Comparative Medicine
Volume 54, Issue 5 (October 2004)
Laboratory Animal Models of Temporal Lobe Epilepsy p. 473-485
Questions:
1)
Temporal lobe epilepsy is a common human disease that is difficult to treat. True or False
2) Seizures are caused by uncontrolled, excess, and hypersynchronous neuronal activity, the
timing of which is unpredictable. True or False
3) Self-limited generalized absence seizures have been identified in ………… rats and genetic
absence epilepsy rats from …………
4)
Reflex seizures occur in ………….. with inherited epilepsy, in which the optimal stimulus is
exposure to a novel environment.
5)
After bilateral temporal lobectomy was performed to treat medically refractory epilepsy; it
caused the immediate and permanent loss of a patient’s ability to form new memories, while
pre-surgical memories remain intact. True or False
6)
Most patients with temporal lobe epilepsy have a history of brain injury. Age at time of the
injury is quite variable, but average about 3 years. True or False
7)
Other precipatating injuries include ……, ……., ………., ………, …….., ………
8)
Some patients with temporal lobe epilepsy have tumors or vascular malformations in the
temporal lobe, the most common lesion, in 70% patients, is hippocampal sclerosis highlighted
by a specific pattern of neuronal loss. True or False
9)
Hippocampal sclerosis has been described in cats with epilepsy, but dogs rarely develop
hippocampal sclerosis even though epilepsy is common in dogs. True or False.
10) Hypothermia has been used to develop several rat models than can be used to investigate
the long term effects of ……….
11) Like the febrile seizure model, the hypoxia model does induce hippocampal sclerosis and is
epileptogenic. True or False
12) The hypoxia plus ischemia treatment is likely to cause a clinical syndrome and
neuropathologic changes extend beyond the temporal lobe and resemble those in patients with
…….. and ………..
13) The use of fluid percussion to generate injuries mechanical force has been done in …………..
a) cats
b) rabbits
c) rats
d) a, and b
e) a, b, and c
14) Tetanus toxin has been used to induce seizures initiated in the neocortex and the
hippocampus of infant rats (9 to 10 days old). True or False
15) Kindling entails repeated, mild, electrical stimulation of the amygdale, olfactory regions,
hippocampus, or other brain regions to induce a progressive and permanent seizure response.
True or False
16) What kind of species has been kindled?
a) frogs
b) lizards
b) rodents
c) cats
d) dogs
e) macaques
f) baboons
g) all of the above
17) …… rats require the most stimulations for amygdale kindling, ……. and
…… rats require the fewest, and ……, ……, ……., and …… rats need an intermediate
number.
18) What are the advantageous of the kindling models?
a) specific brain region can be treated more selectively
b) can animals can help resolve one of the major problems in epilepsy
experiments determining whether a difference between an epileptic and control group is a
cause or an effect of seizures.
c) all of the above
19) Anti-convulsant drugs administered during the latent period do not prevent or delay the
development of epilepsy. True or False.
20) Status epilepticus can be induced by electrically stimulating the lateral nucleus of the
amygdale in adult rats. True or False
21) The pilocarpine-induced status epilepticus rat model may be the most widely used model of
temporal lobe epilepsy. True or False
22) If status epilepticus is blocked or curtailed prematurely, neuronal loss is prevented and
synaptic reorganization and epilepsy fail to develop. True or False
23) Methylscopolamine (1 to 2 mg/kg, i.p) can be used to antagonize the peripheral cholinergic
side effects of pilocarpine. True or False
24) Benzodiazepines and/or barbiturates (e.g., 50 mg of pentobarbital/kg, i.p.) have been used as
anticonvulsant. True or False
25) ……….. injections tends to induce a more focal lesion, longer seizure-free latent periods, and
lower seizure frequencies than do systemic treatments.
26) Advantages of status epilepticus models include robust development of hippocampal
sclerosis and granule cell synaptic reorganization, a latent period, and permanent epilepsy
once it is established True or False
27) The latent period does not present a pre-epileptic state that can help distinguish between
causes and effects of chronic epileptic seizures. True or False
28) Compared with primate status epilepticus models and patients, rodent status epilepticus
models tend to have more neuron loss in regions outside of the hippocampus. True or False.
29) Variability in the mortality and the proportion of animals that develop status epilepticus and
chronic epilepsy can be attributed to:
a) sex
b) strain
c) age differences
d) all of the above
30) After systemic kainite treatment, adult (70 to 90-day-old) male ……… and …… inbred rat
strains, display more sensitivity and more reliable behavior seizures responses than do
……….. outbred rat strains.
31) Aged rats (24 months old) are more vulnerable to kainite induced excitotoxicity than are 3-20month-old-rats. True or False
32) When young rats (up to approx. 25 days old) are subjected to the same pilocarpine or kainite
treatment protocols as adults, they develop acute seizures, but their hippocampal neurons
survive and synaptic reorganization and spontaneous recurrent seizures fail to develop. True
or False
33) After lithium-pilocarpine combination treatment, some rats as young as 20 to 21 days old
develop epilepsy, and some of these do so without hippocampal neuron loss or granule cell
synaptic reorganization. True or False
34) Electroconvulsive stimulation evokes seizures more easily in DBA/2J than in C57BL/6J mice.
True or False
35) Mature (9-10-week-old) DBA/2J mice are more vulnerable than are C57BL/6J mice to
induction of behavioral seizures after systemic kainite treatment. True Or
False
36) The …….., …….., and …….. mice have extensive hippocampal neuron loss and synaptic
reorganization after kainite-induced status epilepticus, whereas ……., ….., ……., ……, ……,
and …… are resistant to neuronal loss.
