. Many of
them also participate in the development of alternative techniques sparing animals. Welfare of animals used in research is also very important for science itself. Animal suffering stress or pain could affect the results of the research.
The official guiding principles of animal research today were defined in 1959 by biologists William Russell and Rex Burch (“Principles of Humane Experimental Technique”) as the so-called “three Rs principle“ - Reduction, Refinement, Replacement:
 Reduction – always reduce the number of animals used to a minimum, obtain information from fewer animals or more information from the same number of animals. Ways to reduce number of animals include
systematic reviews of studies already conducted, careful experimental design (statistics) and paradoxically, development of better animal models closer to the problem studied (inbreeding, transgenic animals
etc.).
 Refinement – the way experiments are carried out has to make sure animals suffer as little as possible
(housing, procedures which minimise pain and suffering).
 Replacement – whenever possible substitute animals with alternative techniques (see further), or avoid
the use of animals altogether.
Most countries of EU and US implemented this concept into legal form (US Animal Welfare Act 1986,
UK Animals Scientific Procedures Act 1986, Czech law ČNR 246/92 Sb. (in full-text 167/93). Both EU and US
legislation similarly specify the following conditions for justifiable animal research:
 the potential results are important enough to justify the use of animals (the cost benefit analysis);
 the research cannot be done using non-animal methods;
 the minimum number of animals will be used;
 dogs, cats or primates are only used when other species are not suitable;
 any discomfort or suffering is kept to a minimum by appropriate use of analgesics;
 researchers and technicians conducting procedures have the necessary training, skills and experience (§17
of the Czech legislation 167/93 specifies necessary qualification for all staff involved in various stages of
animal experiment);
 research premises have the necessary facilities to look after the animals properly.
1.1.3
Commonly used animal species & areas of research
Although animals were used throughout the whole history of medicine only in 20th century we can use the
term “laboratory animal” in its current meaning, i.e. purposefully bred animal not appearing in the nature. First
production farms were established in the United States between the 1st and 2nd world wars producing first inbred
mice, rat and guinea pig strains and subsequently embarking on the process of genetic engineering.
All vertebrate animals used for research purposes and testing are specially bred in production farms (invertebrates such as flies and worms are usually produced locally by each institution and laboratory). No strays,
unwanted pets from animal shelters or poached wild animals can be used. Breeding of laboratory animals very often involves various genetic and microbiologic manipulations with special purposes so these animals are unlikely
survive in wilderness when – even under the good will - freed by activists. Types of animals used in medical research shows table below. Currently, in the EU animal sources are governed by Council Directive 86/609/EEC
(“The protection of animals used for experimental and other scientific purposes”), which requires lab animals to be
specially bred, unless the animal has been lawfully imported and is not a wild animal or a stray.
6
Types of animals used in medical research
Rodents (mice, rats, guinea pigs, hamsters and others)
Fish, amphibians, reptiles, birds, fertilised hen’s eggs
Pigs, sheep, cows
Non-rodent small mammals (mostly rabbits)
Dogs and cats
Monkeys (mostly macaques), great apes (chimpanzees, orangutans and gorillas)
were not used last 20 years and their use is now banned in EU
% (data from UK 2007)
83
15
1.6
0.7
0.2
0.12
Areas consuming most animals are shown in the table below. Biomedical research (including tertiary education of biomedical subjects) is one of the leading areas nowadays and will probably dominate in the future since
number of animals used for other purposes such as safety testing can be broken down further (many testing procedures now employ alternative methods, see further). Testing of certain products like cosmetics/toiletries was abandoned entirely in EU countries. The LD50 test, used for many years to find out how toxic chemicals are, is redundant with better tests today. The same data using fewer animals can be obtained now so that none intentionally
received a fatal dose. The LD50 is now banned in the UK.
Areas consuming laboratory animals
Basic biological and medical research
Development of new treatments (drugs, surgery), diagnostic tools and preventive
measures (vaccines)
Breeding (domestic animals) incl. production of exp. models, veterinary medicine
Safety testing of non-medical products used in households, agriculture, food additives and industry
1.1.4
% (data from UK 2007)
31
30
37
2
Use of animals in education
Use of live animals in carefully designed and properly monitored laboratory exercises is an
indispensable part of training in certain programs of tertiary education (medicine, veterinary, pharmacy,
agriculture). Knowledge, experience, and insights gained through the responsible use of live animals in
the laboratory are believed to be unique, invaluable and irreplaceable elements of education. Similar to research, all
teaching programs involving animals has to be approved by animal ethical committees of particular institutions as
well as national body. Generally, numbers of animals used in tertiary education are very low, majority being rodents.
1.1.5
Laboratory animal genetics & microbiology
Both genetically diverse and genetically defined animals are used in research. For example there are over
3000 genetically defined strains of laboratory mouse available today. Nomenclature with regards to genetics is very
complex depending on the type breeding and particular genetic variations:
Outbred mice: random mating among breeding-age members of the population, results in non-isogenic,
i.e. genetically non-identical offsprings. Outbred mice are useful in experiments where the precise genotype of
animals is not important and will not be used to create new strains. Outbred mice are often used in toxicology and
pharmacology research (e.g. Swiss Webster mice, Sprague Dawley, Zucker or Wistar rats, New Zealand white rabbit etc.).
Inbred mice: results from a minimum of 20 consecutive generations of brother-sister matings. Animals
are virtually identical. The most obvious advantage of working with inbred strains is genetic uniformity over time
and space. Inbred strains serve to eliminate the contribution of genetic variability to the interpretation of experimental results (therefore, fewer animals are needed for the study). They are widely used for biomedical research
(e.g. BALB/c or NOD mice, Dahl salt sensitive (DSS), Goto-Kakizaki (GK) or Zucker Diabetic Fatty (ZDF) rats
etc.)
 Hybrid strains (F1 hybrids) – results from first-generation cross between two inbred strains. Completely
inbred genome is an abnormal condition with detrimental phenotypic consequences. The lack of genomic
heterozygosity is responsible for a generalized decrease in a number of fitness characteristics including
body weight, life span, fecundity, litter size, and resistance to disease and experimental manipulations. It
is possible to generate animals that are genetically uniform without suffering the consequences of whole
genome homozygosity. This is accomplished by simply crossing two inbred strains. All F1 hybrids obtained from a cross between an A strain female and a B strain male will be genetically identical to each
7
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




other over time and space. Both inbred and F1 hybrid are called isogenic, i.e. genetically identical animals. Of course, uniformity will not be preserved in the offspring that result from an “intercross” between
two F1 hybrids; instead random segregation and independent assortment will lead to F2 animals that are
all genotypically distinct and close to outbred animals.
Recombinant inbred strains – are produced when F1 hybrids are further mated together (brother-sister
matings) for a minimum of 20 generations.
Co-isogenic strains – strain that differs from the original strain in a single locus due to a spontaneous
mutation that appeared during the breeding (e.g. Ob-/Ob- mice).
Mutant strains that carry induced mutation – result or either introduction of foreign DNA, chemical or
viral modification or homologous recombination. These strains serve very often as specific models of
human diseases (monogenic as well as complex).
transgenic strains – these animals carry foreign DNA that was intentionally inserted into their own genome. Upon insertion of human mutated gene these can serve as models of human monogenic dominant
diseases.
knock-out strains – a normal gene was rendered non-functional by a process called homologous recombination. These animals can serve as models of human monogenic recessive diseases.
congenic strains – similar to co-isogenic strains, however, genetic dissimilarity between strains is created
through breeding rather than as a result of spontaneous mutation.
Microbiology
Based on the presence or absence of normal, opportunistic and pathogenic microorganisms laboratory animals can be classified into:
 Conventional: animals colonised by resident microorganisms normally associated with its particular species. They are bred in “open” animal facilities. Animals are regularly controlled for the presence of human pathogens.
 Specific pathogen-free (SPF): guaranteed free of particular pathogens (always accompanied by a list of
the absent pathogens). Animals are bred in “barrier” facilities, colonised by defined microflora. The population is checked for the presence of specified pathogens or antibodies against them.
 Gnotobiotic (axenic, completely germ-free): animals can be born through a caesarian section (hysterectomy) then special care is taken so the newborn does not acquire infections, such as use of sterile plastic
bags with a positive pressure differential. Subsequently, animals can be intentionally colonised by one or
several defined microorganisms (mono-, di-, polyxemic animals).
1.1.6
Alternative and complementary techniques
Although vital nowadays, the use of living animals is just one of three main research methods in medicine
and biology. They are not alternatives to each other, more likely they are complementary approaches that are all
equally valid and all contribute to the overall picture of subject studied. The non-animal techniques are:
 In vitro techniques: study of isolated molecules, cells and tissues (originating from humans, animals, micro-organisms or plants) incl. in vitro reconstructed organs (e.g. Episkin, Epiocular, EpiAirway etc.). Provides information about interactions between molecules, within or between cells, or about organ function.
 Microorganisms: simple microorganisms, such as bacteria, fungi, yeasts and algae are being used as early indicators of chemical toxicity (frequently faster and cheaper). Bacteria can be genetically manipulated
to manufacture useful products previously obtained from animals, such as human insulin and monoclonal
antibodies.
 Study of human beings (volunteer studies) and populations: provides information about the body in
health and disease, and about the distribution of diseases in society, however, is limited by what is considered ethical.
 Computer modelling and other high-tech equipments: various knowledge-based expert systems (e.g.
chemical structure-activity relationship (SAR) that can improve the efficiency of animal, non-animal or
human research techniques, limit the numbers and need for replications.
 Isolated perfused organs: although of animal origin, ex vivo settings allowing precise measurements of
input and output parameters usually lead to significant reduction of numbers of animals otherwise needed
and therefore, it is often considered alternative.
It is important to stress that even so widely used alternative models such as in vitro techniques do not obviate animals completely. Growth media for tissue cultures has to be supplemented with bovine serum, mono- and
polyclonal antibodies are raised by immunisation of animals (mice, rats, rabbits or goats commonly) etc.
8
1.2
Work with laboratory animals – principles
1.2.1
Animal husbandry & handling
Animal husbandry is a subjects of specialised field called Animal science. It includes all relevant aspects
from the knowledge of natural habits of different strains of animals, their physiologic parameters, behavioural patterns, nutrient requirements, dark/light cycling etc. including typical diseases, pathogens etc. Certified breeders
have to fulfil stringent criteria defined by authorisation bodies to be certified dealers. Individual research institutions purchase and keep animals for a limited time period defined by the experimental protocol and have to provide
suitable animal facilities to carry out the animal experiments to obtain permission.
During experiment itself, animals have to be handled with maximal care to avoid their unnecessary stress
and eventual injury of experimentator. Rodents are usually kept in cages in groups. Manipulation with mice is best
done holding the animal either at the base of the tail or holding the skin folder at the nape between thumb and forefinger and turning the animal to face abdominal side (for i.p. application). Mice have to be handled very gently
since any gross manipulation can induce stress and panic and increase the aggressivity of animals. Rats are very
easy to handle when approached gently and slowly. To remove animal from the cage it is best to grab its tail in the
middle and slowly elevate and immediately place at the table. For application of anaesthesia i.p. it is best to grab
skin fold approx. in cranial half of the back (immobilising the head so that accidental bite is avoided) and turn the
animal to face abdominal side. Most rabbits are tame and innocuous; however it can bite or graze when scared.
Rabbits should not be stressed since massive release of catecholamines during the stress influences the circulatory
parameters, increases susceptibility to arrhythmia and affects anaesthesia. When approaching rabbits in the cage it
is best to make sounds (e.g. speak monotonously). Rabbits are carried by both hands, one holds skin fold in the
shoulder blade area, the second hand moves along the back towards and under the back legs. Folded to the chest
rabbit can be carried and placed on the table (always rough surface, slippery surface causes panic).
1.2.2
Anaesthesia
Type of anaesthesia depends on the type and length of experimental procedure, knowledge of pharmacokinetics and pharmacodynamics of various anaesthetics in different species. Animals are usually fasted for short
time before anaesthesia to avoid complications (similarly to man) such as vomiting, aspiration etc. Larger animals
are premedicated (atropine) before anaesthesia itself to avoid hypersalivation, bradycardia and bronchospasm.
General anaesthesia can be performed by inhalation or injection anaesthetics, very often in combination.
The ways injection anaesthetics are administered include intravenous (i.v.), intraperitoneal (i.p.), intramuscular
(i.m.) or subcutaneous (s.c.) approach. Dose and rate of absorption depends on the ways of administration. S.c.
administration (free skin on the neck, between shoulder blades or chest) is a common way of premedication. I.m.
administration (m. quadriceps or m. gastrocnemius femoris) is common in anaesthesia of rabbits or guinea pigs. I.
p. administration (upper hypogastrium) is common in rodents (see chapter 2.1.1 for details). I.v. administration
(superficial veins) is common in rabbit (e.g. v. marginalis auricularis, v. saphena magna or v. antebrachii). Tail vein
is a common and easily accessible vein in rodents (esp. rats). The procedure of commonly used anaesthesia (diethylether inhalation + i.p. administration of anaesthetic mixture) is described in detail in chapter 2.1.1. Examples of
routinely used anaesthetics and doses for commonly used animal strains are listed in table below.
Mice
Rats
Sedation
dehydrobenzperidol 3 mg/kg i.m. (~60
min)
dehydrobenzperidol 0.8 mg/kg i.m.
diazepam 2.5 mg/kg i.m.
atropine 0.05 mg/kg s.c. or i.m. 30 min
before surgery
Rabbit diazepam 5 - 10 mg/kg i.m. (~3 - 4 hrs)
Injection
ketamin 100 mg/kg i.m. or i.p. + xylazin 16 mg/kg i.m. or i.p.
(~90 min = 0.1 – 0.2 mL/10 g body weight i.p.) or ketamin
200 mg/kg i.m. + diazepam 5 mg/kg i.m.
pentobarbital 20 mg/kg i.p. or ketamin 100 mg/kg i.m. or i.p.
+ xylazin 16 mg/kg i.m. or i.p. (~60 – 90 min = 0.5 mL/100 g
body weight i.p.) produces increased diuresis!
ketamin 50 – 70 mg/kg i.m. + xylazin 4 – 5 mg/kg i.m. (~45 60 min) or pentobarbital 25 - 40 mg/kg i.v. or
thiopental 25 – 30 mg/kg i.v. (~10 - 20 min)
Inhalatory anaesthetics (diethylether, halotan or methoxyfluran) are administered either by masks (larger
animals), or endotracheal tube (intubation requires training) or by inhalation in an enclosed space (glass tanks rodents). There are several minor disadvantages of inhalatory anaesthesia, mainly the reflexive holding of breath
which affects circulatory parameters.
After general anaesthesia it is necessary to administer an adequate care, extubation (in rabbits) has to be
9
performed after return of the swallowing reflex. It is necessary to prevent hypothermia, sometimes by warming-up
by infrared lamps. After arousal animals should be isolated to prevent injury and mutual assault. Self-injury can be
prevented by restrainers. When back in normal position and physical activity water and chow can be given. Analgetics are administered during healing as long as necessary after surgical procedures.
1.2.3
Resuscitation
General anaesthesia can be complicated by several events, most often apnoea. It is necessary to act immediately (especially in rats since they do not tolerate apnoe well and soon respond with heart arrest) - to pull the
tongue out to free the airways and gently massage the chest (emergency intubation is possible in rabbit). Warning
signs include central cyanosis (visible on tongue, ears and interdigitally).
1.2.4
Euthanasia
Every experiment has to be terminated by euthanasia to minimize physical and mental suffering of the animal after experiment. This is required by the law! Euthanasia can be carried out by mechanical or pharmacological
methods depending on the type of laboratory animal, aim of the experiment (interference with measured parameters) and subsequent procedures planned (e.g. tissue specimens for histology, blood for analysis etc.).
Physical methods are applied in rodents and include decapitation or cervical dislocation. Pharmacological
methods are basically based on anaesthesia overdose, often by inhalatory (ether or halothan) or injection anaesthetics (barbiturates).