37) Pilocarpine-induced status epilepticus results in high mortality in C57BL/6 mice from The
Jackson Laboratories, for reasons that are unclear, not in C57BL/6 mice from Charles River
Laboratory. True or False
38) Young adults rhesus macaques were given 0.1 to 0.2 ml of alumina gel injection
stereotaxically into one to three sites in a temporal lobe. After 12 to 14days, animals given
injections into hippocampus developed …….. , ……… seizures that required euthanasia.
After 2 to 3 weeks, animals given injections in the entorhinal cortex developed ……. Seizures.
39) A status epilepticus model of temporal lobe has been develop in infant (6 to 7 months old)
………… monkeys.
40) Studies in nonhuman primates may be useful in learning why the human hippocampus is
predisposed to epileptic injuries and in identifying epileptogenic factors that develop during the
long latent period. True or False.
Answers:
1)
True
2)
True
3)
Wistar Albino Glaxo rats (WAG/Rij), from Strasbourg (GAERS).
4)
Mongolian Gerbils
5)
True
6)
True
7)
Head trauma, infections, status epilepticus, hypoxia/ischemia, birth trauma, and toxins.
8)
True
9)
True
10) Febrile seizures
11) False. Hypoxia model does not induce hippocampal sclerosis and is nor epileptogenic
12) Epilepsy and cerebral palsy
13) E. Cats, rabbits and rats.
14) True
15) True
16) G. All of the above
17) Lewis rats; Sprague –Dawley and Brown Norway rats; Wistar, Fischer 344, ACI and WistarKyoto rats.
18) C. All of the above
19) True
20) True
21) True
22) True
23) True
24) True
25) Intracerebral
26) True
27) False. It presents a pre-epileptic state that can help distinguish between causes and effects
of chronic epileptic seizures
28) True
29) D. All of the above
30) Wistar-Furth and Fisher 344; Sprague-Dawley and Long Evans Hooded.
31) False. Less vulnerable
32) True
33) True
34) True
35)
36)
37)
38)
39)
40)
True
DBA/2J, FVB/N, and 129/SvEMS mice; C57BL/6, BALB/c, C3H, ICR, 129/SvJ, and SJL
True
Severe, spontaneous; less severe
Pigtailed macaque
True
Animal Models of Ischemic Stroke: Balancing Experimental Aims and Animal Care p. 486-496
Abstract: Animal models of ischemic stroke are examples of an induced model that can present
challenges from the perspectives of protocol review and animal management. The review presented
here will include a brief summary of the current state of knowledge about clinical stroke; a general
synopsis of important unanswered research questions that justify use of animal stroke models; an
overview of various animal models of ischemic stroke, including strengths and limitations; and a
discussion of animal care issues relative to ischemic stroke models. Good communication and
interactive education among primary investigators, laboratory animal veterinarians and caretakers,
and institutional animal care and use committee members are critical in achieving a balance between
research objectives and animal care issues when using animal stroke models.
Questions:
1. What is a stroke (cerebral vascular accident)?
2. What are the three leading causes of death in the U.S.?
3. What are the three types of stroke seen in clinical patients?
4. What is the most common type of stroke?
5. What causes ischemic stroke?
6. What is the difference between intracerebral hemorrhage and subarachnoid hemorrhage?
7. True or False: Stroke research has focused prevention, treatment, management, and
underlying mechanisms.
8. True and False: Exploring sex-specific difference in brain injury and the role of sex steroids in
these difference has become an emerging area of stroke research.
9. Most animal stroke models are based on ______________ stroke.
10. What are some limitations of animal models of stroke?
11. Animal models allow investigators to study ________ about stroke.
12. Stroke models can be characterized in terms of the animal species used. What are the two
general categories based on animal species?
13. Name an advantage and limitation of each.
14. Another way to characterize animal stroke models is by type of cerebral injury created. What
are the three types?
15. Which animal is an important model of global cerebral ischemia?
16. What is unique about the vascular anatomy in this animal (from Q15)?
17. How can complete global cerebral ischemia be induced in this model (from Q15)?
18. True or False: There is no variability in rodent models to stroke induction.
19. Most focal stroke models involve which artery?
20. What are some methods used to induce MCAO?
21. What type of stain is used to detect infarct injury in rodent models? How does it work?
22. For many stroke studies, how are animals housed post-surgery?
23. Does environmental enrichment affect experimental outcomes?
24. True or False: Animals with risk factors for stroke may have specific housing needs.
25. What tests are recommended in the guidelines published by the Stroke Therapy Academic
Industry Roundtable (STAIR) for animal stroke models?
26. True or False: Anesthetic choice has not been documented to affect ischemic outcome in
experimental stroke.
27. True or False: During the initial postoperative period “stroked” animals generally require
intensive care.
28. What are some of the poststroke complications that can occur in stroke models?
29. True or False: In animal ischemic stroke models, observed clinical signs of pain are more likely
related to surgical preparation and manipulation than to neuronal damage.
30. True or False: In human patients, pain after acute stroke and brain injury often remains
untreated.
31. Stroke scoring systems are __________ (helpful/not helpful) in establishing intervention
criteria.
32. Are analgesics beneficial/detrimental/or show no effect in experimental stroke models?
33. Is placement of a telemetry transmitter within the abdominal cavity a major or minor surgery?
34. True or False: Criteria for humane endpoints for a given stroke model should be based on
clinical assessment of physical and behavioral parameters before and after experimentally
induced stroke.
Answers:
1. A stroke or cerebral vascular accident is defined as loss or alteration of bodily function that
results from an insufficient supply of blood to part of the brain.
2. Cancer, heart disease, and stroke
3. ischemic, intracerebral hemorrhage, and subarachnoid hemorrhage
4. Ischemic stroke
5. In general, a clot or other blockage in an artery leading to the brain causes ischemic stroke.
6. Intracerebral hemorrhage and subarachnoid hemorrhage are types of stroke caused by
sudden rupture of an artery within the brain. Intracerebral hemorrhage is often related to
hypertension and occurs when the ruptured artery releases blood into the brain, compressing
brain structures. Subarachnoid hemorrhage occurs when the location of the ruptured artery
leads to blood filling the space surrounding the brain rather than inside of it.
7. True
8. True
9. Ischemic stroke
10. Limitations of animal models include physiological variability, substantial mortality in acute and
chronic survival studies, variability of injury or infarct size, and completeness of vascular
occlusion.
11. Animal models allow investigators to study pathophysiologic and neuroprotective mechanisms,
therapeutic responses, immediate and early ischemic events. Histopathologic, biochemical,
and physiologic measurements can also be done in animals.
12. nonrodent and rodent
13. Advantages of nonrodent models include concurrent and multiple measurements (i.e., blood
gas, blood glucose), physiological measurements (i.e., blood pressure), regional imaging
techniques and regional cerebral blood flow (CBF) and metabolism are more easily
determined. Many nonrodent models also have a gyrencephalic or convoluted brain, which is
similar to that in humans. Limitations of nonrodents models include species-specific
anesthesia requirements that may affect outcome measurements, public animal welfare
concerns, cost and labor.
Advantages of rodent models include smaller brain size (allows for more extensive and
comprehensive evaluation of the entire brain), anatomic similarities (especially rats) to human
anatomy of cranial circulation, availability of genetically homogeneous inbred rodent strains,
and examination of functional outcomes. Limitations of rodent models include limitations of
concurrent and multiple measurements over time, limited physiological monitoring, and unlike
humans, rodents have lissencephalic or unconvoluted brains.
14. global, focal, or multifocal cerebral ischemia
15. gerbil
16. Gerbils do not have a posterior communicating artery, which results in an incomplete Circle of
Willis and no redundant circular vascular supply at the ventral aspect of the brain.
17. Complete global ischemia can be induced by bilateral occlusion of the common carotid artery.
18. False.
19. the middle cerebral artery
20. Middle cerebral artery occlusion (MCAO) can be induced by injection of performed fibrin or
blood clots, photochemical thrombosis, electrocoagulation, surgical vascular clips, ligatures,
cuff inflation, etc.
21. 2,3,5-triphenyltetrazolium chloride staining. Viable brain tissue appears red as a result of the
reduction of the stain by mitochondrial enzymes, whereas injuried areas appear white.
22. For many studies, animals are singly housed after surgery.
23. Environmental enrichment may or may not cause confounding on experimental outcomes.
24. True
25. neurobehavioral evaluations, functional tests, and other long-term outcome measures
26. False
27. True
28. pulmonary edema, hypothermia, hypovolemia, generalized shock,
29. True
30. True
31. Helpful
32. Depends on the analgesic, the time points evaluated and the research questions being
studied.
33. major
34. True
1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Lesioned Model of Parkinson’s Disease, with
Emphasis on Mice and Nonhuman Primates p. 497-513
(1.)
The principal neuropathologic feature of Parkinson’s Disease (PD) is selective degeneration
of what portion of the midbrain?
A: Dopamine-producing neurons of the substantia nigra pars compacta (SNpc)
(2.)
The pathological hallmark of PD is the presence of eosinophilic inclusions called ________?
A: Lewy bodies
(3.)
What is the causative agent and the risk factor for PD?
A: Unknown; Age
(4.)
What are four specific Mutations in kindreds with familial PD?
A: a-synuclein (PARK1), parkin (PARK2), ubiquitin carboxy-terminal hydrolase L1 (UCH-1) and DJ1 (PARK 7)
(5.)
A.
What neurotoxicant was focused in this review and what species were used?
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), nonhuman primate and mouse
(6.)
Who developed an acute form of Parkinsonism with an inadvertent self-adminstration of
MPTP?
Heroin addicts
A.
(7.) Were Lewy bodies observed in this group of heroin addicts?
A:
No, Lewy bodies may be age dependent in humans. The same is true in nonhuman
primates, present in aged MPTP-lesioned monkeys.
(8.)
List the six steps in the Neurotoxic mechanism of MPTP.
A: 1.
MPTP crosses the blood brain barrier after system administration.
2.
3.
4.
5.
6.
MPTP converted to 1-methyl-4-phenyl-2,3-dihdropyridinium (MPDP+) by monoamine
oxidase B primarily in astrocytes.
MPDP+ spontaneously oxidizes to 1-methyl-4-pyridium (MPP+).
MPP+ has a substrate dopamine transporter (DAT) and is taken up by the SNpc neurons.
MPP+ leads to inhibition of mitochondrial complex I and depletions of ATP.
Generation of reactive oxygen species (ROS) and death of dopaminergic neurons.
(9.) Which species is very sensitive to MPTP?
A: Nonhuman primates, old world more so than new world.
(10.) Which species are less sensitive and which one is least sensitive?
A:
Mouse, cat, dog, and guinea pig are less sensitive. Rat is least sensitive. There are
differences within strains. CS7BL6 mouse is the most sensitive and CD-1 and BALBC appear
almost resistant. Swiss Webster sensitivity varies among vendors.
(11). Does the age of animals used have an effect on MPTP neurotoxicant effects?
A:
Yes, older mice and nonhuman primate cells contain higher MAO-B activity than in
younger animals.
(12.) What safety factors should be considered in MPTP use?
A: - MPTP and MPP+ should be supplied in secure septum-sealed vials.
- Used by highly trained and experienced personnel.
- Personnel should wear protective clothing to include gowns, double gloves, face
shields/goggles and filtered mask.