1.3
Reference values of selected physiological parameters in commonly
used adult laboratory animals
Weight (g)
Body temperature (°C)
Heart rate (/min)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Breathing rate (/min)
Volume of blood (% of body weight)
Blood clotting (min)
Erythrocyte count (mil/mm3)
Haematocrite (%)
Leucocyte count (thousands/mm3)
- neutrophils (%)
- basophils (%)
- oeosinophils (%)
- lymphocytes (%)
- monocytes (%)
Thrombocyte count (thousands/mm3)
Haemoglobin (g/L)
Sexual maturity /gestation period
Life span (years)
Mouse
Mus musculus var.
alba
20 – 90
37.1
480 – 740
95 – 140
20 – 90
84 – 230
7.6
1–3
4.7 – 12.3
41
10
20 – 30
0–1
0–1
35 – 80
0–3
350
105 – 160
7 weeks / 20 days
1-2
Rat
Rattus norvegicus
var. alba
200 – 500
37.3
260 – 600
90 – 100
80
66 – 210
5–8
2–4
5.5 – 10
46
12.5
18 – 36
0–1
1–4
65 – 75
1–6
600
130 – 150
13 weeks / 20 days
2-3
10
Rabbit
Oryctolagus cuniculus
1 500 – 6 000
38.5
180
100 – 140
70 - 90
53
5
5–7
6.3
39.8
5.2 – 12
8 – 50
1–3
1–8
20 – 90
1–4
130 – 1 000
140 - 150
6 months / 31 days
5–6
2
Experimental practical manual
2.1
Animal experiments
2.1.1
Explorative laparotomy in laboratory animal
Kateřina Kaňková
Aims:
 To demonstrate methods of restraint, anaesthesia and surgical techniques in laboratory animal (rats).
 To examine the topography of abdominal cavity and other regions commonly used in subsequent experiments.
 To acquire the basic practical skills necessary for subsequent experimental work.
Materials, equipment & animals:
Animals: adult Wistar rats, both genders, weight 250 – 300 g.
Glass container with a lid, diethylether, anaesthetic mixture 10 mL 1% Narkamon (ketamin) and 0.5 mL
2% Rometar (xylazine) to be administered in a dose 0.5 mL/100 g i. p., syringes with needles, scales, operating
table, straight scissors, anatomical forceps (2 pairs), needle holder, blepharostat, suture material, surgical needles,
paper tissue, gauze (cotton), swabs, disinfectant.
Procedure:
The rat must be handled calmly and with confidence. Sudden and jerky movements may frighten the animal. To transfer the animal from its cage it should be held at the base of its tail with the first and second fingers,
lifted from the cage head down and quickly placed into the glass container.
To administer inhalatory anaesthesia, paper tissue on the bottom of the glass container should be moistened with about 20 ml diethylether prior to placement of the animal. At first, the animal will exhibit excitation
then it will gradually fall asleep. The pupils dilate, nystagmus develops, breathing becomes irregular due to irritation of the respiratory tract with ether and skin sensitivity is reduced. With deepening anaesthesia, first eyelid, then
corneal reflexes disappear and the pupils narrow. The stage at which surgery can be carried out is indicated by the
cessation of movement of tactile bristles on the animal’s muzzle. Immediately, the animal must be removed from
the glass container. Longer stay in the ether atmosphere could result in respiratory arrest and subsequent death.
Such danger is indicated by irregular, superficial breathing and dilatation of pupils.
The intraperitoneal anaesthesia covering the whole experimental period planned should be administered
during the 2 min following the inhalatory introduction. Animal must be weighed and the correct dose of intraperitoneal anaesthetics calculated (0.5 mL/100 g of body weight). I. p. administration is carried out with the animal
being held by the assistant by the dorsal skin fold – abdominal site facing the person administering the anaesthesia.
Needle is inserted first approx. 3 mm lateral to the umbilicus perpendicularly through the skin and abdominal muscles (into ~0.5 cm depth) and then horizontally inserted further 1 - 2 cm into peritoneal cavity. Only then the whole
volume of anaesthetics can be slowly administered. When the necessary level of anaesthesia is reached, the animal
is restrained in the supine position on the operating table, its extremities and the
upper incisors immobilised under the rubber straps.
The surgical procedure starts with shaving and disinfecting the skin at
the future operation field (in the case of rat, aseptic conditions are not necessary
due to extremely efficient immunity). To perform laparotomy, a transverse skin
fold is lifted by pair of forceps at the level of the umbilicus and skin is cut
through. The cut is then extended in the medial line for the whole length between the caudal end of the sternal bone (processus xiphoideus) and cranial end
of pelvic bone (symphysis). The muscle layer will be opened along the linea
alba to the same extent by scissors as well, the wound edges are protected with
gauze and held apart by means of a blepharostat. If necessary, anaesthesia can
be prolonged by administering ¼ - ½ of the original volume of anaesthetics
mixture.
Following the inspection of abdominal cavity (i.e. topography of the
abdominal organs), the wound will be closed in two layers. First, the abdominal
wall and peritoneum will be sutured continuously beginning at the cranial end
of the wound (the length of thread required is approx. 40 cm), see figure on the
right. Needle (different kinds for muscle and skin!) is held in the needle holder.
11
The skin is sutured with individual stitches ~5 mm apart placed ~3 mm from the
edge of the wound (see figure on the right). All knots are made at the same side
of the suture and tightened firmly. After the operation, the animal should be
placed in the transport cage and kept in a warm environment.
Results & Conclusions:
Describe briefly the reason for equipment used, relevant steps of the
procedure and comparison of topography of the human vs. rat abdominal cavity.
12
2.1.2
Experimentally induced acute radiation syndrome in laboratory animal
Lydie Izakovičová-Hollá
Aims:
 To assess changes in the peripheral blood count induced by ionizing radiation in two different time periods - 3 and 20 days - following the single dose of 4 Gy-radiation, i.e. in the phases of the deepest depression of bone marrow and in the phase of its regeneration.
 To assess changes in the body and spleen weight of the irradiated animals in particular time-periods.
 To learn principles of methods for determining the counts of individual blood elements (erythrocytes, leucocytes and thrombocytes), measurement of haemoglobin concentration and haematocrit.
 To practically prepare blood smears for the counting different white blood cells and reticulocytes.
Introduction:
Acute radiation syndrome (ARS) develops in a multicellular organism after whole-body irradiation. Manifestation of ARS depends on a total received dose. Sensitivity to ionizing radiation varies between different tissues. It is generally highest in intensively dividing cells such as lymphatic tissue, bone marrow or intestinal epithelia. Following irradiation the blood count exhibits characteristic changes – (1) during the earliest phase (hours to 1
day after irradiation) numbers of leukocytes (granulocytes) increase in the periphery as a result of the release of
immature leucocytes from the bone marrow and spleen reserve, later on, their numbers rapidly decline due to depletion of their precursors in bone marrow. (2) Lymphocytes usually decline immediately due to their higher sensitivity to DNA damage, stress-activated apoptosis (in bone marrow as well as in the periphery) and glucocorticoidmediated lysis. (3) Platelets and red blood cells (RBC) decline slowly (RBC’s life span in the circulation is longer
and therefore it takes the longest time before the deficit in erythropoiesis in the peripheral circulation becomes evident). The amount of RBCs in peripheral blood is given by the relationship between their “onflow” (here given by
the number of reticulocytes) and their “outflow” (the dose used for radiation does not affect RBCs in the periphery), i.e. 100/56 = 1.79% a day in the rat (average life span of rat’s RBC is 56 days).
Because blood elements in the rat spleen are produced also postnatally, weight and cellularity of the
spleen corresponds with changes in the bone marrow (after the initial depletion, the weight of the spleen increases
above the original level and hemopoiesis in the spleen quickly compensates for a minor persisting deficit of hemopoiesis in the bone marrow).
Note: There are considerable differences between humans and rats both in radiosensitivity (rats are much
more resistant and therefore able to tolerate higher doses of radiation) and blood count (rats have a reverse neutrophils/lymphocytes ratio compared to a man and a shorter life span of RBCs in the periphery ~approx. 56 days).
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300g) per group will be
divided into 3 groups: 1) controls (non-irradiated, 1/3 of animals), 2) animals irradiated 3 days prior the experiment
(1/3 of animals) and 3) animals irradiated 20 days prior to the experiment (1/3 of animals). Glass container with lid,
scales, immobilisation table for small animals, anaesthetics, surgical instrumentarium, Petri dish (for the excised
spleen).
Material for (1) heamoglobin and (2) haematocrit measurements [cuvette (thickness 1 cm), spectrophotometer with a color filter for 530 - 550 nm, transformation solution according to Drabkin, glass cappilaries, plasticine in Petri dish, centrifuge], (3) material for the manual method of cell counting [flasks with hematological solutions - Hayem´s (for RBCs), Türk’s (WBCs), procain solution (for platelets), counting chamber, light microscope,
micropipette 25 L, sterile mouthpiece, cotton wool], material for (5) peripheral blood smear: set of dyes
LEUKODIF 200 (Lachema) in several different staining cuvettes [color solution 1 (Eosin Y + phosphate buffer pH
6.8) color solution 2 (Azur II + phosphate buffer pH 6.8), rinsing solution (phosphate buffer pH 7.2)], glass slides,
glass slide with brilliant cresyl blue, light microscope, immersion oil.
Procedure:
Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture of ketamine/xylazine
0.5 mL/100 g i.p.) and immobilisation of animal on the table in the supine position perform thoracotomy and take
~1.5 mL blood sample from the cardiac ventricle into the 2 mL plastic heparinised syringe. See the figure on the
top right of the next page for the technique of blood sampling from the cardiac ventricle. Animals will be sacrificed
this way (combination of hypovolemia and pneumothorax after opening the thoracic cavity). Immediately after
sampling, place two drops of blood on two glass slides to prepare a peripheral blood smear for counting white
blood cells (WBCs) and reticulocytes (see further). Then transfer the rest of the blood sample into a tube with anticoagulant (oxalate mixture) and stir gently before further processing. Perform laparotomy, remove the spleen and
weight it.
13
1. Haemoglobin (Hb) concentration (the haemiglobin cyanide method): In principle, Hb is oxidized to haemiglobin (methemoglobin) by potassium cyanoferrate. Potassium cyanide is then used to
convert haemiglobin to hemiglobincyanide. Resulting red-brown
complex is stable and suitable for photometric determination by spectrophotometer. Add 25 uL of blood into a tube containing 7 mL of
transformation solution (Drabkin’s), stir and left for 10 min. Measure
the sample (in the photometric cuvette 1 cm thick) versus blank
(Drabkin’s solution). Simultaneously, measure the extinction of the
standards with known concentrations for a calibration curve. Hb concentration will be expressed in g/L of blood.
2. Haematocrit measurement (the microhaematocrit method): Fill capillaries (75 mm long) with the blood column (horizontally) and centrifuge for 3 min at 100 000 RPM. The separated column
of blood cells will be measured using a reading device with the scale.
3. Manual cell counting (using counting chamber):
The principle of the cell counting consists in the incubation
of the blood with particular hematologic solution whose composition
causes lysis of all other corpuscles but the specified one (e.g.
Hayem’s solution leaves only RBCs intact to count). Such a suspension in the counting chamber with known volume can be then easily
quantified using light microscope (10× or 20× magnification) and
numbers expressed per volume unit.
 RBC count: use a rubber tip to fill a glass micropipette with a scale by blood slightly over the 25 μL
mark. Clear the tip of blood and check the blood column again. Mix the blood with 4975 μL of the
Hayem’s solution (i.e. 200-fold dilution) and incubate for 2 - 3 minutes. Afterwards, shake the solution
gently (to avoid artifact due to sedimentation of cells) and fill the micropipette again. Keep the pipette in
a horizontal position. Let a few drops of blood suspension drain away, then place the tip of the pipette at
the margin of the glass cover of the counting chamber and let the suspension fill the chamber evenly and
form a continuous film between the glass cover and chamber bottom. After ~3 minutes, count RBCs in 20
rectangle fields: RBC =  20 rectangles × 10 000 (number/uL).
 WBC count: Blood (25 μL) is taken analogically as in RBCs (only the solution is different - Türk’s). After 5 minutes WBCs are counted in 50 central squares: WBC =  50 squares × 100 (number/uL).
 Thrombocyte count – according to Piettes: Using a micropipette, add 25L of blood to a flask with 475
L of procain solution (i.e. 20-fold dilution). Allow the diluted blood to stand for at least 20 minutes so
that haemolysis of erythrocytes has time to occur and thrombocytes can stabilize. Then shake the suspension again and pipette it into the counting chamber. Thrombocytes are allowed to sediment for additional
10 min and then counted in 20 rectangles: Platelets =  20 rectangles × 1 000 (number/uL).
4. Additional haematologic parameters (RBC indices) such as mean corpuscular volume (MCV), mean
corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) can be calculated
from the RBC count, concentration of Hb in blood and haematocrit according to these formulas:
 MCV (m3) = [Hct (L/L) × 10] / [RBC count (mil/mL)], indicates an average RBC volume (changed in
normo- or macrocytic anemia),
 MCH (ng) = [Hb (g/L) × 10] / [RBC count (mil/mL)], indicates the average mass of Hb in RBC,
 MCHC (%) = [Hb (g/L)] / [Hct × 100], indicates the average concentration of the Hb in RBC (both MCH
and MCHC changed in normochromic anemia).
5. Preparation of a peripheral blood smear (for the assessment of the white blood cell (WBC) differential
and reticulocyte counts):
WBC differential count: Place a small drop of whole blood on a clean slide about 1 cm from the right
side. Hold the upper (spreader) slide on top of the bottom slide with a drop at the angle of ~30 - 40 (the sharper the
angle, the thinner the smear) on the left side from the blood drop. While maintaining the contact with the bottom
slide pull the top slide back until it touches the drop (which will spread along the edge by the capillary action).
Maintain the firm contact with bottom slide and push the spreader slide forward in one motion (at ~30 angle)
evenly but quickly to produce the smear (the drop of blood must be spread within seconds or the cell distribution
will be uneven). See figure on the top left of the next page for details. The smear should be homogenous, even and
appropriately thin, this requires a certain skill. The proper smear should be thicker the one end, getting thinner and
smoother (feather-like) towards the other edge. Allow the blood smears to air dry for 0.5 - 4 hours. Staining is performed using the LEUKODIF 200 kit (Lachema Brno). Pour the reagents into staining cuvettes. Fix the smear by
dipping it 5-times for 1 sec. into the fixing solution 1. Then, dip the smear 6-times for 1 sec. into the staining solu14
tion 2. Let the solution drain away after every dip. Rinse the slide with the rinsing solution and let it air dry. Numbers of individual WBC types is determined at least per 100
(or better 200) nucleated cells. Inspect the smear systematically (serpentinely) in one
direction. Finally, express the number of the individual types of WBCs (see table below) in percentage.
Reticulocyte count (indirect method with brilliant cresyl blue): Number of
immature RBCs with remnants of RNA in cytoplasm (so called substantia reticulofilamentosa) will be determined by brilliant cresyl blue that will stain RNA remnants to
dark blue, so that reticulocytes can be distinguished from mature RBCs. Prepare the
blood smear similarly to the WBC on the slide covered with a dried drop of 1% of brilliant cresyl blue. Place the blood drop within the drop of dye, mix well, smear and allow it to air dry. Count with
10× magnification and immersion oil. Number of reticulocytes per 1000 RBCs will be counted. Give the result in
‰ (promile).
Results:
The data obtained from all the groups will be compared (i.e. control group vs. animals 3 vs. 20 days following irradiation) and dynamics of changes of peripheral blood parameters will be evaluated. Using the summary
results of all study groups per week perform the statistical analysis using appropriate tests (e.g. Kruskal-Wallis
ANOVA, Mann Whitney, Spearman correlation etc.).
Conclusions:
Based on the results obtained explain the hematologic manifestation of the ARS and its consequences.
Explain the time course of changes in the blood parameters.
15
2.1.3
Experimentally induced atherosclerosis in laboratory animal
Kateřina Kaňková
Aims:
 To explain the pathogenesis of atherosclerosis (AS) with emphasis on the causal role of endothelium.
 To demonstrate cumulative effect of major cardiovascular risk factors – hypertension and hypercholesterolemia – on the progression of AS.