- 10 mg. of Eldepryl (2- 5mg. Tablets) availability in case of needle stick.
- A neurologist familiar with PD to supervise human needle stick injuries.
- Animals administered MPTP should not be returned to the colony for 72 hours, bedding should
be incinerated and cages decontaminated with a 1% bleach solution.
(13.) Name four compounds mentioned in the potentiate MPTP.
A: diethyldithiocarbamate (DDC), probenecid, acetalaldehyde and ethanol.
(14.) Mice may receive single injections (sc, ip) , series of weekly injections (ip,sc), addition bolus 30
days later or weekly injections of MPTP with probenecid. (True or False)
A: True
(15.) Nonhuman primates may receive s.c., i.v., i.p., i.m., unilateral and/or bilateral intracarotid
injection. (True or False)
A: True
(16.) What are the effects of diethyldithiocarbamate (DDC) on MPTP when co-ministered in mice?
A: DDC is a copper-chelating agent that increases the bioavailability of MPP + and enhances the
toxicity of MPTP when co-ministered in mice.
(17.) What are the effects of probencid on MPTP when co-ministered?
A: Probencid delays MPTP metabolism and reduces urinary and neuronal clearance of MPTP
and its metabolites.
(18.) What are the effects of acetaldehydes and ethanol on MPTP when co-ministered in mice?
A: Enhance MPTP susceptibility by altering dopamine and glutamine receptor _expression and
dopamine transporter and mitochondrial function.
(19.)
A:
(20.)
Nine species of nonhuman primates have been in the MPTP model; most species do not
exhibit two characteristics of PD. What are these characteristics?
(1). A resting tremor (2) Lewy bodies
What two groups of nonhuman primates appeared to respond to L-DOPA and dopamine
receptor agonists when treated with MPTP?
A: Aged animals and Old World monkeys such as rhesus macaques or African greens.
(21.) List the three stages of PD and some characteristics of each stage and where they occur in
nonhuman primates.
A: 1. Acute – develops within hours – sedation, hyperadrenergic state, hypersalivation,
emesis, exaggerated startle, seizure-like activity dystonic posturing of the trunk.
2.
Subacute – develops within hours to days due to MPTP on the autonomic nervous
system and peripheral organs such as the liver, kidney and heart. Also included are weight
loss, altered blood pressure, hypothermia. Animals may require a feeding tube and
placement in an incubator to stabilize body temperature.
3. Chronic – develops within days to weeks with a stabilization of body weight and
temperature, normalization of certain blood biochemical analysis and
(22.) The systemic lesioned nonhuman primate model with MPTP administered IM, IV, IP and SC
may be used for:
A: - Titrating of lesions
- L-DOPA administration and animals developing motor complications
- Suited for therapeutics, neuroprotective agents and dopamine modulation.
(23.) Intracarotid administration of MPTP allows for the development of:
A: - A hemiparkinsonian state in primates – unilateral administration
- Multiple bilateral carotid administration develops but debilitating model with prolonged stability
- Animals are more self sufficient.
(24.) What was the first species in which behavioral recovery MPTP-lesioning was studied.
A: Cat
(25.) List some of the other species used to study MPTP-lesioned models.
A: rainbow trout, snake, lizard, chicken, rabbit, dog, and pig
(26.)
The MPTP-lesioned mouse and nonhuman primate models have allowed the following
advances in PD to be made.
A: - MPTP disruption leads to depletion of ATPA production and formation of highly deleterious
reactive oxygen species (ROS)
- Neuroprotective strategies depending on the type of cell death proposed.
- Dopamine replacement therapy is one means to overcome the neurochemical deficit.
- Novel neurorestoactive therapies have been developed to include the use of
transplantation of human stem cells into MPTP lesioned mouse.
- Development of specific gene products
- Method to study L-DOPA treated MPTP-lesioned animals which exhibit “wearing off” effect.
- Surgical targeting in patients with idiopathic PD
- MPTP-lesioned mouse and nonhuman primates have an important property of intrinsic
recovery.
-The mouse and nonhuman primate have specific advantages and disadvantages which
may be used in the study of PD.
Lymphoglandular Complexes are Important Colonic Sites for Immunoglobulin A Induction
against Campylobacter jejuni in a Swine Disease Model p. 514-523
Lots of histo pictures in this articleCampylobacter jejuni is the most common reported food-borne infection in the US and is
emerging disease throughout the world. Children are the main group at risk for abdominal pain,
diarrhea, fever, and frank blood in the stool. These symptoms are associated with colitis and cecitis.
Children infected in early life have less severe disease than children infected later in childhood.
Severe infection may allow C. jejuni to become extra-intestinal, particularly in immunocompromised
hosts. Microscopic changes associated with C. jejuni include acute colitis with crypt abscesses,
depletion of goblet cells and inflammatory infiltrates of the lamina propria composed of
polymorphonucuclear neutrophils, lymphocytes, and plasma cells. The role of concurrent infections
of C. jejuni and other bacteria or parasites on the severity of clinical or histologic lesions is unknown.
A lymphoglandular complex (LCG) is a circumscribed submucosal lymphoid nodule, distributed
most densely in the rectum and distal portion of the colon in humans, swine, cattle, rats, horses and
other animals. Grossly they appear as raised nodular domes with a central pore. The nodule is
histologically and ultrastructurally similar to Peyer's patches. The LCG is presumably a lymphoid
follicle with entrapped mucosal crypts opening on the central pore. They have been suggested to
recognize and initiate immune responses against foreign agents. The authors hypothesize that LCG's
are antigen sampling structures that will respond to the presence of C. jejuni.
Cesarean section derived germfree pigs were delivered into germfree incubators
and maintained throughout the study on sterilized complete milk diet. At 3 days of age the pigs were
infected with C. jejuni alone, T. suis alone, or a combination of both, and were compared to noninfected control pigs. The pigs were necropsied at day 27.
Dually infected pigs have been previously shown to have more frequent and severe diarrhea
and more severe pathologic changes than contol pigs, or those singly infected with either T. suis or C.
jejuni.
At day 27, C. jejuni could be detected in LCG's by immunochemical staining and electron
microscopy. Results also showed that the size of the LCG's , particularly the B cell numbers
secreting IgA, in the LGC germinal centers (LCG GC) was increased. This only occurred in pigs
infected with C. jejuni (either single or duel infection), but not in control or T. suis infected pigs. C.
jejuni infected pigs had more LCG GC expansion than did GC's in ileocecal peyer's patches or
mesenteric lymph nodes. Cell surface markers were used for immunostaining and demonstrated the
presence of MHC-II in the basal region of the germinal center and either anti-C. jejuni IgA or IgM (but
not both) in the germinal center core of the LGC. CD4 and CD8 + T cells were found in the mantle
areas of the LGC GC. Macrophages/monocytes were found in all examined tissues, but were more
associated with the ileocecal peyer's patches (ICPP) than LGC's. The lamina propria, follicle domes
and interfollicular areas were heavily infiltrated with macrophages/monocytes.
IgA+ B-cells were present in the follicles and lamina propria of all three groups of infected
pigs. However, only C. jejuni infected pigs displayed IgA+ germinal centers in LCG's. The authors
suggest that C. jejuni infection stimulated a switch to IgA LGC's since discrete IgA+ germinal
centers were seen only in those pigs infected with C jejuni. The greatest total cell numbers
identified were IgM + B cells - found in LGC follicles but not germinal centers. C. jejuni infection in
germfree pigs does cause a change in LCG structure as the authors hypothesized. On the basis of
lesion distribution and quantitative comparisons of IgA specific staining between groups, the LCG was
an important site of C. jejuni immune sensitization compared to the ICPP or mesenteric lymph node.
Questions:
1. Lymphoglandular complexes are found in all mammals (T/F)
2. Swine have been used for experimental models of Campylobacter jejuni infection (T/F)
3. LCG's enlarge after co-infection with C. jejuni and T. suis. (T/F)
4. LCG's don't enlarge after infection with T. suis (T/F)
5. Clinical lesions are worse in C. jejuni singly infected germfree pigs (T/F)
Answers:
1. F. 2. T. 3. T 4. T 5. F.
Microsatellite Analysis in FVB/N Mice p. 524-527
Summary: The purpose of this study by the authors was to identify by size a set of microsatellite
markers the diagnostic genetic monitoring of FVB/N mouse colonies. Using microsatellite technology,
the allele sizes for 27 microsatellite markers in the FVB/N strain of mice were identified. The authors
give an excellent overview on the history and current use of the FVB/N mouse strain on page 524.
Methods: The technology employed in this paper was to evaluate simple sequence length
polymorphisms (SSLP), also called microsatellites. These simple sequence repeats are of variable
length and are randomly distributed throughout the mammalian genome. The identification of these
polymorphisms is use for genetic monitoring. The advantages of microsatellite markers for genetic
monitoring include the following: 1) easy to perform, 2) Large panels of primers have been already
identified for the analysis of markers on all mouse chromosomes 3) Can be more sensitive than
alloenzyme genetic monitoring analysis, 4) DNA can be harvested from tissue for tail snip (2 mm), no
necropsy for tissue harvest is required. Additionally, DNA amplified by PCR technique can be
evaluated using semi-high throughput agarose gel electrophoresis to determine the size of the 27
FVB/N microsatellite marker alleles tested.
Results: In a comparative study, 3 mouse strains were tested: FVB/N, C57BL/6, and C3H/HeN
mouse strains. Of the 27 marker alleles evaluated, 14 have been previously reported in the literature
concerning FVB/N mice. Of the 14 markers previously evaluated, only 1 marker (D8MIT215)
evaluated in this study was not consistent with previously published allele size. The authors reported
that the D8MIT215 locus in the FVB/N mice was equal in size to that of the C3H strain at 164 bp, and
not greater in size that had been previously reported. The sizes of the alleles reported in this study
were consistent with < 6 bp of previously published results.
Conclusion: With standard PCR and gel electrophoresis protocols microsatellite markers can be used
to monitor the genetic integrity of the FVB/N mouse strain
Questions:
1. Define SSLP
2. Define Allele
3. Define Chromosome
4. What are the advantages of using microsatellite technology for genetic monitoring?
Answers:
1. Simple sequence length polymorphisms (SSLP), also called a microsatellite. These simple
sequence repeats are of variable length and are randomly distributed throughout the
mammalian genome.
2. One of the possible alternative forms of a gene usually distinguished from other alleles by its
phenotypic effects.
3. The threadlike structures in the nucleus that carry genetic information.
4. The advantages of microsatellite markers for genetic monitoring include the following: 1)
easy to perform, 2) Large panels of primers have been already identified for the analysis of
markers on all mouse chromosomes 3) Can be more sensitive than alloenzyme genetic
monitoring analysis, 4) DNA can be harvested from tissue for tail snip (2 mm), no necropsy
for tissue harvest is required.