 To demonstrate the longitudinal progression of AS process (vessel remodelling).
Introduction:
Endothelial dysfunction represents crucial moment and probably
causal etiologic factor in the development of AS. Endothelial cells can be
chronically damaged by mechanical (e.g. hypertension, shear stress), chemical (e.g. hyper-cholesterolemia [esp. oxidised LDL particles], reactive oxygen
species, homocystein etc.) or biological agents (some microorganisms). Initially functional impairment gradually starts vascular remodelling involving
all parts of vascular wall (see figure on the right). Increased IMT is a surrogate marker of AS process. Impaired vascular flow and thus perfusion of certain region, or even complete obstruction of the vessel by AS process or, more
often, thrombosis of the AS plaque, can be clinically manifested as a chronic
coronary artery disease (or acute myocardial infarction), chronic ischemic
brain disease (or acute stroke) or peripheral artery disease (or acute artery
occlusion, most often on the lower limbs).
Materials, equipment & animals:
Material: glass container with lid, anaesthetics, scales, immobilisation table for small animals, surgical instrumentarium, physiologic solution, syringes and needles, Fogarty arterial embolectomy catheter (= catheter with
inflatable balloon near its tip, 120 602F, 60cm, balloon volume max. 2ml   4mm, see figure on the left), 0.5%
Evans blue, Bouin’s fixation, alcohol, xylen, paraffin, microtome, haematoxylin-eosin dyes, monoclonal antibodies against endothelial markers
(anti-vWf).
Animals: total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be divided into 2 groups: 1)
controls (catheterisation only, ~1/4 of animals), 2) AS animals (catheterisation of carotid artery + denudation of endothelium, ~3/4 of animals).
The AS groups will be further sub-divided into three models of AS:
 A: catheterisation of carotid artery + denudation of endothelium,
 B: experimentally induced hypertension (by sub-complete ligature of abdominal aorta 1 week in advance)
followed by catheterisation of carotid artery + denudation of endothelium,
 C: experimentally induced hypercholesterolemia (by diet, i.e. enrichment of butter in chow 1 week in advance and continued throughout the experiment) followed by catheterisation of carotid artery + denudation of endothelium.
Procedure:
The whole experiment consists of three steps: 1) experimental induction of atherosclerosis, 2) euthanasia
in different time-periods and processing of vessels for histology, 3) microscopic assessment of slides and image
analysis.
Ad 1) Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture of ketamine/xylazine 0.5 mL/100 g i.p.) and immobilisation of animal on the table in the supine position the large arteries
of the neck (a. carotis communis and its branches) will be accessed by incision on the neck (gently by scissors!)
slightly laterally from the middle line. Schematic topography of the area is shown on the following page figure.
The procedure itself is demonstrated in the next page figure below. A. carotis ext. will be ligated (A) to allow better
manipulation with catheter. Fogarty catheter will be inserted into a. carotis ext. (B) and pushed retrogradely into a.
carotis communis (approx. 1 cm). After inflation of the balloon endothelium will be denudated by gentle pulling
the catheter back with simultaneous rotation (C). A. carotis externa will be ligated below the insertion point (D) to
prevent bleeding. Control animals will be sham operated, i.e. the procedure will be the same except balloon inflation. Finally, incision will be closed by suture (individual stitches). The animals in each groups (controls and A, B
and C models of AS) will be sacrificed in different time points – at 7, 14 and 28 days following the endothelial
denudation. In the meantime, adequate care and nutrition will be provided.
16
Ad 2) Euthanasia will be performed in specified time points by
anaesthesia overdose. Half of the animals in each control or AS group
will be administered 1 hr before euthanasia Ewans blue into the tail vein
(1mg/kg) in order to prepare “native staining” (Ewans blue “leaks” in
sites with incomplete endothelial lining into the deeper layers of vessel
wall and thus marks damaged endothelium). Immediately after euthanasia
pressure perfusion will be established in order to prevent vessels from
collapsing (instillation of physiol. solution via large artery and vein puncture). Subsequently, fixation by the Bouin solution will be carried out for
16 hrs. the same way.
Carotid arteries will be then excised and embedded in paraffin.
Using microtome ~4μm cross-sectional slices will be cut, stained in haematoxylin-eosin and by anti-von Willebrand factor monoclonal antibody
(endothelial marker).
Ad 3) Microscopic evaluation of vessel histology (mounted
slides) will be performed using light (H-E slides) and fluorescent microscope (anti-vWf / DAB / fluorescein). Following parameters will be assessed: (i) intima/media thickness (mm), (ii) lumen diameter (mm), (iii)
lumen area (mm2),
see figure at the bottom of the page.
Results:
Inspect the histology slides stained for endothelium
markers (von Willebrandt factor), collagen and elastin using
light microscope and describe the overall pattern. Measure and
calculate means and standard deviations (SD) for parameter
listed in the table below in each of the four groups (controls,
denudation only, hypertensive and hypercholesterolemic animals) and each of the four time-points (show mean  SD if n >
3 per group). Express data graphically (e.g. box-and-whisker
plots). Using the summary results of all study groups per week
perform the statistical analysis using appropriate tests (e.g.
Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation etc.).
Conclusions:
Interpretation of results – did the denudation of
endothelium started the AS process and why? Was the AS
progression accelerated in the presence of additional risk
cardiovascular factors? What are the major roles of endothelium under the physiologic circumstances and what are
its relevant tools for maintaining the normal morphologic
and functional properties of vessels?
Controls
Model A (normo BP/CH)
IMT (mm)
Lumen 
(mm)
Lumen
area (mm2)
17
Model B (TK)
Model C (CH)
days
7
14
28
7
14
28
7
14
28
2.1.4
ECG monitoring of experimentally induced arrhythmias in laboratory animal –
models of hyperkalemia, hypocalcemia and enhanced adrenergic stimulation
Kateřina Kaňková
Aims:
 To describe and explain the pathogenesis and time course of ECG changes during
adrenergic stimulation, hypocalcemia and hyperkalemia, induced experimentally
by epinephrine, Ca-channel blocker (verapamil) and KCl administered as a i. p.
infusion.
 To compare these model situations with true potentionally life threatening pathophysiological situations in human medicine.
Introduction:
Autonomic nervous system (sympathetic via ß1 adrenergic receptors and parasympathetic via M2 cholinergic receptors) modulates generation of action potential in pacemaker cells of the SA node (chronotropic effect)
and conduction through AV junction (dromotropic effect). Adrenergic stimulation affects also myocardial contractility (inotropic effect).
There are three major pools of calcium in the body. (1) A vast majority of body calcium is in bone. Within bone, 99% of the calcium is tied up in the mineral phase, but the remaining 1% is in a pool that can rapidly exchange with extracellular calcium. (2) A large majority of calcium within cells is sequestered in mitochondria and
endoplasmic reticulum. Intracellular free calcium concentrations fluctuate greatly, from roughly 100 nM to greater
than 1 uM, due to release from cellular stores or influx from extracellular fluid. These fluctuations are integral to
calcium's role in intracellular signalling, enzyme activation and muscle contractions. (3) Extracellular, total plasma
Ca (2.2 - 2.7 mM/L) consists of ~45-50% of ionised Ca (acutely physiologically relevant), the rest is bound to
plasma proteins (mainly albumin) and in complexes with hydrogencarbonate, phosphate, citrate etc. Ca ionisation
is pH-dependent. Plasma Ca is also greatly affected by phosphate concentration (phosphorus binds to Ca, thus elevated phosphates decrease Ca and the product of both concentrations remains constant). Calcium balance in organism is maintained by its appropriate resorption from the intestine (food content + vitamin D), regulated excretion in
the kidney (parathormon) and equilibrium between formation and resorption of the bone (parathormon, calcitonin).
Decrease of plasma ionised Ca leads to the increase of nerve and muscle excitability. Hypocalcemia prolongs repolarisation, hypercalcemia, on the contrary, accelerates it. Ca is also positively inotropic.
Potassium balance in organism (3.8 - 5.5 mM/L in ECF) is influenced by its intake by food vs. renal excretion. Regulated renal excretion is crutial for maintenance of physiological K values and is regulated by the action of several hormones: aldosterone (kidney), insulin (ubiquitous effect) and epinephrin (ubiquitous effect). Under the normal kidney function, even excessive oral intake of K does not produce hyperkalemia. Serious hyperkalemia (>7 mM/L) develops usually during oligoanuric phase of acute renal failure and during chronic renal failure providing K intake is not restricted by the diet. Retention of K is present also in hypocorticalism (M. Addison)
and can be iatrogenically induced by potassium-sparing diuretics. Arrhythmia is the most significant sign of hyperkalemia (development of ECG changes largely depends on the time course and absolute value of potassium) which
can eventually lead to a heart arrest.
Materials, equipment & animals:
Material: glass container with lid, scales, anaesthetics, immobilisation table for small animals, surgical instrumentarium, physiologic solution, syringes and needles, suture material, ECG monitors, solutions: epinephrine 1
mg/mL (Epinefrin Léčiva inj., 1 mg/mL), verapamil 20 mg/mL (Isoptin Abbot, 40 mg/tbl.), KCl 12.5% (Kalium
chloratum, plv.).
Animals: total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be
divided into 3 groups: 1) adrenergic stimulation, 2) hyperkalemia, 3) hypocalcemia. ECG record before administration of exp. substance (epinephrine, KCl or verapamil) will be used as a control recording.
Procedure:
Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture of ketamine/xylazine
0.5 mL/100 g i.p.) and immobilisation of animal on the table in the supine position initial pre-stimulation ECG is
recorded (leads are placed on the limbs using subcutaneous needles). Then canyle will be introduced i.p. (periumiblically) to provide access for slow administration of substances tested. Syringe will be filled with 2mL of the particular solution (epinephrine, KCl or verapamil) and the whole volume slowly administered in fractions of ~ 0.5mL
via the i.p. canyle every 5 – 7 min. ECG record and its changes during the experimental period are continuously
monitored.
18
Results:
Description of the initial (control) ECG record and temporal pattern of changes of the ECG record upon
the exposition to the active substance tested.
Conclusions:
Explain the pathophysiological mechanism of the electrophysiological changes during adrenergic stimulation, hypocalcemia and hyperkalemia and pathogenesis of the relevant medical situations in human medicine.
19
2.1.5
Experimentally induced renal ischemia in laboratory animal as a model of
secondary (renovascular) hypertension
Julie Bienertová-Vašků
Aims:
 To induce renal ischemia in laboratory animal as a model of one of the common types of secondary hypertension.
 To evaluate macroscopic changes of the kidneys.
 To perform microscopic observation of the renin granules in immunohistochemistry specimens taken
from ischemic vs. control kidney.
Introduction:
The restriction of perfusion within the renal parenchyma is associated with activation of the reninangiotensin-aldosterone system (RAAS). While sudden critical decrease of blood flow to kidney may result in the
acute renal failure (ARF), gradual decline of renal perfusion leads – after the failure of compensatory mechanisms to the development of chronic kidney disease (CKD) and secondary renovascular hypertension. Such situation
leads to the disturbances of the local autoregulatory mechanisms - decreased production of vasodilation agents
(NO, prostacyclins) and release of vasoconstrictive agents, such as endothelin – as well as changes of the systemic
haemodynamics since renal hypo-perfusion is falsely interpreted as a systemic hypovolemia. Activation of RAAS
in the underperfused kidney results in the increase of the systemic blood pressure (effect of the angiotensin II)
which increases glomerular filtration pressure and thus GFR in the opposite kidney providing it is intact. Resulting
secondary hypertension is therefore of pressoric normovolemic type.
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be
divided into 2 groups: 1) controls (sham operated), 2) unilateral ligation of a. renalis.
Material: glass container with lid, scales, anaesthetics, immobilisation table for small animals, surgical instrumentarium, light microscope.
Procedure:
The whole experiment consists of two steps: 1) experimental induction of unilateral renal ischemia followed by 2) euthanasia and processing of kidneys for macroscopic evaluation and histology 3 weeks later.
Ad 1) Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture of ketamine/xylazine 0.5 mL/100 g i.p.) and immobilisation of animal on the table in the supine position laparotomy is
performed in the middle plane from the processus xiphoideus to the symphysis. V. cava inferior should be separated from the aorta in the middle of the branching of a. renalis dx. and sin. (note: left a. renalis stems from aorta more
distally than the right one, therefore, to make it easier, experiment is
modified in that way that ligation is placed on the aorta between the
origin of both renal arteries). For details see figure on the right. Using
semicircular forceps carefully pull the suture thread below the aorta. Before fixing lay a blunted needle (0.5 mm diameter) carefully along the
aorta parallely with the axis of the vessel and fix the ligature of the aorta
together with the needle using a surgical knot. Needle is then removed.
This procedure should cause partial ischemia of the distal left kidney
leaving the proximal right kidney intact. The abdominal cavity is closed
in two layers.
During the 3 weeks of follow-up compensatory mechanisms
will take place. While the ischemic kidney decreases in size and initiates
the development of the secondary hypertension (hypertrophy of the juxtaglomerular apparatuses with stimulated production of the renin from the
granular cells), intact kidney becomes hypertrophic.
Ad 2) After three weeks, animals will be euthanized (anaesthetic overdose). Perform laparotomy and bilateral nephrectomy. Both kidneys are cleansed and weighted along with the myocardium. The histological analysis for the presence of the granular renin-producing cells in
the biopsy specimens taken from kidneys of control and experimental
animals is performed using slides stained with anti-renin antibody and
light microscope.
20
Results & Conclusions:
Describe the changes induced by the unilateral stenosis of. a. renalis in the kidneys and the heart. Compare the parameters – organ weights – between control and exp. animals. Using the summary results of all study
groups per week perform the statistical analysis using appropriate tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation etc.). Explain the pathophysiological mechanism of this type of secondary hypertension
and explain the other possible types of secondary hypertension due to kidney disease.
21
2.1.6
Experimentally induced acute renal insufficiency in laboratory animal –
measurement of GFR based on kinetics of renal inulin excretion
Michal Jurajda
Aims:
 To demonstrate the principle of GFR measurement using the single-bolus inulin clearance test.
Introduction:
Glomerular filtration rate (GFR) reflects functional capacity of the kidneys. The decrease of number of
functional glomeruli can be detected as a GFR decrease. GFR can be estimated by the renal clearance of certain
substances – those that are solely filtered, but not reabsorbed or secreted by the kidneys. Most commonly used endogenous substances for such measurement is creatinin. There are also several exogenous substances available
(such as polysaccharide inulin). Creatinin clearance measurement consists of one venous blood sample withdrawal
following a 24-hour (or shorter) urine collection. The clearance is then calculated using simple equation [cplasma ×
Vplasma = curine × Vurine; where Vplasma equals to the volume of plasma cleared of creatinin, i.e. approx. GFR]. When
using exogenous substances (e.g. inulin) for GFR measurement single bolus or continuous infusion with several
venous blood samples are required but no urine collection is needed. Excretion of such substance follows exponential curve and rate of clearance is calculated using regression analysis.
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be
divided into 2 groups: 1) unilateral ligation of a. renalis to acutely decrease GFR – at least 5 animals per group for
the time-point measurement, 2) controls (sham operated) – at least 5 animals per group for the time-point measurement.
Material: glass container with lid, anaesthetics, scales, immobilisation table for small animals, surgical instrumentarium, centrifugation tubes, micropipettes, tips, centrifuge, water bath, spectrophotometer. Solutions:
physiologic saline solution, inulin 25 mg in Eppendorf tube, inulin test kit (75 mM solution of beta-indolacetic
acid, concentrated HCl, 0.5 % TRITON X-100).
Procedure:
Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture ketamine/xylazine 0.5
mL/100 g i.p.) and immobilisation of animal on the table in the supine position laparotomy is performed in the
middle plane from the processus xiphoideus to the symphysis. A. and v. renalis on one side should be isolated and
ligature placed underneath both vessels. The ligature should be tightened in experimental group, in control group
the ligature will be left loose.