Simple Duplex Fecal PCR Assay that Allows Identification of False-Negative Results in
Helicobacter sp.-Infected Mice p. 528-532
Summary: /Helicobacter/ species are gram-negative, microaerophilic, spirochetal bacteria that
colonize the GI tract of mammals. In the laboratory mouse, currently identified /Helicobacter/ species
include /Helicobacter hepaticus/ (associated with hepatocellular adenomas and carcinomas in A/J
mice, inflammatory bowel disease in SCID, nude and other immunocompromised mice), and /H. bilis,
H. muridarum, H. rappini, H. rodentium, H. typhlonius/ and /H. ganmani/. The principal mouse
pathogens are thought to be /H. hapaticus, H. bilis/ and /H. typhlonius/. Most /Helicobacter/ species
colonize the distal portion of the GI tract in mice, although /H. hepaticus/ and /H. bilis/ are also
localized in the liver. /Helicobacter/ are thought to be transmitted by the fecal-oral route.
Traditionally, infection with /Helicobacter/ has been diagnosed by microaerobic bacteriologic
culture, or histologic evaluation of tissue sections using Seiner's silver stain. In recent years, PCR
analysis has been used as a more specific and less expensive test compared to these earliers means
of diagnosis. Typically, 6-8 fecal pellets are collected per mouse cage, the fecal DNA is extracted
and amplified using either species- or genus-specific oligonucleotied primers. A major concern with
all fecal PCR tests is the potential of obtaining false-negative results due to PCR inhibitors transferred
from feces with the fecal DNA into the reaction that prevent amplification. To detect these falsenegative results, negative samples would have to be re-run spiked with /Helicobacte/r DNA or all
samples tested run in duplicate with the second sample spiked, etc. This report describes a simple
duplex PCR assay in which /Lactobacillus/ species (normal flora of rodent intestine) specific probes
are combined with the Helicobacter specific probes for co-amplification in a single assay. Samples
that are negative for both /Lactobacillus/ and /Helicobacter/ are then re-purified and re-tested to
determine true /Helicobacter/ status. This duplex assay was performed in two different laboratories
using somewhat different PCR conditions, equipment and personel with perfect concordanence of
results. In a series of 202 samples tested at M.D. Anderson, the false-negative rate was <2%,
however without this internal quality control, positive animals would have been mis-diagnosed as
/Helicobacter/ free.
Questions:
1. /Helicobacter/ organisms are typically identified diagnostically in the following mouse tissues:
A) Liver
B) Lung
C) Spleen
D) Feces
E) Liver and Feces
2. T/F /Helicobacter hepaticus, bilis /and /typhlonius/ are considered the major /Helicobacter/
pathogens of mice.
Answers:
1. E
2. True




Involvement of Calpain Isoforms in Retinal Degeneration in WBN/Kob Rats p. 533-542
The Wistar Bonn/Kobori (WBN/Kob) rat originated from a Wistar rat colony in the Institute of
Experimental Gerontology in Basel. With these rats, hyperglycemia is maintained for a long period
and is associated with various secondary diseases included cataracts, nephropathy, and neuropathy.
This rat strain is a model for spontaneous retinal degeneration and is considered useful as a model
for human diabetes mellitus and exo-endocrine pancreatic impairment.
Calpains are calcium activated cysteine proteases. There are two major classes of calpains,
ubiquitous calpains (calpain 1/µ-calpain, calpain 2/m-calpain, and calpain 10), and tissue-specific
calpains. Proteolysis by calpains has been suggested to play an important role in visual functions.
The experiment reported in the article had the following set-up:
Rats were sacrificed at 5, 12, 24, and 48 weeks of age so that the enucleated eyes could be
assessed for morphological changes.
Electroretionograms (ERGs) were recorded at 3, 5, 12, 24, and 48 weeks of age. ERGs were
taken after the rats were adapted to the dark for 2 hours. ERGs were recorded using platinum wire
electrode coils that were positioned on the cornea while the animals were anesthetized.
Dry weights of the retinas were measured and then digested in HCL to allow for calcium
content measurement.
Retinas were also homogenized to allow for protein preparation for immunoblot analysis for
calpain 2, calpain 10 and alpha spectrin (one of the substrates for activated calpain).



Casein zymography was performed on the retinal soluble proteins as well. The intensity of the
zymogram gels of calpain 2 were determined by use of densitometric image analysis.
Isolation of RNA and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was
performed to explain changes in calpain activities.
In situ hybridization was performed for calpain 2mRNA detection.
Light microscopy demonstrated the progression of retinal degeneration as the rats
aged. Substantial changes were noted at 12 weeks. While changes were noted at 5 weeks, they
were not substantially different from the control rats. At 48 weeks, only a single outer nuclear cell
layer remained and the photoreceptor cells were almost gone. Retinal thickness did decrease in the
control rats as they aged, but retinal layers did not disappear. At 48 weeks, male WBN/Kob rats
developed diabetes mellitus and associated cataracts.
Amplitudes of the ERG a waves were significantly less in 12 week old male WBN/Kob rats
compared to the age-matched controls. The amplitudes of the females had a tendency to decrease.
Amplitudes of a waves from the 24 and 48 week old WBN/Kob rats were significantly decreased
compared to age and sex matched controls. A waves originate from photoreceptor cells and the
wave amplitude decreases are associated with the histological changes seen. B waves in 5 week or
older WBN/Kb rats were significantly less than in the age and sex matched controls. B waves
originate from bipolar cells in the inner nuclear layer and the wave amplitude decreases are
associated with the histological changes seen.
Proteolysis studies revealed that there was an increase in alpha spectrin breakdown products
in 12, 24, and 48 week old WBN/Kob rats. Calcium content was increased in the WBN/Kob rats at 5
weeks of age and older compared to the controls. Calcium content increases coincided with the
increase in calpain-induced proteolysis.
Casein zymography was used to measure calpain activities in the retinas. Compared with the
matched control Wistar rats, activities of calpains 1 and 2 increased in 5 week old WBN/Kob rats and
remained so through till 48 weeks.