The inulin will be administered to both exp. and control animals in a single bolus to the jugular vein. The
v. jugularis will be accessed by incision on the skin (gently by scissors!) slightly laterally from the middle line (see
figure on the right). The needle is introduced intravenously in the caudal
part of v. jugularis covered by m. pectoralis (compression prevents
bleeding) and inulin suspension is administered slowly in the volume of
0.1 mL/100 g body weight. The vessel is fragile and should not be
traumatised (not even by aspiration which is otherwise necessary when
administering i.v.). The time will be measured from that point. Animals
in each group will be sacrificed successively in a defined time point (5,
10, 15, 20 and 30 min) by thoracotomy (pneumothorax) and cardiac
puncture to obtain sample of blood for the inulin measurement. Blood
samples will be centrifuged for 10 minutes at 3000 RPM. The inulin
concentration will be determined in the sera using the kit (50 μL of beta-indolacetic acid + 1.5 mL of conc. HCl + 50 μL of sera). Specimens
will be gently mixed, incubated for 10 min in 60°C and then mixed
with 500 μL of 0.5 % TRITON X-100. Absorbance will be measured at
520 nm using spectrophoptometer.
Results:
Place the results in the table (show mean  SD if n > 3 per
group) and express them graphically. Half-time of inulin excretion will
be calculated using semi-logarithmic paper or by fitting exponential
curve using MS Excel. Using the summary results of all study groups
per week perform the statistical analysis using appropriate tests (e.g.
22
Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation, etc.).
Inulin concentration (given as an absorbance at 520 nm)
5 min
10 min
15 min
20 min
30 min
Control group
Exp. group
Conclusions:
Compare variable methods applicable to determine GFR in human medicine. Discuss their interpretation,
sensitivity, specificity and diagnostic potential. What are the major physiologic factors determining GFR in a given
subject?
23
2.1.7
Experimentally induced renal failure - peritoneal dialysis in laboratory animal
Julie Bienertová Vašků
Aims:
 To demonstrate that normal peritoneum has diffusion properties that can be used as an administration/excretion way to/from the body (e.g. for administration of drugs or dialysis in case of end-stage renal
disease).
 To demonstrate that kidneys are an important organ in the potassium homeostasis and peritoneum might
be an interface affecting potassium level in the organism when solutions with variable potassium concentration are administered intraperitoneally.
 To demonstrate the efficiency of peritoneum in therapeutic management of hyperkalemia in nephrotoxic
model of acute renal failure (ARF).
Introduction:
Peritoneum is a semipermeable membrane with the surface which approximately equals the body surface
area and blood flow of roughly 70 mL/min. Therefore, peritoneal dialysis represents an alternative tool to conventional (hemo)dialysis in which similar principles are employed (diffusion and filtration). The great advantage of the
peritoneal dialysis is the better quality of life of the patient, more stable homeostasis and the absence of blood losses; however, the greater disadvantage/risk of peritoneal dialysis consists in an increased risk of bacterial peritonitis.
Properties of the peritoneum as a dialysis membrane will be demonstrated in the animal model of ARF
induced by nephrotoxic substance (ethylene glycol). Following the infusion of solution with different concentration
of K+ (hypo-, normo- and hyperkalemic) animals capable of handling the potassium concentration (controls with
healthy kidneys) and exp. animals (ARF) will be monitored using electrocardiograph (ECG) to observe eventual
changes of heart rate, automacy and conductivity (arrhythmia) corresponding to hyperkalemia.
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be
divided into 2 groups: 1) nephrotoxin-induced acute renal failure, 2) controls (healthy), at least 3 animals per group
are necessary for the 3 kalemia solutions (hypo-, normo- and hyperkalemic).
Material: glass container with lid, scales, anaesthetics, immobilisation table for small animals, surgical instrumentarium, cannula (for human peripheral vein access), ECG monitor with limb leads, ethylene glycol, physiological solution, solution for peritoneal dialysis, 7.5% solution of KCl, 0.1M solution of HCl, bromphenol violet
(pH indicator 5.0 – 6.8), glass capillary for acid-base balance examination.
Procedure:
Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture of ketamine/xylazine
0.5mL/100g i.p.) and immobilisation of animal on the table in the supine position the blood sample will be withdrawn (from the superficial incision of the tail vein) for determination of
the initial values of acid-base balance (pH). Subsequently, either solution of ethylene glycol (15 mL/100 g body weight) or physiological solution (equal volume) will be administered to exp. or control animals,
respectively, in a single bolus to the jugular vein. The jugular vein in the
neck will be accessed by incision of the skin (gently by scissors!) slightly laterally from the middle line. Schematic topography of the area is
shown in the figure on the right. The needle is introduced intravenously
in the caudal part of v. jugularis covered by m. pectoralis (compression
prevents bleeding) and ethylene glycol solution is administered slowly.
The vessel is fragile and should not be traumatised (not even by aspiration which is otherwise necessary when administering i.v.). Control animals are operated exactly the same way but physiologic saline solution
of the same volume is administered instead.
After 15 minutes, a cannula with needle will be introduced intraperitoneally, i.e. in approx. half a distance between spina iliaca anterior and navel. After having penetrated the abdominal wall, the guide
needle will be removed and the cannula will be inserted further (approx.
1 cm) into the abdominal cavity and fixed with adhesive tape. ECG record will be monitored using limb leads placed subcutaneously in usual
setting. Heart rate and traditional ECG characteristics (e.g. PQ interval,
el. axis, QRS interval, ST segment denivelisation, etc.) will be de-
24
scribed.
Dialysis solutions will be prepared from the stock solution for
peritoneal dialysis and KCl to final concentrations of 0 (hypokalemic),
4.0 (normokalemic) and 10.0 mM/L (hyperkalemic). Different animals
in both control and ARF groups will be given dialysis solutions with
different concentration of K+ (i.e. 0, 4.0 and 10 mM/L) via the intraperitoneal access (the total amount of solution infused into abdominal cavity is up to 30 mL). ECG will be continuously monitored and all changes
observed recorded. After the onset of sustained arrhythmia (most likely
in the hypo- and hyperkalemic subgroup of ARF) another blood sample
will be withdrawn from the tail vein and analyzed again for the acidbase balance parameters.
Finally, the thoracotomy will be performed and sample of
blood taken by intracardial punction (see figure on the right) to obtain at
least 1.5 mL of blood to determine alcalic reserve. The blood is centrifuged (10 minutes at 3000 RPM), plasma then separated and acidometric measurement of alcalic reserve performed: 500 L of plasma + 10
L of indicator (bromphenol violet) titrated with 0.1M HCl (stepwise
by 1.0 L). Consumption of HCl is compared with that used for standards of known alcalic reserve.
Results:
Compare the initial and end-stage values of acid-base balance parameters between particular models and
groups (show mean  SD if n > 3 per group). Using the summary results of all study groups per week perform the
statistical analysis using appropriate tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation
etc.).
Conclusions:
Summarize the overall physiological mechanisms involved in the potassium homeostasis. Explain the
pathophysiological mechanisms of hyperkalemia in ARF and its effect of the excitable tissues such as heart and
skeletal muscle. Compare the effect of exogenous manipulation of potassium homeostasis in both control vs. exp.
animals. Explain the mechanism of nephrotoxicity of ethylene glycol and other medically-relevant substances.
25
2.1.8
Experimentally induced peptic ulcer in laboratory animal
Michal Jurajda
Aims:
 To study the pathogenesis of the gastroduodenal (peptic) ulcers using animal model of experimentally
(pharmacologically) induced ulcers by non-steroidal anti-inflammatory drugs (NSAID).
 To elucidate the effect and mode of action of the H2 blockers on the development of peptic ulcers.
Introduction:
Gastric juice contains very aggressive mixture of enzymes and hydrochloric acid (HCl). Physiological
mucosal surface of the gastric lumen provides a protective barrier preventing stomach wall from being selfdigested. This barrier is formed predominantly by mucus produced by stomach itself. Increased concentration of
the hydrogencarbonate ions in the gastric circulation (due to the opposite active secretion of the acid into the lumen) plays an important role too. Mucus is produced by epithelial cells lining the entire luminal surface of the
stomach. Production of mucus is influenced by many factors, among others by prostaglandins. NSAID decrease
mucus production by inhibiting cyclooxygenase and thus production of prostaglandins. Therefore, chronic use of
NSAID can lead to the development of gastric erosions or even gastric ulcers. Unlike erosion, gastric ulcer reaches
and penetrates muscularis mucosae layer of stomach wall. The gastric ulcer can lead to the erosion of blood vessels
producing life threatening bleeding or even penetrate through all layers of the stomach wall resulting in acute peritonitis.
Gastric ulcer development can be provoked in animal model by administration of the NSAID. This is also
one of the most common contributing factors in humans. Such model can further serve to demonstrate the effect of
anti-ulcer drugs with various mode of action, e.g. H2-blockers (antagonists).
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be
divided into 2 groups: 1) controls (without H2-blocker), 2) exp. animals given anti-ulcerative protection by H2blocker prior to the induction of the peptic ulcer.
Material: glass container with lid, anaesthetics, scales, immobilisation table for small animals, surgical instrumentarium, gastric tubes. Solution & drugs: physiological saline solution, H2-blocker (ranitidine), NSAID (indomethacin), cork pad, pins, glass plates, fixation solution.
Procedure:
The whole experiment consists of two steps: 1) experimental induction of gastric ulcer, 2) euthanasia after
24 hrs. and processing the tissue for histology.
Ad 1) Animals will be fasting 12 hrs prior to experiment. Following slight inhalatory anaesthesia (diethyl
ether) an H2-blocker (25 mg/mL ranitidine, dose 0.4 mL/100 g of body weight) or physiological saline solution
(dose 0.4 mL/100 g) will be applied by gastric tube to either experimental or control animals, respectively. After
0.5 hour, animal will be narcotised again and 1.0% suspension of NSAID (capsule of indomethacin diluted in 5 mL
of distilled water, dose 1 mL/100 g) will be administered to both groups using the stomach tube.
Ad 2) 24 hours later, all animals will be sacrificed by diethylether overdose. Following laparotomy, stomach will be removed and cut along the greater curvature. The mucosa will be washed to remove the mucus and
event. blood. The stomach will be flattened by pressing between two glass plates, lesions will be inspected and
counted. Stomach will be then spread on a cork pad, fixed by pins, mucosa upwards, and fixed in 10% formaldehyde for 24 hrs. Fixed material can be processed for an image analysis the next day (digital camera, software ImageJ).
Results:
Using digitalised pictures in standardised resolution and original size following parameters will be assessed (show mean  SD if n > 3 per group): 1) number of lesions per stomach, 2) total area of the stomach mucosa
(cm2) and 3) area of the stomach mucosa damaged (occupied) by lesions (cm2). Percentage of affected area can be
thus calculated. Compare the results between groups with and without H2-blocker protection. Using the summary
results of all study groups per week perform the statistical analysis using appropriate tests (e.g. Kruskal-Wallis
ANOVA, Mann Whitney, Spearman correlation etc.).
Conclusions:
Explain the mechanism of secretion of HCl into the gastric lumen and concurrent physiologic protective
mechanisms. Explain the mode of action of the NSAID as a risk factor and H2-blocker as a protection agents.
26
2.1.9
Quantification of gastric juice secretion after its pharmacological manipulation
in laboratory animal
Michal Jurajda
Aims:
 To elucidate mechanisms involved in the regulation of gastric acid secretion.
 To investigate the ability of H2-receptor antagonist (ranitidine) to reduce gastric secretion of HCl in a
dose-dependent fashion.
Introduction:
The gastric acid secretion is regulated by various mediators acting in endocrine and/or paracrine fashion
(acetylcholine, gastrin, histamine, somatostatin, etc.). Absolute or relative hyperacidity plays an important role in
the development of gastric ulcer. HCl production can be modulated by various drugs (antacids) used to treat dyspepsia, gastroduodenal ulcers, gastroduodenal reflux and Zollinger-Ellison syndrome or to prevent their recurrence
and allow healing. Gastric HCl production can be most efficiently blocked by direct proton pump inhibitors (e.g.
omeprazole). Omeprazole directly and irreversibly inhibits the transport of protons by the proton pump (H+/K+
ATPase) in a dose- and pH-dependent fashion (i.e. organ-specific for stomach). Part of the effect is probably due to
the carboanhydrase inhibition as well.
Another therapeutic possibility involves interference with the signal substances which activate parietal
cells to produce HCl, namely histamine released from ECL cells. H2-receptor antagonists (such as cimetidine,
ranitidine or famotidine) are widely used as a prophylactic and long-term treatment of the above mentioned gastrointestinal conditions.
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be
divided into 2 groups: 1) controls (without H2-blocker), 2) exp. animals given anti-ulcerative protection by H2blocker prior to the induction of the peptic ulcer.
Material: glass container with lid, anaesthetics, scales, immobilisation table for small animals, surgical instrumentarium, gastric tubes. Solution & drugs: physiological saline solution, H2-receptor antagonist (ranitidine),
0.01M NaOH, 1% phenolphthalein in alcohol, micropipette 0.1 mL, burette, Erlenmeyer flask, calibrated centrifugation tubes, centrifuge.
Procedure:
Animals should be fasting for 48 hour prior to the experiment. Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture of ketamine/xylazine 0.5 mL/100 g i.p.) an H2-receptor antagonist (25
mg/mL ranitidine, dose 0.4 mL/100 g) or physiological saline solution (dose 0.4 mL/100 g) will be applied to their
dorsal region by the subcutaneous injection. After immobilisation of an animal on the table in the supine position
laparotomy is performed in the middle plane from the processus xiphoideus to the symphysis. The stomach will be located, slightly pulled
out and lifted using surgical forceps to visualize pylorus around which
the ligature should be placed and tightened. The immediate content of
the stomach will be aspired using 5 mL syringe to prevent the introduction of the uncontrolled error.
Meantime the animal is left in anaesthesia with occasional
moistening of intestinal loops by physiologic saline solution. Then, 1
hour after the ligation of the pylorus, the oesophagus entry into the
stomach in the cardia is accessed and ligated and the whole isolated
stomach removed (see figure on the right). The cumulated content of
the stomach will be emptied into calibrated centrifugation tubes. The
volume of the gastric juices will be measured after centrifugation (Vstomach). The HCl concentration in the gastric juice will be measured using
alkalimetric titration.
Titration: an aliquot of 0.1 mL of the gastric juice will be diluted in 0.9 mL of the distilled water and one drop of indicator (phenolphthalein) will be added to this mixture. Mixture will be titrated with
0.01M NaOH (adding a small drop at the time) until the pink colour
appears and stays permanent. The consumptions of NaOH will be recorded (VNaOH).
27
Results:
Concentration and production of HCl can be calculated using ascertained variables as follows:
A) Known/measured variables:
 Volume of the content of the stomach: Vstomach (mL)
 Consumption of NaOH: VNaOH (mL)
 Weight of the rat: m (g)
B) Calculated parameters:
 cHCl (mM/L)
 production of HCL per hour per 100 g of body weight: p (M/h/100 g)
Calculation:
cHCl 
p
VNa OH  cNa OH

Vstomach
....... mL  ....... M / L
 ................M/L =.................mM/L
....... mL
Vstomach  cHCl ....... L  ..... mM / L

 .............mM/h/100g =..........M/h/100g
tm
1hour  ....... 100g
Express the results in the table and graphically (show mean  SD if n > 3 per group). Compare the two
groups with respect to the H2-receptor antagonist presence/absence. Using the summary results of all study groups
per week perform the statistical analysis using appropriate tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney,
Spearman correlation, etc.).
Weight (g)
Vstomach (mL)
VNaOH (mL)
cHCl (mM/L)
pHCl (M/h/100 g)
Controls
H2-blocker
Conclusion:
Discuss the observed results and the effect of H2-receptor antagonists. Explain the physiologic regulation
of gastric acid secretion and modalities available for its therapeutic management.
28
2.1.10 Experimentally induced diabetes mellitus in laboratory animal – principles of
diagnostics by glucose tolerance test
Kateřina Kaňková
Aims:
 To demonstrate the principle of glucose tolerance test (GTT) as one of the essential diagnostic and
screening tools for diagnosis of diabetes mellitus (DM).
 To demonstrate the interpretation of GTT in experimental settings upon induction of insulinopenia (i.e.