The _expression of calpain 2 mRNAs was slightly up-regulated at 24 weeks in WBN/Kob rats
compared to controls, and values increased at 48 weeks.
The activation of ubiquitous calpains 1 and 2 was temporally associated with retinal
degeneration in WBN/Kob rats. Retinal degeneration with age was observed histological and
functionally in male and female WBN/Kob rats. The photoreceptor cell layer appeared thin, and the
number of cells in the outer and inner nuclear layers was reduced in 12 week old WBN/Kob rats.
These changes became severe with age. Proteolysis of alpha spectrin was strongly associated with
retinal degeneration. Caseinolytic activity of calpains 1 and 2 increased in male and female
WBN/Kob rats after the age of 12 weeks. Increased calpain was also confirmed by results for
immunoblot analysis. Calpain activity was temporally associated with proteolysis of alpha spectrin.
These results were different than those of previous studies which indicated a decrease in caseionlytic
activity in retinas after ischemia-reperfusion. Total calcium concentrations were increased in retina
from WBN/Kob rats at five weeks and gradually increased.
A positive relationship between
increased calcium content and activation of calpain was observed during retinal determination of
WBN/Kob. _Expression of calpain 10mRNA was only slightly down-regulated in WBN/Kob rats but
the _expression patterns of calpain 10 proteins were different in male and female WBN/Kob and agematched Wistar rats. These changes were associated with retinal degeneration in WBN/Kob rats.
Questions
1) Male WBN/Kob rats develop hyperglycemia due to what disease state?
2) Calpains are __________ activated _________ proteases.
3) The two types are calpains are:
a.
Ubiquitous and organ-specific
b. Tissue and organ-specific
c.
Ocular and tissue specific
d. Ubiquitous and tissue-specific
4) ERG stands for what diagnostic measurement?
5)
6)
7)
8)
A waves of ERGs originate from what type of retinal cell?
What is alpha spectrin?
Calcium content is in increased due to the increase in calpain-induced ___________.
T or F Activation of ubiquitous calpains 1 and 2 was not temporally associated with retinal
degeneration in WBN/Kob rats.
Answers
1) Diabetes mellitus
2) Calcium, cysteine
3) D
4) Electroretinography
5) Photoreceptor cells
6) Substrate for calpain
7) Proteolysis
8) F
Scanning Electron Microscopy of the Infundibulum, Ampulla, and Eggs of Mice p. 543-548
Summary: Scanning electron microscopy (SEM) was conducted to discover the details of 3dimentional profiles of infundibulum, ampulla, and eggs in the mouse. The topographic
understanding of the infundibulum facilitates quicker identification of these organs to achieve a more
satisfactory transfer of microinjected mouse eggs through the small opening of the infundibulum to
the ampulla of the oviduct of a pseudopregnant (foster) mouse. It will reduce the chance of damaging
the infundibulum. The infundibulum is a small organ embedded inside the bursa membrane.
Bleeding, which occurs once the bursa membrane is surgically opened to expose the infundibulum,
makes it difficult to find the organ in a short time for successful transfer. In this SEM study, the bursa
membrane covering the ovary and oviduct was carefully and surgically opened to expose the natural
location of the infundibulum between the ovary and oviduct. The SEM preparation preserved the
similar or same appearance and the natural/geographic/topographic location of the infundibulum in
relation to the ovary and oviduct. This will help investigators find it quickly and be able to transfer
more microinjected mouse eggs into the infundibulum in a shorter period. It will also help reduce
bleeding of the bursa membranes, which should lead to a higher percentage of pups being born.
In this study, many fertilized eggs accumulated in a swollen ampulla region of the oviduct. The
detailed appearance of each crown of the infundibuluar head differed in SEM. Observation of the
same or similar type of cilia on the surface of the crown folds of the infundibular head as those on the
surface of the internal linings of the infundibulum/oviduct walls provides direct evidence that the
crown folds of the infundibular head are the evagination extension of the internal surface of the
infundibulum/oviduct walls. Also the new observation of the narrowing region of the crown in the
infundibular head after surgical removal of the crown supports the theory of so-called evatination
process of the internal cilia of the infundibulum/oviduct wall that forms the crown of infundibuluar
head.
The overall diameter of the mouse egg at the one-celled stage is approximately 80 to 120 um. The
size of eggs from the CD-1 and FVB/N mouse strains was usually >100 um in diameter, and they
were easy to microinject. Each egg was enclosed in a mucoprotein envelope, the zona pellucida,
which is composed of 3 major acidic sulfated blycoproteins – Zp1, Mr200,000; ZP2, Mr 120,000; ZP3,
Mr 80,000. ZP3 functions as a spermatozoa receptor and initiates the acorosome reaction. There
were one or two polar bodies from which one or two projections were protruding. The shape of each
egg ranged from sherical to oval, and the surface of the fertilized egg had a rougher appearance than
that of the on-fertilized egg. The rough surface of the fertilized egg is an important sign to observe
before selecting an egg for microinjection.
Questions:
1. What supports the theory of the so-called “evagination process” of the internal cilia of the
infundibulum/oviduct wall that forms the crown of the infundibular head in this SEM study?
2. What is an envelope called that covers each fertilized egg?
3. Which glycoproteins of the zona pellucida functions as a spermatozoa receptor and initiates
the acrosome reaction?
a)
Zp1
b)
Zp2
c)
Zp3
4. T/F: The surface of the fertilized egg had a smooth appearance.
Answers:
1. Observation of 1) the same or similar type of cilia on the surface of the crown folds of the
infundibular head as those on the surface of the internal linings of the infundibulum/oviduct
walls, 2) the narrowing region of the crown in the infundibular head after surgical removal of
the crown.