T1DM-like state) in laboratory animal.
glycemia (mmol/L)
Introduction:
DM is a collective name for the syndrome characterized by deficient action of insulin. There are two possible reasons for the deficient action: (1) absolute lack of insulin due to autoimmune destruction of β-cells of Langerhans islet of pancreas (clinically equals to Type 1 DM) and (2) relative deficiency due to concomitant insulin
resistance in peripheral tissues normally sensitive to insulin and (i.e. muscle, liver and adipose tissue) and impaired
insulin secretion due to gluco- and lipotoxicity to pancreatic β-cells (Type 2 DM). T1DM and T2DM are the most
common primary forms; however, there are other forms of DM such as monogenic, secondary and gestational
forms.
Fasting plasma glucose (FPG) should be below 6.1 mM/L (100 mg/dL) in normal subject. Manifest DM
is characterised by FPG ≥ 7 mM/L (126 mg/dL) in venous blood after approx. 12-hr fasting. Repeated finding (at
least twice on two independent occasions) is usually sufficient for DM diagnosis. However, there are early “borderline” stages of DM with FPG above normal (6.1 – 7 mM/L) and yet with still preserved regulation of postprandial
glucose metabolism (i.e. impaired fasting glucose, IFG) or normal FPG (< 6.1 mM/L) but delayed postprandial
normalisation (i.e. impaired glucose tolerance, IGT). GTT can help to distinguish between physiological glucose
tolerance, IFG, IGT and DM. It is ideal tool for screening and diagnostic purposes. The test is normally performed
orally (oGTT), i.e. subject tested receives the
12
standard load of 75 g of glucose diluted in water
diabetes
11
or tea which should be drank within 5 minutes,
IGT
11.1
11.1
10
venous blood is sampled after 60 and 120
IFG
9
minutes following ingestion. Glycemia in the
normal
th
120 minute is decisive for the DM diagnosis 8
glucose level should be normally below 7.8
7 7.0
7.8
7.8
mM/L (140 mg/dL). Levels between this and
6 6.1
11.1 mM/L (200 mg/dL) indicate IGT; glucose
5
levels above 11.1 mM/L (200 mg/dL) at 2-hours
4
confirms a diagnosis of DM (see figure on the
3
right). Note: values given hold for venous blood,
FPG
60 min
120 min
should capillary blood be used for glucose measurement the reference values would be different.
In case of exp. animals, the procedure suited for humans will have to be slightly modified – we would of
course not succeed to convince the animal to drink such sweet solution orally in a defined time period so intraperitoneal application of 20% solution of glucose will be used instead. Also, owing to the faster metabolic rate in small
rodents, blood will be sampled from the tail vein in 0, 30th and 90th minutes.
There are several possibilities available how to induce DM in experimental settings in animals. T1DM is
commonly modelled by administration of substances toxic for insulin-producing cells such as alloxan1 or streptozotocine2. Both substances exhibit selective toxicity for pancreatic β-cells and eventually lead to the absence of
insulin secretion (insulinopenia) and therefore T1DM (after the short period of stimulation – marked by hyperinsulinemia and hypoglycaemia – cause selective destruction of β-cell mediated by the enhanced production of reactive
oxygen species, thus oxidative damage of insulin producing cells).
1
Alloxan and streptozotocin are toxic glucose analogues that preferentially accumulate in pancreatic beta cells via the GLUT2 glucose transporter. In the presence of intracellular thiols, especially glutathione, alloxan generates reactive oxygen species (ROS) in a cyclic redox reaction with its reduction product, dialuric acid. Autoxidation of dialuric acid generates superoxide radicals, hydrogen peroxide and, in a final iron-catalysed reaction step, hydroxyl
radicals. These hydroxyl radicals are ultimately responsible for the death of the beta cells, which have a particularly low antioxidative defence capacity, and
the ensuing state of insulin-dependent ‘alloxan diabetes’. As a thiol reagent, alloxan also selectively inhibits glucose-induced insulin secretion through its
ability to inhibit the beta cell glucose sensor glucokinase.
2
Following its uptake into the beta cells, streptozotocin is split into its glucose and methylnitrosourea moiety. Owing to its alkylating properties,
the latter modifies biological macromolecules, fragments DNA and destroys the beta cells, causing a state of insulin-dependent diabetes. The targeting of
mitochondrial DNA, thereby impairing the signalling function of beta cell mitochondrial metabolism, also explains how streptozotocin is able to inhibit glucose-induced insulin secretion.
29
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be divided into 2 groups: 1)
controls (healthy), 2) diabetic - alloxan induced DM.
Material: glass container with lid, anaesthetics, scales, immobilisation table for small animals, surgical instrumentarium, glucometers (developed to be used for home self-monitoring of glycemia
by diabetics or for quick glycemia measurement in outpatient clinics or
terrain) with reagent strips for glycemia measurement, reagent strips
for the detection of glucose and ketone bodies in the urine (MelliPhan,
GlukoPhan, KetoPhan PLIVA Lachema), desinfection, Petri dish.
Solutions: alloxan in the physiological saline solution (13 mg/mL),
glucose (20%).
Procedure:
The whole experiment consists of two steps: 1) experimental
induction of DM, 2) GTT after one week of development of insulinopenia.
Ad 1) Experimental induction of DM: Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture ketamine/xylazine 0.5 mL/100 g i.p.) and immobilisation of animal on the
table in the supine position the jugular vein on the neck will be accessed by incision of the skin (gently by scissors!) slightly laterally
from the middle line. Schematic topography of the area is shown in the figure on the right. The needle is introduced
intravenously in the caudal part of v. jugularis covered by m. pectoralis (compression prevents bleeding) and alloxan solution is administered slowly in the volume 0.1 mL/100g body weigh. The vessel is fragile and should not be
traumatised (nor even by aspiration which is otherwise necessary when administering i.v.). Control animals are
operated exactly the same way but physiologic saline solution is administered instead of alloxan. Skin incision is
sutured by individual stitches. In the meantime, adequate care and nutrition will be provided (adequate hydration is
essential due to developing hyperglycaemia and thus osmotic diuresis).
Ad 2) GTT: One week later animals are anesthetised as usually. Immediately after the onset of general
anaesthesia FPG is measured in the venous blood obtained from the tail vein incision (gently with razor after disinfection of the tail) using glucometer (following the manufacturer’s instructions). Subsequently, a dose of 1mL/100g
of 20% glucose will be applied i.p. Glycemia will be measured the same way as FPG in the 30th and 90th minute
after the administration. In the meantime animal is placed ventrally over the Petri dish to collect urine. Finally,
presence of glucose and ketone bodies in the urine will be detected using reagent strips.
Results:
Place data (GTT) in the table and calculate means for the different time points and groups (show mean 
SD if n > 3 per group). Express data graphically as “glycemic curves” representing the profile of glycemia over the
test time. Using the summary results of all study groups per week perform the statistical analysis using appropriate
tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation, etc.).
FPG (mM/L)
30th min (mM/L)
90th min (mM/L)
U-glucose
U-ketone bodies
Controls
Diabetics
Conclusions:
Explain the pathophysiological mechanism of DM development following the administration of the substance toxic for β-cells. Discuss the possible causes (differential diagnosis) of hyperglycaemia, glycosuria and ketoacidosis.
30
2.1.11 Experimentally induced haemolytic, hepatotoxic and obstruction
hyperbilirubinemia/jaundice in laboratory animal
Lukáš Pácal
Aims:
 To explain the ethiopathogenesis of various types hyperbilirubinemia.
 To demonstrate the principle of differential diagnostics of various types hyperbilirubinemia.
 To explain the terms hyperbilirubinemia vs. icterus.
Introduction:
Bilirubin is the final product of haem degradation. Bilirubin metabolism begins with the breakdown of
red blood cells in reticuloendothelial system. Red blood cells contain haemoglobin, which is broken down to haem
and globin. Haem is converted to bilirubin, which is then carried bound to albumin in the blood to the liver. Since
bilirubin is highly insoluble in water, it must be converted into a soluble conjugate prior to elimination from the
body. In the liver, enzyme uridine diphosphate-glucuronyl transferase converts bilirubin to a mixture of monoglucuronides and diglucuronides, referred to as conjugated bilirubin, which is then secreted via the bile to the small
intestine. This process is normally highly efficient; therefore plasma unconjugated bilirubin concentration remains
low. Unconjugated bilirubin used to be called indirect bilirubin. Total serum bilirubin consists of unconjugated
(indirect) bilirubin and conjugated (direct) bilirubin (reference values: total Bi 5 – 17 μM/L, conjug. Bi 1 – 5
μM/L). Conjugated bilirubin is released into the bile by the liver and stored in the gallbladder, or transferred directly to the small intestine. Bilirubin is further broken down by bacteria in the intestine (urobilinogen and further stercobilinogen) and those breakdown and oxidised products (stercobilin) contribute to the colour of the faeces. A
small percentage of the urobilinogen are reabsorbed back again by intestinal cells and eventually appear as an oxidised urobilin in the urine. The kidneys do not filter unconjugated bilirubin because of its firm binding to albumin.
For this reason, the presence of bilirubin in the urine indicates the presence of conjugated hyperbilirubinemia. Hyperbilirubinemia refers to the finding of total Bi > 17 μM/L. Unconjugated hyperbilirubinemia is a result of impaired or overloaded conjugation (hemolysis, drug-induced, neonatal, hereditary diseases such as Crigler-Najjar or
Gilbert syndromes). Conjugated hyperbilirubinemia results from reduced secretion of conjugated bilirubin into the
bile or damage of hepatocytes (e.g. in patients with hepatitis) or from impaired flow of bile into the intestine (e.g.
biliary obstruction). For distinction between ethiopathogenic types of hyperbilirubinemia see table below. Icterus is
an objective symptom and means the yellow discoloration of skin, mucose membranes and the sclera of the eye,
which occurs when bilirubin accumulates in the blood over the threshold approximately 30 – 50 μM/L.
Total bilirubin
Conjugated bilirubin
Unconjugated bilirubin
Urobilinogen
Urine/stool colour
Biliary enzymes (ALP)
Liver enzymes (ALT, AST)
Pre-hepatic

normal


normal / normal
normal
normal
Hepatic

normal / 
normal / 
normal / 
dark / normal
normal

Post-hepatic


normal
 (negative)
dark / pale

normal
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be
divided into 2 groups: 1) controls (~1/4 of animals), 2) three icterus models rendered hyperbilirubinemic 1 week
prior the bilirubin determination (~3/4 of animals) by: A) the obstruction of bile flow (i.e. by ligation of ductus
choledochus), B) chemically induced acute hemolysis (phenylhydrazine, C6H5NHNH2), and C) toxic damage of
liver parenchyma (tetrachlormethan, CCl4).
Material: Glass container with lid, scales, anaesthetics, immobilisation table for small animals, surgical
instrumentarium, physiologic solution, syringes and needles, centrifugation tubes, phenylhydrazine, tetrachlormethane, set for the determination of plasma bilirubin, Heptaphan reagent strips.
Procedure:
The whole experiment consists of two steps: 1) experimental induction of hyperbilirubinemia, 2) objective and biochemical evaluation of hyperbilirubinemia/icterus.
Ad 1) Approximately 1 week prior the experiment itself animals will be rendered hyperbilirubinemic (icteric) by the following procedures to obtain 3 models: A) obstructive, B) haemolytic and C) hepatotoxic icterus.
31
Model A: Following general anaesthesia (initial diethyl ether
inhalation + anaesthetic mixture of ketamine/xylazine 0.5 ml/100 g i.p.)
and immobilisation of animal on the table in dorsal position the middle
laparotomy will be performed. Upon opening the abdominal cavity,
liver, stomach and duodenum will be located. Ductus choledochus can
be found (together with vena portae) in the junction of the liver undersurface and duodenum. Carefully separate ductus choledochus from the
vena portae and ligate it (see figure on the right). Laparotomy will be
closed by suture (in 2 layers), wound disinfected and rats will be placed
in the cage until the end of anaesthesia. In the meantime, adequate care
and nutrition will be provided. Next stage of the experiment follows
after 1 week.
Model B: Hemolysis will be induced by i.p. application of
phenylhydrazine (0.2 mL of 3% phenylhydrazine solution, 6-times during the 3-week period in regular intervals).
Model C: Single-dose application of tetrachlormethan (0.75
mL 48 hours before the determination of bilirubin) into the stomach by
nasogastric tube will elicit damage of liver parenchyma and subsequent
hyperbilirubinemia and icterus.
Ad 2) Diagnostics of hyperbilirubinemia/icterus (all experimental groups): Following general anaesthesia (initial diethyl ether
inhalation + anaesthetic mixture of ketamine/xylazine 0.5 mL/100 g
i.p.) observe the colouring of ears, paws and tail in experimental animals and compare with controls. Compare the
weight of the animal before and after intervention. Restrain the rat on the operating table in the dorsal position and
perform urinary bladder punction to obtain a urine sample for determination of bilirubin and urobilinogen using the
HeptaPhan reagent strips. Further, perform the middle laparotomy and thoracotomy (this way animals will be euthanized by pneumothorax) and collect blood from the heart chambers into the 2 mL syringe. Transfer the blood
from the syringe (without a needle!) to a plastic centrifugation tube and centrifuge for 10 minutes to obtain serum
for further analyses. Note the colouring of the serum in experimental and control animals. The total and conjugated
bilirubin concentration in serum will be measured spectrophotometrically. Finally, remove the liver and weigh it.
Results:
Put the results in the table and compare the various models (show mean  SD if n > 3 per group). Using
the summary results of all study groups per week perform the statistical analysis using appropriate tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation, etc.).
Controls
A. Obstructive
B. Haemolytic
C. Hepatotoxic
P
Weight - baseline (g)
Weight - end-point (g)
U-bilirubin
U-urobilinogen
S-total Bi (μM/L)
S-conj. Bi (μM/L)
Liver weight (g)
Conclusions:
Describe the pathophysiological mechanisms behind the observed changes. Summarize the diagnostic algorithm for the differential diagnostics of hyperbilirubinemia/icterus.
32
2.1.12 Experimentally induced venous thrombosis in laboratory animal
Anna Vašků
Aims:
 To demonstrate the role of the following common situations in medicine - a) blood stasis or its slower circulation, b) increased coagulation or decreased fibrinolytic potential, c) endothelial dysfunction or damage – as precipitating factors for venous thrombosis.
 To elucidate the effect of exogenous heparin (a homolog of endogenous antithrombin III cofactor) as a
powerful anticoagulant substance with broad therapeutic and prophylactic potential in the model of experimentally induced thrombosis.
 To explain principles of routine coagulation tests.
Introduction:
When a blood vessel is injured, platelets and fibrin form a blood clot (i.e. primary and secondary hemostasis) to prevent a loss of blood. Hemostasis is also the first step in tissue repair and healing. If that mechanism
causes too much clotting, becomes initiated without vessel injury or the clot breaks free, this is called thrombosis.
Thrombosis is the unwanted, pathologic formation of a blood clot (thrombus) inside a blood vessel, obstructing the
blood flow. Thromboembolism is a general term describing both thrombosis and its main complication – embolism
(i.e. migrating part of the thrombus). When a thrombus occupies more than ~75% of the surface area of the vessel
lumen, perfusion becomes seriously compromised to cause symptoms related to hypoxia and metabolic acidosis
(ischemic pain). More than ~90% of obstruction can result in anoxia followed by tissue infarction (due to necrosis
type of cell death).
According to hypothesis formulated by Virchow (known as Virchow’s triad) venous thrombosis occurs
via three mechanisms: decreased flow rate of the blood, damage to the vessel wall (esp. endothelium) and an increased tendency of the blood to clot (hypercoagulability). Generally, thrombosis can happen in any part of the
circulatory system, however, there are different precipitating factors. Venous thrombosis can affect deep veins
(usually on the lower extremities such as v. femoralis or poplitea) leading to the condition called deep vein thrombosis (DVT). Several medical conditions and situations can lead to DVT, such as compression of the veins, trauma,
cancer, infection, surgery, immobilization (hospitalization, locally when limb casts are used, or during long-haul
flights), certain drugs (such as estrogens or erythropoietin) or inborn abnormalities of coagulation known as familial thrombophilia (for example, in carriers of the Leiden mutation in factor V – see chapter 2.2.5 for further details).
Superficial venous thrombosis is usually manifested as a thrombophlebitis. Other commonly affected veins comprise portal vein, jugular vein or hepatic veins (Budd-Chiari syndrome). Arterial thrombosis is the formation of a
thrombus within an artery which, in most cases, follows the rupture of atheroma (typically resulting in the myocardial infarction or stroke). The thrombus may become detached and enter circulation as an embolus, finally lodging
in and completely obstructing a blood vessel at a distant site, which unless treated very quickly will lead to tissue
necrosis in the area past the occlusion (e.g. pulmonary embolism).
Thrombosis and embolism can be treated and also partially
prevented with anticoagulants (such as heparin or vitamin K inhibitors).
For diagnostic and monitoring purposes many coagulation tests are
available (such as activated partial thromboplastin time (APTT), prothrombin time (INR), platelet count, fibrinogen and fibrin degradation
products (D-dimers), etc.). The most commonly used diagnostic method
for verification of thrombosis when accessible to imaging is Doppler
ultrasound (see chapter 2.2.1 for further details).
Materials, equipment & animals:
Animals: Total number of x rats (adult Wistar rats, both genders, weight 250 – 300 g) per group will be divided into 2 groups: 1)
controls, 2) exp. animals with thrombosis induced by a combination of
endothelial damage (by hypotonic solution) and blood stasis (by ligature).
Material: Glass container with lid, scales, anaesthetics, immobilisation table for small animals, surgical instrumentarium, Petri dish
with filtration paper, syringes and needles, centrifugation tubes, centrifuge, hypotonic physiologic saline solution (25%), physiological saline
solution, heparin (Heparin Léčiva diluted in the Michealis buffer), coagulation test kits: Sevatest APTT kit, coagulometer, tubes, automatic
micropipette (0.1 mL), kaolin reagent, 0.025 M CaCl2.
33
Procedure:
A) Exp. induction of deep vein thrombosis: Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture
of ketamine/xylazine 0.5 mL/100 g i.p.) and immobilisation of animal
on the table in the supine position the jugular vein will be accessed by
incision of the skin (gently by scissors!) slightly laterally from the
middle line. Schematic topography of the area is shown in the figure
on the bottom right of the previous page. The needle is introduced
intravenously in the caudal part of v. jugularis covered by m. pectoralis (compression prevents bleeding) and hypotonic physiologic saline solution (25% diluted) is administered slowly in the total volume
of 2 mL. The vessel is fragile and should not be traumatised (not even
by aspiration which is otherwise necessary when administering i.v.).
Control animals are operated and administered the hypotonic solution
exactly the same way but the anticoagulant (heparin 4 U/kg) is given
at the same time. Since the volume of prophylactic dose is extremely
low for such small animal (requiring the excessive dilution) heparin
will be used to wash (i.e. coat) the syringe prior its filling with hypotonic solution. Although not exactly quantifiable, such approach provides empirically well documented effect.
Subsequently, laparotomy will be performed in the middle
plane from the processus xiphoideus to the symphysis. V. cava inferior
should be separated from the aorta and from surrounding and underlying tissues and ligated cranially (to let the
blood stagnate caudally). Then, 10 min later, a second ligature will be placed and tied 2 cm below the first one.
Additional ligatures will also be tied on other larger branches of the v. cava inf. and the whole segment will be
excised and placed on a moist Petri dish for 30 min to allow an eventual thrombus to develop.
Finally, thoracotomy will be performed and ~1.5 mL of blood collected from the cardiac ventricle (see
figure on the top right) into the 2 mL syringe containing 0.2 mL sodium citrate (3.8%). Blood should be well mixed
in the syringe and (without the needle) transferred into a plastic centrifugation tube and centrifuged for 15 min at
3000 g to provide plasma for the measurement of coagulation rate (see below). The excised segment of v. cava inf.
will be weighed as it is, then opened (cut along), thrombus rinsed with physiological saline solution, carefully dried
with filtration paper and weighed again (i.e. the difference between the two values represents the weight of thrombus itself.
B) Measurement of coagulation parameters (APTT): kaolin reagent (0.1 mL aliquots) will be warmed up
to 37°C for 3 min in coagulometer and incubated with 0.1 mL of plasma for further 3 min. Subsequently, 0.1 mL of
0.025 M CaCl2 will be added and, from this point onward, the coagulation time will be measured by the coagulometer.
Results:
Place the results into table (show mean  SD if n > 3 per group). Compare the two groups based on variables analyzed. Using the summary results of all study groups per week perform the statistical analysis using appropriate tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation, etc.).
Controls
Heparin-medicated
P
Thrombus weight (mg)
APTT (s)
Conclusions:
Discuss the observed differences. Explain the pathogenic effects of interventions used to cause thrombosis and the mechanism of the action of heparin as an anticoagulant agent.
34
2.1.13 Effect of selected pharmacologically active substances on microcirculation in
laboratory animal
Lukáš Pácal
Aim:
 To observe the changes in the microcirculation after application of medically relevant vasoactive substances - epinephrine and histamine – as a model of microvascular alterations seen in shock, oedema, etc.
Objective:
Part of the circulatory bed consisting of arterioles, capillaries, venules and small lymphatic vessels is together referred as the microcirculation. The function of the microcirculation is to enable the transfer of nutrients,
gases, electrolytes and metabolites between the extracellular fluid (mainly blood) and the tissues. Spatial and temporal regulation of blood flow in the microcirculation is carried out by the smooth muscle cells in the vascular wall
of arterioles and venules exposed to local and systemic vasoactive substances. Regulation of vascular tone represents the major site of systemic vascular resistance.
Blood flow in the microcirculation is controlled by the nutritional needs of the tissues. In some tissues
(e.g. skeletal muscle), a substantial part of capillary net remains closed for long periods due to contraction of the
precapillary sphincters. This provides a reserve flow capacity and can be opened quickly in response to local conditions such as a fall in partial tension of oxygen.
An increase in the local blood flow is called hyperemia. Active (work-induced) hyperemia results from
the arteriolar dilatation caused by exercise (in skeletal muscle) or by the release of inflammatory mediators. Reactive hyperemia is the transient blood flow increase following the period of ischemia. One of the most important
vasoactive substances is histamine. Histamine, produced mainly by tissue-resilient mast cells, acts via type 1 histamine receptors expressed on endothelial cell surfaces. Subsequent changes in cellular signalisation lead to arterial
dilatation and increased vascular permeability.
Materials, equipment & animals:
Animals: Total number of x mice (ICR strain, 6 - 8 weeks old, both genders, weight 25 - 30g).
Material: glass container with lid, scales, anaesthetics, immobilisation table for small animals, surgical instrumentarium, 1% solution of histamine, 1% solution of epinephrine, light microscope.
Procedure:
Following general anaesthesia (initial diethyl ether inhalation + anaesthetic mixture ketamine/xylazine 0.1
– 0.2 mL / 10g i.p.) and immobilisation of animal on the table in the supine position laparotomy is performed in the
middle plane from the processus xiphoideus to the symphysis (max. 2 – 2.5 cm). Then rotate animals sideways to
the right side, evacuate the intestinal loops with free mesenterium extra-abdominally and carefully spread the intestine loops on the small cork board using pins.
Put the whole pad with the mouse on the microscope stall and view the intestinal loops using 10 × magnification objective. Locate suitable arteriole and venule and then change to the higher magnification (40 ×). Wash
the mesenterium occasionally with physiological solution during observation. Following the initial observation
apply 1 drop of epinephrine and monitor the reaction. Leave the effect to regress (wash-out the mesenterium with
physiological solution between applications) and after 10 – 15 minutes after the first application apply 1 drop of
histamine and monitor the reaction analogically.
Results & Conclusions:
Describe the pattern and time-course of changes observed. Compare the two substances. Give examples
of pathophysiologically-relevant situations in vivo.
35
2.1.14 Experimentally induced anaphylactic response in laboratory animal
Lydie Izakovičová-Hollá
Aims:
 To elucidate the dynamics of sensitization by foreign material (protein) as a pathogenic mechanism of
type 1 hypersensitivity.
 To demonstrate the acute manifestation of anaphylactic reaction upon its exp. induction in the model organism (rabbit).
Introduction:
Type 1 (immediate) hypersensitivity reaction is one of the several pathologic reactions of immune system
that instead of fighting foreign health-threatening agent (e.g. microorganism) harms the host itself. Its manifestation
is called allergy (the genetic disposition to this reaction is often referred as atopy). Allergic reactions occur to environmental substances known as allergens (proteins and substances that are tolerated with majority of population).
Allergic reactions –quite rapid – can be limited to certain exposed areas (airways, skin, GIT) or can be systemic
(anaphylaxis). Pathogenesis of type 1 hypersensitivity involves excessive activation of mast cells and basophils by
IgE antibodies resulting in a fast response due degranulation of mast cells and basophils during which they release
histamine and other inflammatory chemical mediators, and a delayed response due to migration of immune cells
into the site. The acute clinical symptoms are mediated by histamine (vasodilation, bronchoconstriction, etc.) and
inflammation. Common allergic reactions include eczema, hives, hay fever, asthma, food allergies, and reactions to
the industrial materials (e.g. washing powders or latex) or venom of stinging insects such as wasps and bees.
Anaphylaxis is a clinical syndrome that represents the most severe systemic allergic reaction. It results
from an immunologically mediated reaction in which the vasodilator substance histamine becomes released into the
blood in excessive amounts. Profound systemic vasodilatation of arterioles and venules together with a marked
increase in capillary permeability results in circulatory shock. The vascular response is often accompanied by lifethreatening bronchospasm, laryngeal oedema, contraction of gastrointestinal and uterine smooth muscles, urticaria
or angioedema. Reactions to various drugs (such as antibiotics or protein-based drugs), contrast materials used in
radiology, foods (e.g. nuts and shellfish) and insect venoms are among the most frequent causes of anaphylactic
shock. The onset of anaphylaxis depends on the sensitivity of the person and the quantity and way of antigen exposure. Anaphylactic shock often develops suddenly, respiratory and circulation collapse can occur within a matter of
minutes unless appropriate medical intervention is promptly administered.
Type 1 hypersensitivity reaction can be induced experimentally in model animal (rabbit) by repeated exposure to foreign protein (e.g. serum proteins of different species). The titre of anti-species antibodies gradually
builds up over the time and when the same proteins administered intravenously later on, systemic and fatal anaphylactic reaction occurs.
Materials, equipment & animals:
Animals: Total number of x rabbits (New Zealand strain, both genders, weight 2.5 – 3 kg).
General material: glass container with lid, scales, anaesthetics, disinfection, foreign species (horse) serum,
syringes with needles.
Material for the manual method of cell counting: flasks with hematological solutions Türk’s (for WBCs),
Dunger’s (for eosinophils), procain (for platelets), counting chamber, light microscope, micropipette 25 L, sterile
mouthpiece, cotton wool.
Material for peripheral blood smear: set of dyes LEUKODIF 200 (Lachema) in several different staining
cuvettes [color solution 1 (Eosin Y + phosphate buffer pH 6.8], color solution 2 (Azur II + phosphate buffer pH
6.8), rinsing solution (phosphate buffer pH 7.2)], glass slides, glass slide with brilliant cresyl blue, light microscope, immersion oil.
Procedure:
The whole experiment consists of two steps: 1) experimental induction of type 1 hypersensitipity by
gradual sensitisation to foreign serum, 2) induction of acute anaphylactic reaction by systemic administration of
foreign serum.
Ad 1) Prior to the whole experiment, initial sample of heparinised venous blood as a control for haematological examination will be collected from the vena marginalis auricularis of the rabbit’s ear and stored at -80°C
until the further use. Sensitisation of the rabbit will be achieved by repeated (3-day interval) subcutaneous injections at several sites of the horse serum in a total received dose of 3 mL. After ~3 weeks animals will be rendered
hypersensitive.
Ad 2) Blood sample at the end of sensitisation period will be taken from the vena marginalis auricularis.
Subsequent intravenous injection of 3 mL of the horse serum used for sensitisation will elicit the anaphylactic re36
sponse manifested by the following symptoms and signs: restlessness, bronchospasm, expiration dyspnoe, coughing, dubbing the muzzle with the front extremities, urinary and feacal incontinence. In the most pronounced cases
animal dies in convulsions due to cerebral hypoxia [the anaphylactic response itself will be shown at film since the
number of animals per week is limited and used solely to obtain the blood samples for further evaluation]. In case
animal dies during the anaphylaxis, blood for examination is taken from the right ventricle into a heparinised syringe, otherwise, blood will be taken from the vena marginalis auricularis. Blood samples taken at the presensitisation (control), post-sensitisation and post-anaphylaxis periods will be analysed for WBC, platelet and eosinophil granulocyte counts by manual cell counting (using counting chamber) and for assessment of peripheral
WBC count.
The principle of the manual cell counting consists in the incubation of the blood with particular hematologic solution composition of which causes lysis of all other corpuscles but the specified one (e.g. Türk’s solution
leaves only WBCs intact to count). Such a suspension in the counting chamber with known volume can be then
easily quantified using light microscope (10× or 20× magnification) and numbers expressed per volume unit.
 WBC count: use a rubber tip to fill a glass micropipette with a scale by blood slightly over the 25 μL
mark. Clear the tip of blood and check the blood column again. Mix the blood with 4975 μL of the Türk’s
solution (i.e. 200-fold dilution) and incubate for 5 minutes. Afterwards, shake the solution gently (to
avoid artifact due to sedimentation of cells) and fill the micropipette again. Keep the pipette in a horizontal position. Let a few drops of blood suspension drain away, then place the tip of the pipette at the margin of the glass cover of the counting chamber and let the suspension fill the chamber evenly and form a
continuous film between the glass cover and chamber bottom. After ~5 minutes WBCs are counted in 50
central squares: WBC =  50 squares × 100 (number/uL).
 Thrombocyte count – according to Piettes: Using a micropipette, add 25L of blood to a flask with 475
L of procain solution (i.e. 20-fold dilution). Allow the diluted blood to stand for at least 20 minutes so
that haemolysis of erythrocytes has time to occur and thrombocytes can stabilize. Then shake the suspension again and pipette it into the counting chamber. Thrombocytes are allowed to sediment for additional
10 min and then counted in 20 rectangles: Platelets =  20 rectangles × 1 000 (number/uL).
 Eosinophil granulocyte count: similarly to WBC, add 25 L of blood to a flask with 475 µL of Dunger’s
solution, mix and allow to stand for at least 15 min. Afterwards, shake the solution gently (to avoid artifact due to sedimentation of cells) and fill the micropipette again. Keep the pipette in a horizontal position. Let a few drops of blood suspension drain away, then place the tip of the pipette at the margin of the
glass cover of the counting chamber and let the suspension fill the chamber evenly and form a continuous
film between the glass cover and chamber bottom. After ~3 minutes eosinophils are counted over the
whole area of the grid (9 mm2). Eosinophils =  grid × 11.1 (number × 103/µL).
Preparation of a peripheral blood smear for the assessment of the WBC differential count: Place a small
drop of whole blood on a clean slide about 1 cm from the right side. Hold the upper (spreader) slide on top of the
bottom slide with a drop at the angle of ~30 - 40 (the sharper the angle, the thinner the smear) on the left side from
the blood drop. While maintaining the contact with the bottom slide pull the top slide back until it touches the drop
(which will spread along the edge by the capillary action). Maintain the firm contact
with bottom slide and push the spreader slide forward in one motion (at ~30) evenly
but quickly to produce the smear, the drop of blood must be spread within seconds or
the cell distribution will be uneven (see figure on the top left). The smear should be
homogenous, even and appropriately thin, this requires a certain skill. The proper
smear should be thicker the one end, getting thinner and smoother (feather-like) towards the other edge. Allow the blood smears to air dry for 0.5 - 4 hours. Staining is
performed using the LEUKODIF 200 kit (Lachema Brno). Pour the reagents into staining cuvettes. Fix the smear by dipping it 5-times for 1 sec. into the fixing solution 1.
Then, dip the smear 6-times for 1 sec. into the staining solution 2. Let the solution drain
away after every dip. Rinse the slide with the rinsing solution and let it air dry. Numbers of individual WBC types is determined at least per 100 (or better 200) nucleated cells. Inspect the smear systematically (serpentinely) in one direction. Finally, express the number of the individual types of WBCs in percentage.
Results & Conclusions
Tabulate the results (chapter 2.1.2 for the example). Compare the pre-sensitisation (control), postsensitisation and post-anaphylaxis parameters ascertained. Discuss the mechanisms of the type 1 hypersensitivity
and its effect on the parameters analysed. Using the summary results of all study groups per week perform the statistical analysis using appropriate tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation, etc.).
37
2.2
Experiments using alternative models – humans & in vitro
techniques
2.2.1
Principles and demonstration of peripheral blood flow examination using
Doppler ultrasonography
Michal Jurajda
Aims:
 To demonstrate the available methods and principles of physiological examination of the extremity arteries (as diagnostic methods for peripheral arterial disease) using blood pressure measurement and Doppler
phenomenon.
Introduction:
Arterial occlusive disease of extremities is caused predominantly by atherosclerosis (AS) seen most
commonly in vessels with turbulent pattern of blood flow (branching, meandering) with higher BP (large arteries)
and shear stress. Vessels typically affected by AS comprise coronary, carotid and brain, renal and lower extremity
arteries. In case of AS of arteries in the lower extremities – peripheral arterial disease (PAD) - so called intermittent
claudication (i.e. pain when walking) is the primary symptom of chronic arterial obstruction. Other signs of ischemia of the tissues in affected limb include atrophic changes and thinning of the skin and subcutaneous tissues, muscle atrophy and lower temperature of the foot. With the disease progression pain at rest, trophic ulcers and gangrene
may develop.
Normally, the systolic arterial blood pressure measured on the lower extremities is higher compared to
systolic arterial blood pressure taken on the upper extremities (even in the supine position). The so called anklebrachial index (ABI) is  1 in a healthy person. ABI can be measured at rest or repeated after 5 minutes of walking
on a treadmill (measurement performed usually at both sites). ABI in PAD patient < 0.95 usually indicate narrowing of one or more blood vessels in lower extremity, ABI < 0.8 is usually associated with the pain in the foot, leg or
buttock during exercise, with ABI < 0.4 symptoms may occur when at rest and with ABI < 0.25 severe limbthreatening PAD is probably present. The exact site of the obstruction can also be determined by the segmental
blood pressure measurement. The segmental blood pressure is measured using manometer with appropriate cuff
and Doppler ultrasound system. The principle employed to locate the level of stenosis lie in the search for the systolic blood pressure drop occurring distally from the stenosis.
A Doppler ultrasound is a non-invasive test that can be used to evaluate blood flow and pressure by
bouncing high-frequency sound waves (ultrasound) off moving corpuscles, i.e. red blood cells. The Doppler effect
is a change in the frequency of sound waves reflected by a moving object - the observed frequency is increased if
the source is moving towards the observer/probe and, vice-versa, it is decreased if the source is moving away. Using this principle, Doppler ultrasound can estimate how fast blood flows through certain areas accessible to examination by measuring the rate of change in its frequency. This test may be done as an alternative to more invasive
procedures such as arteriography and venography, which involve injecting dye into the blood vessel to enhance Xray images. Doppler ultrasound is routinely used to diagnose many conditions such as blood clots (thrombosis),
incompetent valves in leg veins (chronic venous insufficiency), heart valve defects and congenital heart diseases,
blocked arteries (arterial occlusion), narrowing (stenosis) of an artery (PAD), etc.
Chronic venous insufficiency (CVI) is an extremely common condition affecting 2-5% of adult population. Historically, CVI was called post-phlebitic or post-thrombotic syndrome, both of which refer to the etiology
of some cases. However, most common cause of the CVI the congenital absence or dysfunction of venous valves in
the superficial and communicating systems. Resulting venous hypertension retrogradely affects capillary flow and
tissue nutrition. CVI manifests as varicose veins, leg discomfort and ache (precipitated by prolonged standing),
oedema, poorly healing ulcers and lipodermatosclerosis. Doppler ultrasound is used in patients with CVI to assess
venous flow, its direction, and the presence of thrombus.
Materials & equipment:
Doppler ultrasound equipment, 5-8 MHz Doppler probe, speaker, inflatable cuff, manometer, stethoscope.
Procedure:
1) Evaluation of arteries: Measure brachial arterial blood pressure at both upper extremities using auscultatory method (sphygmomanometer, cuff and stethoscope). Then measure brachial arterial blood pressure at both
upper extremities using Doppler ultrasound. Similarly, measure arterial blood pressure at both lower extremities
38
using both auscultation and Doppler ultrasound.
2) Evaluation of veins: Locate v. jugularis and – using Doppler ultrasound – observe the changes of blood
flow induced by the breathing and Valsalva manoeuvre3. Further, examine the superficial venous system of the
lower limbs.
Results:
Tabulate the results (show mean  SD if n > 3 per group). Compare values of systolic blood pressure obtained with stethoscope with that obtained with Doppler ultrasound (i.e. correlation). Calculate ABIs. Using the
summary results of all study groups per week perform the statistical analysis using appropriate tests (e.g. KruskalWallis ANOVA, Mann Whitney, Spearman correlation, etc.).
Left arm
Right arm
Left ankle
Right ankle
ABI left
ABI right
Systolic BP
Conclusion:
Explain the principle of Doppler measurement of blood flow and pressure and its practical applicability.
Comment on the results of ABI measurement and its interpretation. What are the major factors influencing BP values in different parts of circulatory system.
3
The Valsalva manoeuvre is performed by forcibly exhaling against a closed airway. Variations of the manoeuvre can be used either as a test of cardiac
function and autonomic nervous control or to normalize middle ear pressure when ambient pressure changes, as in diving or aviation. It can be used to arrest
episodes of supraventricular tachycardia. Valsalva is also used by dentist following extraction of a maxillary molar tooth to determine if a perforation or antral
communication exists. A modified version is done by expiring against a closed glottis. This will elicit the cardiovascular responses described below but will
not force air into the Eustachian tubes. The normal physiological response consists of 4 phases: [1] initial pressure rise (pressure rises inside the chest forcing
blood out of the pulmonary circulation into the left atrium causing a mild rise in blood pressure); [2] reduced venous return and compensation (cardiac output
is reduced and blood pressure falls reflexively causing blood vessels to constrict with some rise in BP and the pulse rate); [3] pressure release (pressure on the
chest is released, allowing the pulmonary vessels and the aorta to re-expand causing a slight fall in BP due to decreased left ventricular return and increased
aortic volume, respectively; [4] return of cardiac output (blood return to the heart is enhanced causing a rapid increase in cardiac output and BP which usually
rises above normal before returning to a baseline level).
39
2.2.2
Measurement of resting and ambulatory blood pressure and heart rate, postural
changes, effect of isometric and aerobic exercise
Anna Vašků
Aims:
 To summarize physiologic factors involved in the short- and long-term regulation of BP and its circadian
pattern as plausible pathophysiological mechanisms of arterial hypertension.
 To determine the effects of light aerobic vs. isometric physical exercise on changes in blood pressure and
heart rate as prototypical examples of two situations with different pattern of blood distribution and therefore different overall effect on arterial BP.
 To demonstrate the principle and interpretation of 24-h ambulatory blood pressure measurement as a useful diagnostic and monitoring tool.
Introduction:
Blood pressure (BP) refers to the force exerted by circulating blood on the walls of blood vessels. BP decreases as blood moves through arteries, arterioles, capillaries, and veins; so – in a medical context - the term BP
generally refers to arterial pressure in the larger arteries (such as brachial artery commonly used for measurement).
BP can be measured either invasively (inside the blood vessels upon catheterisation restricted to a hospital settings)
or non-invasively. The latter can be commonly done by auscultatory or oscillometric methods in order to determine
random momentary BP. There is also possibility to measure continuously using beat-to-beat BP measuring finger
device based on a method described by Penaz. In the doctor’s office arterial BP is most commonly measured via a
sphygmomanometer with Riva-Rocci inflatable cuff4 and stethoscope to determine Korotkoff sounds. The cuff is
inflated to a pressure above that of the arterial systolic BP. At this point, the walls of the artery are opposed preventing blood flow. The cuff is then deflated below systolic pressure allowing blood flow to resume; this flow can
then be detected using various means by auscultation (most often), oscillometry, palpation or Doppler. Historically,
this method uses the height of a column of mercury in millimetres to reflect the BP. Today, BP values are still
commonly reported in mmHg, though electronic devices do not use mercury (unit kPa is sometimes used, where
1 kPa = 7.50 mmHg). Oscillometric methods are often used in general practice and also for the long-term ambulatory BP measurement (ABPM). The equipment is similar to that of the auscultatory method, but with an electronic
pressure sensor with a numerical readout of BP instead of using the stethoscope (the pressure sensor has to be calibrated periodically to maintain accuracy). In most cases the cuff is inflated and released by an electrically operated
pump and valve, which may be fitted on the wrist, although the upper arm is preferred.
Systolic and diastolic arterial BPs are not static but undergo natural variations from one heartbeat to another and throughout the day (circadian rhythm). Physiologic regulation of BP is a complex network of short- and
long-term acting feedbacks. BP also changes in response to stress, nutritional factors, medication, disease, exercise,
and with postural changes. Due to the complex rhythmicity of BP it might be desirable to assess its daily pattern
(using ABPM) in some situations - spurious measurements of random BP, unusual BP variability, “white coat”
hypertension (from anxiety related to an examination by a health care professional), control of anti-hypertensive
treatment (resistance, multiple medication, over-treatment), pregnancy-induced hypertension etc. ABPM involves
BP measuring usually for 24 hours as the subject goes about his/her daily routine and when asleep. Subject wears a
device that measures BP at regular intervals. The information is recorded on a chip in the device and allows the
doctor to get a detailed picture of BP variation in a normal environment. Average day-time and night-time ABPM
blood pressure should be lower than equivalent random BP readings (percentage of above-the-threshold readings is
considered, usually 15 - 20% per day). A high reading using ABPM are: > 135/85 for the general population or >
130/80 for people with diabetes (however, this is a necessary simplification; in reality the interpretation of ABPM
by cardiologists is much more detailed).
While average BP could be determined for any given population, there is a large variation from person to
person (interindividual variability) and BP also varies in individuals from moment to moment. In the elderly, blood
pressure tends to be higher, largely because of reduced flexibility of the arteries (arterial stiffness). Factors such as
age, gender and race also influence BP. Average BP of any given population can be taken as a reasonable correlation with its general health (reference interval defined solely as a mean  2 SD, i.e. 95% percentile), however, as
documented by multiple clinical and epidemiological studies, the risk of cardiovascular disease increases progressively throughout the range of BP and begins at values as low as ~110/70 mmHg. Therefore, current reference intervals and guidelines for the BP management (< 140/90 or < 130/80 in subjects with multiple morbidity) reflect
also the long-term outcome of average BP, especially for the cardiovascular health. In general, hypertension refers
4
The cuff should be 20% wider than the diameter of the part of the limb being used (or cover two-third its length). Cuffs that are too small will
lead to overestimation of blood pressure and vice versa. Therefore, there are different cuffs for arms, wrists or ankles and also for adults and children.
40
to arterial BP - determined by current methods - being abnormally and chronically high due to either 1) complex
interplay of multiple homeostatic, genetic and environmental factors (i.e. primary (essential, idiopathic) hypertension) or 2) well defined etiologic cause (i.e. secondary hypertension). The principal medical debate concerns the
aggressiveness and relative value of methods used to lower BP and also the issue of changing environment and
lifestyle, so the future modification of recommendations and guidelines might logically be expected.
Materials & equipment:
Sphygmomanometers, cuffs, stethoscopes, manometers (hand grip manoeuvre), stop watches, ABPM device, PC with operating software installed.
Procedure:
The group of students will be working in pairs (both students will alternate for the exercise and measurements) to measure the systolic and diastolic BP (SBP and DBP, respectively) using auscultatory method during 1)
aerobic and 2) isometric exercises. Moreover, one student per group will be monitored by ABPM device throughout the whole period (in a combined pre-defined and manual regimen). BP and heart rate will be measured at rest at
the beginning of each type of exercise.
 Aerobic exercise: each examined student will perform 50 squats with maximum speed possible. SBP,
DBP and heart rate will be measured immediately after the end of the exercise and again after 5 min at
rest.
 Isometric exercise: each examined student will press the manual manometer to achieve his/her maximum
pressing force. The pressure will be then relaxed to a half of the maximum value and will be held at this
level for a period of 2 min. At the end of the second minute, SBP, DBP and heart rate will be measured
and again after 5 min at rest.
Results:
Tabulate the results (show mean  SD if n > 3 per group). Compare values of SBP, DBP and heart rate
during different types of exercise. Using the summary results of all study groups per week perform the statistical
analysis using appropriate tests (e.g. Kruskal-Wallis ANOVA, Mann Whitney, Spearman correlation, etc.).
Before (at rest)
Aerobic exercise
(“hand-grip”)
Isometric exercise
(“squatting”)
Immediately after
After 5 minutes
SBP (mmHg)
DBP (mmHg)
heart rate (/min)
SBP (mmHg)
DBP (mmHg)
heart rate (/min)
Conclusions:
Comment on different mechanisms of BP regulation during aerobic and isometric exercise. Summarize
the mechanisms responsible for BP regulation. Compare the pros and cons of different methods available for blood
pressure measurement.
41
2.2.3
Pathophysiology of ventilation disorders – pulmonary function tests: spirometry
Michal Masařík
Aims:
 To demonstrate the principle and interpretation of the spirometry and its use for the classification of prevailing pathophysiological mechanisms of common ventilation disorders.
Introduction:
Spirometry is the most common of the pulmonary function tests, measuring the lung functions, specifically the measurement of the amount (volume) and/or speed (flow) of the air that can be inhaled and exhaled. Lung
volumes are absolute changes of amount of air and thus lung size during inhalation and exhalation while lung capacities represent different combinations of lung volumes (see figure below). Spirometry is an important diagnostic
tool used to assess various respiratory conditions producing obstructive pattern (e.g. bronchial asthma or chronic
obstruction pulmonary disease (COPD)), restrictive pattern (e.g. pulmonary fibrosis) or others (combined diseases,
cystic fibrosis etc.). Spirometry is also very important tool for monitoring the efficiency of bronchodilation therapy.
Using spirometry, we can determine both static
and dynamic parameters. Static parameters cannot be
changed and can be assessed after deep inhalation and exhalation in the rest: tidal volume (TV), inspiratory reserve
volume (IRV) and expiratory reserve volume (ERV). Their
sum (IRV + ERV + TV) is called vital capacity (VC). Residual volume (RV) is the amount of air left in the lungs
after the maximal exhalation. This is the amount of air that
is always in the lungs and can never be expired, therefore,
we cannot measure RV and thus total lung volume (TLC) by
spirometry.
Dynamic parameters include forced expiratory
volume in the 1st sec (FEV1), forced vital capacity (FVC)
and forced expiratory flow or maximum expiratory flow
(FEF resp. MEF). Definitions of basic static and dynamic
parameters are summarized in the table bellow. A decrease of dynamic ventilation parameters usually indicates an
obstructive ventilation disease while a decrease of static ventilation parameters usually indicates a restrictive ventilation disease. The expected values for the healthy population are often adjusted for age, sex and body size. Dynamic parameters are excellent indicators of lung function. Based on comparison of actual FEV1 compared to the
reference value severity of obstructive disease can be classified into: 1) normal (80%), 2) mild obstruction (60 80%), moderate obstruction (40 - 60%) and severe (<40%).
Different spirometers display the results in two possible ways: 1) as a time-volume curve showing the
time (s) along the X-axis and volume (L) along the Y-axis or 2) as a flow-volume loop, which graphically shows
the total volume inspired or expired (L) on the X-axis and the rate of airflow (L/s) on the Y-axis.
Besides spirometry there is another test of lung function called peak-flowmetry (measuring peak expiratory flow, PEF). A peak-flow meter is a small, hand-held device used to determine a maximal expiratory speed.
This is very handy for home management of asthma by monitoring airflow through the bronchi and thus the degree
of restriction in the airways any time during the day. The peak-flow meter measures the patient’s maximum ability
to exhale, (peak expiratory flow rate, PEFR). Peak-flow readings are higher when patients are well, and lower
when the airways are constricted.
FVC (L)
FEV1 (L)
PEF (L/s)
FEV1/FVC
MVV
(L/min)
FEF
Forced Vital
Capacity
Forced Expiratory
Volume in 1
Second
Peak Expiratory
Flow
Tiffeneau index
Maximum Voluntary Ventilation
Forced Expiratory
Flow
= total amount of air that can be forcibly blown out after maximal inspiration (using
IRV)
= the amount of air that can be forcibly blown out in one second, along with FVC
FEV1 is considered one of the best indicators of lung function
= the speed of the air moving out of lungs at the beginning of the expiration (good
indicator of the degree of obstruction)
= the ratio of FEV1 to FVC which, in healthy adults, should be approximately 75 - 80%
= a measure of the maximum amount of air that can be inhaled and exhaled in one
minute, measured in litres/minute
= the average flow (or speed) of air coming out of the lungs during the middle portion
of the expiration
42
Materials & equipment:
Spirometer, peak-flow meter, sterile mouthpiece.
Procedure:
A. Spirometry: Although there are various spirometer devices made by different companies, they all
measure the same thing generally in the following way - person examined takes the deepest breath he/she can manage and then blows out through the mouthpiece into the device as hard as possible for as long as possible. It is
sometimes directly followed by a rapid inhalation (inspiration), in particular when assessing possible upper airway
obstruction.
B. Peak-flowmetry: The procedure is quite the same as in spirometry measurement, briefly: examined
person takes the deepest breath he/she can manage and then exhales into the sensor as hard as possible for as long
as possible. The measurements are usually repeated 3 times and the highest value is considered.
Results:
reference
value
mean measured
value (males)
% of reference
value (males)
mean measured
value (females)
% of reference value (females)
PEF
FVC
FEV1
FEV1/FVC
Conclusions:
Compare the results measured for males and females. Identify eventual ventilation disturbances.
43
2.2.4
Use of enzymes as diagnostic markers – evaluation of lactate dehydrogenase
(LDH) isoenzymes by agarose gel electrophoresis
Lukáš Pácal
Aims:
 To discuss the principles and interpretation of enzyme diagnostics.
 To compare electrophoregrams of LDH isoenzymes from normal and pathological sera.
Introduction:
LDH (EC1.1.1.27) is a tetrameric enzyme consisting of two subunits: H (for heart muscle, encoded by the
LDHA gene on chromosome 11) and M (for skeletal muscle, encoded by the LDHB gene on chromosome 12),
which combine to form an active enzyme. LDH catalyses the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+. The efficiency of the conversion increases with the number of M chains.
Five isoenzymes (i.e. different molecular forms catalyzing the same reaction): LDH1 (H4, heart), LDH2 (H3M,
reticuloendothelial system), LDH3 (H2M2, lungs), LDH4 (HM3, kidneys) and LDH5 (M4, liver) represent the
various combinations of the two subunits. LDH1 and LDH2 are the predominant forms of the enzyme in cardiac
muscle while LDH4 and LDH5 prevail in liver. Therefore, the high concentration of LDH5 (4 M subunits) in the
skeletal muscle rapidly converts pyruvate to lactate while the high concentration of LDH1 (4 H subunits) in heart
tissue favours conversion of pyruvate to acetyl CoA which enters the citric acid cycle.
Physiologically, LDH2 is a predominant isoform found in plasma. Elevated plasma LDH levels can be
found in several pathological situations and indicates the cellular damage since LDH is a cytoplasmic enzyme: (1)
Myocardial injury: elevated isoenzymes LDH1 and LDH2, also ratio LD1/ LD2 is > 1 while in healthy people it is
< 1 (e.g. acute myocardial infarction, however, quite late peak in 3 - 4 days after MI). (2) Liver injury: elevated
isoenzymes LDH4 and LDH5 (e.g. acute viral hepatitis, cirrhosis, organic solvent intoxication). (3) Hemolysis:
elevated LDH2 (e.g. hemolytic anemias, incompatible blood transfusion). (4) Others: increased tissue turnover (e.g.
acute tumor breakdown after chemotherapy), analysis of exudates vs. transudates (eLDH > tLDH), analysis of cerebrospinal fluid in bacterial and viral meningitis.
LDH isoenzymes differ in charge hence they can be separated by electrophoresis. Because the H polypeptide contains more acidic amino acid residues than the M polypeptide, the
electrophoretic mobility of the LDH isoenzymes are as follows: LD1 > LD2 > LD3 > LD4 > LD5.
LD1 migrates towards anode (positive electrode), LD5 towards cathode (negative electrode), see
example of possible findings in figure on the right (left column – normal finding, right column –
liver damage, e.g. acute viral hepatitis). During the incubation of the sample with LDH in the solution containing substrate (lactate) and colour marker, isoenzymes can be visualized.
Principally, the reaction works as follows: LDH catalyzes the removal of hydrogen atoms from the lactate. The products (NADH+ and H+) react with the hydrogen transmitter phenazine methosulfate (PMS) and, finally, with the hydrogen acceptor nitroblue tetrazolium (NBT)
present in solution. NBT is reduced to dark blue formazan. Therefore, every zone containing LDH isoenzyme is
stained blue and can be seen. Colour intensity is directly proportional to the amount of particular isoenzyme (intensity can be quantified). Schematically:
 Lactate + NAD+ → Pyruvate + NADH+ + H+ followed by NADH + H+ + NBT → NAD+ + formazan
Materials & equipment:
Glass microscopic slides (7.6 x 2.6 cm), electrophoretic apparatus, power supply, automatic pipettes and
tips, thermostat, agarose (powder), TBE buffer, normal and pathological sera, barbital buffer, staining solution
(lithium lactate (= enzyme substrate), coenzyme NAD+, nitrobluetetrazolium (NBT), phenazinmethosulphate
(PMS), buffer (pH 8.6)).
Procedure:
To prepare agarose slides for separation carefully pour pre-heated 1% agarose onto slide and let it solidify
in the room temperature. Load 2 samples (10l of sera per each sample) onto the gel in one slide (via diffusion
through filtration paper for 15 minutes). Afterward rinse the gel and put it in the electrophoretic apparatus. Connect
the slides with barbital buffer using filter paper, close the apparatus and start electrophoresis (U = 200 V, I = 32
mA, t = 55 min, T = 10C). After electrophoretic separation place the gel into the container with staining solution
and allow the gel to stain for 45 minutes in 37°C. Observe the results visually, then scan the stained and dried gels
and analyse images using software TotalLab.
Conclusions:
Describe the differences in isoenzyme distribution between normal and pathological sera and discuss it.
44
2.2.5
Molecular biological methods in haematology – diagnostics of familial
thrombofilias: detection of the factor V Leiden mutation by PCR
Lukáš Pácal
Aims:
 To summarize the current molecular-biology methodology available for genetic analysis and diagnostics
of diseases.
 To explain principle and demonstrate the technique of polymerase chain reaction, restriction fragment
length polymorphism and gel electrophoresis.
 To practically perform detection of the specified pathogenic variant (Leiden mutation of Factor V) in the
responsible for common form of familiar thrombophilia (Activated protein C resistance) in DNA samples
of healthy and affected subjects.
Introduction:
Genetic predisposition (i.e. carrier state of defined genetic variant(s) or (allele(s)) to certain disease can
either confer the subject’s risk almost completely in spite of the effect of environment (i.e. monogenic diseases) or
variably increase the risk of disease development compared to non-carriers (i.e. complex diseases). Providing
“risk” variant are identified, carriers can be easily detected by many of the current molecular biology methods, one
of them being polymerase chain reaction (PCR). PCR is a method enabling enzymatic synthesis of DNA – a copy
of the given template. PCR uses specifically designed oligonucleotides called primers that are complementary to
the DNA sequence to be amplified. The primers provide a starting point for the extension of the DNA by the enzyme called DNA polymerase. It is a cyclic reaction (newly synthesized DNA serves as a template for DNA synthesis in subsequent cycles), therefore the number of copies increases progressively. PCR is carried out in a cycle
consisting of three steps (cycles are typically repeated 25 to 35 times): (1) denaturation – usually 93 - 95°C at
which hydrogen bonds between DNA strands are interrupted and DNA strands separate; (2) annealing – in this step
the reaction temperature is lowered so that the primers can attach (anneal) to the single-strand DNA template, the
temperature depends on the melting temperature of the primers and is usually between 50 - 64°C; (3) extension –
during this step the DNA polymerase copies the DNA template starting at the primers annealed to both of its
strands, the temperature depends on the DNA polymerase used (Taq polymerase has a temperature optimum of 70 74°C), the extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be
amplified. The PCR reaction mixture therefore consists of: template (human, animal or bacterial genomic DNA),
primers (commercially synthesized oligonucleotides 20 – 30 bases long), DNA polymerase (thermostabile enzyme
catalysing DNA synthesis, the most commonly used is Taq polymerase isolated from Thermus aquaticus), dNTP
(mixture of deoxynucleotidetriphosphates), MgCl2 (Mg2+ forms a complex with dNTP which is recognised by the
polymerase and incorporated into the newly synthesized strand), PCR buffer (to ensure an optimal pH) and distilled
water. PCR is carried out in the instrument called thermocycler, which is designed to automatically, quickly and
accurately change the temperature of the PCR mixture during the cycling (the whole procedure – i.e. temperature
and duration of each step – is programmed by the operator and can be quickly accessed from the menu). At the end
of the procedure, the PCR mixture contains millions of copies of defined size (bp) of the original template which
can be subsequently used for determination of the exact nucleotide sequence (DNA sequencing), determination of
the product size (gel electrophoresis), detection of the presence of restriction sites (digestion by bacterial endonucleases, see further), cloning onto the vectors (plasmids, viruses), etc.
Restriction Fragments Length Polymorphism (RFLP) methodology involves cutting (digestion) of a particular region of DNA (PCR product) with known sequence variability with bacterial restriction enzyme(s) at specific recognition nucleotide sequence known as restriction site (often palindrome). Such enzymes are found in bacteria and archea and are thought to have evolved to provide a defense mechanism against invading viruses. There
are thousands of restriction endonucleases (produced by recombinant technology nowadays) specific for various
double stranded DNA sequence and thus representing a powerful tool of molecular biology. Cleaved DNA fragments are then separated by the agarose gel electrophoresis. Determining the number of fragments and respective
sizes allows discriminating e.g. the wild-type and mutated alleles (since mutation can alter or create restriction site
for a given enzyme). Agarose gel electrophoresis is a method for separating and visualisation of DNA fragments
produced by RFLP. DNA has a negative charge, therefore it migrates to the positive pole in an electric field. The
fragments are thus separated according to their size, large pieces of DNA move slower than small pieces of DNA.
Activated protein C resistance (APCR) is the most common inherited hypercoagulable (thrombophilic)
disorder and also the most common inherited predisposition to the deep vein thrombosis and subsequent thrombembolic disease. In 90% of cases APCR is due to a single nucleotide exchange in the gene encoding for the factor
V where guanine (G) is replaced by adenine (A) in the position 1691. This nucleotide exchange leads to the substitution of the amino acid glutamine for arginine at position 506. The altered factor V is referred to as factor
45
V Leiden (FVL). This amino acid change alters the cleavage site
of FV where activated anticoagulant protein C normally binds to
inactivate FV. It results in prolonged conversion of prothrombin to
thrombin. The prevalence of the FVL mutation is 2 – 5 % in general population and molecular diagnostics should be considered in
cases of unexpected or recurrent deep vein thrombosis or pulmonary embolism, recurrent spontaneous miscarriage, female infertility and some other rarer situations.
H2O
buffer
MgCl2
dNTP
primer FW
primer R
Taq DNA polymerase
PCR mastermix
DNA template
Total volume
per 1 sample (l)
13.4
2.5
1.5
0.5
1
1
0.1
20
2
22
Materials & equipment:
PCR: H2O, reaction buffer (10× concentrated), MgCl2
(25 mM), dNTP (mixture of dATP, dTTP, dCTP, dGTP, 10 mM),
primers (10 mM), genomic DNA (50 ng/l), Taq polymerase (5
U/l).
RFLP: H2O, restriction endonuclease (enzyme) MnlI (Moraxella nonliquefaciens), buffer specific for the
restriction enzyme.
Electrophoresis: 1×TBE buffer, loading dye (bromphenol blue/ficol, 1:1), ethidium bromide (10 mg/mL).
Procedure:
1) PCR: Prepare the PCR mastermix mixing the aliquots shown in table on the top of the page (volume
per 1 sample) multiplied by the number of samples to be amplified, mix well by vortexing and shortly centrifuge.
Prepare the appropriate number of 0.2 mL plastic microtubes and mark them with the sample codes. Pipette the 2
μL of each DNA sample (template) into the appropriate PCR microtubes (place the drop on the bottom). Add 20 l
aliquots of the PCR mastermix into each microtube containing DNA sample. Finally, overlay the mixture with a
drop of mineral oil into each microtube. Cover the lids and place all microtubes into the thermocycler. Run the
“Factor V Leiden” programme.
2) RFLP: A to G nucleotide substitution in the position 1691 in the gene encoding for factor V alters the
restriction site for enzyme MnlI. In the homozygous “wild type” individuals (non-carriers), incubation of amplified
DNA fragment with MnlI yields 3 fragments of 142, 45 and 37 bp. In homozygous carriers of the Leiden mutation,
the restriction enzyme recognition site is lost yielding only 2 fragments of 142 and 82 bp. Heterozygotes exhibit
both restriction patterns. To perform RFLP pipette 10 l of amplified PCR product into PCR microtube. Prepare
the restriction mixture (see table on the bottom left) for a given number of samples to be digested (mix and centrifuge) and pipette 10 l aliquots into microtubes containing PCR product. Place your tubes into the thermostat
(37°C) and incubate for at least 2 hours.
3) Electrophoresis: To prepare 3% gel weight 1.8 g of agarose in a small flask with a stir bar in it and add
60 ml 1×TBE buffer. Heat the agarose in microwave oven until the agarose dissolves into the buffer and the consistency is uniform. Assemble the gel casting tray and comb (the comb should not touch the bottom of the tray).
Pour the slightly cooled agarose (50°C) mixed with ethidium bromide (2 l per 10 ml of the gel) into the prepared
casting tray trying to avoid introducing bubbles into the gel and allow to cool and solidify (~20 minutes). Once the
gel has solidified, pour a small amount of 1×TBE buffer on top of the gel and carefully pull the comb out of the gel.
Place the tray holding the gel into the electrophoresis apparatus. Make sure that the end of the gel with the wells is
positioned towards the negative (black) electrode. Pour 1x TBE buffer into the tank up to ~2 to 3 mm above the top
of the gel. The gel is now ready to be loaded with your DNA samples. Mix your DNA samples with loading dye
containing glycerol and blue dye (the high density of the glycerol ensures that the samples will layer smoothly at
the bottom of the wells and the dye enables to see the progress of an electrophoresis run). Also load a molecular
weight marker onto one well. Once all samples are loaded, place the lid on the electrophoresis apparatus, connect
the leads to the power supply (red to red and black to black) and turn the power on. Adjust the power supply to an
output of 90 V constant voltage and allow to run until the loading dye has migrated approximately 75% of the distance of the gel. At this point, turn off the power supply, disconnect the electrodes, and remove the top of the electrophoresis apparatus. Carefully remove the gel and put it under the UV transluminator. The DNA in the gel is visualized under the ultraviolet light. Ethidium bromide, a fluorescent dye that intercalates into DNA, excites and
emits the visible fluorescence. Size of the DNA bands may be assessed by comparison with molecular weight
standards. Take a picture of the gel under the UV light using built-in camera.
H2O
restriction buffer
MnlI (restriction enzyme)
DNA
Total volume
per 1 sample (l)
7.3
2
0.7
10
20
Results & Conclusions:
Describe the results of mutation detection and document it with the picture taken. What measures has to be taken to avoid false positive or negative results? Offer other examples of the use of PCR in medicine.
46