2. zona pellucida
3. c) Zp3
4. F
Pathogenicity of Helicobacter rodentium in A/JCr and SCID Mice p. 549-557
Helicobacter rodentium was first recognized as a potential pathogen when it was isolated, along with
Helicobacter bilis, from a colony of scid/Trp53 knockout mice with diarrhea. Clinical disease in these
mice was more severe than that previously reported in mice infected with H. bilis alone, thus
suggesting that H. rodentium contributed to the pathogenesis of enteritis. The purpose of the study
reported here was to address two questions: is H. rodentium pathogenic in mice, and when coinfection with a pathogenic helicobacter occurs, does H. rodentium augment disease? To this end,
A/JCr and C.B-17/IcrCrl-scidBr mice were inoculated with H. rodentium and/or H. hepaticus. Twelve
weeks after inoculation, mice were euthanized. The cecum and liver were evaluated microscopically
for evidence of disease. Cecal interferon-inducible protein 10 (IP-10), macrophage inflammatory
-10), and interferon gamma (IFNmeasured as an indicator of mucosal immune response. Hepatic lesions were not identified in mice
mono-infected with H. rodentium; likewise, cecal lesion scores were not significantly different from
those of uninfected controls. With the exception of an increased IL-10 mRNA value in SCID mice,
mean immune-related gene _expression in H. rodentium mono-infected and uninfected control mice
was not significantly different. In contrast, all mice infected with H. hepaticus developed moderate to
severe hepatitis, significant increase in cecal lesion scores, and increased immune-related gene
_expression. The C.B-17/IcrCrl-scidBr mice co-infected with H. hepaticus and H. rodentium had liquid
cecal contents and low terminal body weight. Further, compared with mice infected with H. hepaticus
alone, co-infection was associated with significant increases of IL-10, MIP-10 mRNA
values in C.B-17/IcrCrl-scidBr and IFNsuggested that H. rodentium alone does not cause hepatitis or enteritis in A/JCr or C.B-17/IcrCrlscidBr mice; however, co-infection with H. hepaticus and H. rodentium was associated with
augmented cecal gene -expression and clinical manifestation of disease in immunodeficient mice.
Take Home Message:
H. hepaticus is the only species that consistently causes enteritis and chronic active hepatitis
in immunodeficient as well as susceptible strains of immunocompetent mice.
H. rodentium has only been convincingly associated with disease in immunodeficient mice coinfected with H. bilis .
H. rodentium is a spiralshaped, fastidious, microaerophilic bacterium. It has bipolar, single,
nonsheathed flagella, and was the first urease-negative Helicobacter sp. isolated from the intestine
of mice.
Helicobacter hepaticus and H. rodentium infection can be diagnised using a species-specific
PCR assay designed to amplify non-conserved regions of the 16S rRNA gene.
H. hepaticus-infected mice had moderate, multifocal, non-suppurative, necrotizing hepatitis.
There is no significant difference in the severity of histologic lesions in mice co-infected
with H. rodentium and H. hepaticus compared with that in mice infected with H. hepaticus
alone. These results alone suggest that H. rodentium is nonpathogenic in mice.
The implication of this study for rodent facility managers and veterinarians is that H. rodentium
may be an acceptable contaminant in most conventional mouse colonies.


Evaluation of Liposome-Encapsulated Oxymorphone Hydrochloride in Mice after Splenectomy
p. 558-563
This article discusses a method for delivery of slow release oxymorphone using a subcutaneous
injection of liposome encapsulated oxymorphone (LE oxymorphone). The model used for analgesic
evaluation is splenectomy under isoflurane anesthesia in six week old ICR mice. Post operative
responses were compared between Buprenorphine, LE oxymorphone, and no analgesia following
splenectomy with controls that were anesthetized but no surgery was done and given the LE drug or
no analgesic. Comparisons are also made to previous studies of the LE drug in rats. Parameters
evaluated after surgery included food and water consumption, body weight, an ethnographic score
(see Table 1), and voluntary exercise on a running wheel. The LE oxymorphone provided more rapid
return to baseline presurgery values of these parameters than the buprenorphine or no analgesic
groups, especially as measured by running wheel activity. A large advantage of LE oxymorphone is
that repeat dosing is not required, unlike the commonly used buprenorphine. Both analgesics
provided for a faster recovery than controls receiving only saline. Oxymorphone also does not have a
ceiling effect, meaning a maximum level of analgesia above which increased dose has no additional
effect, while buprenorphine does have a ceiling effect. Buprenorphine is thus more applicable to
procedures leading to mild to moderate pain where a pure agonist opioid such as oxymorphone is
recommended for moderate to severe pain.
This study provides needed scientific evaluation of analgesia for mice undergoing major
surgical procedures, for which there is currently limited information in the literature. Mice do not
display many of the behaviors evaluated in larger animals to indicate pain. Giving repeated injections
involves handling which in itself is a stress for these animals. This group noted that running wheels
have been used to assess voluntary exercise in mice, and used it here to evaluate lack of pain
indicated by willingness/ability to engage in voluntary exercise.
Analgesics for rodents include NSAIDs and opioid analgesics. Buprenorphine is most widely
used and has been dosed by injection or oral administration. It is believed to have a duration of
action of 6-12 hours while the other opioids such as morphine sulfate, butorphanol tartrate, or
oxymorphone which must be given at very short intervals or by continuous IV infusion. In previous
work with rats by this same group, buprenorphine did not demonstrate effective analgesia in rats after
intestinal resection. Long acting tablets, transdermal patches, and other long acting forms are not
easily adapted to mice, and the oral forms must be swallowed intact to achieve long release action.
Liposome-encapsulated oxymorphone was developed by Douglas Pharmaceuticals Ltd., Auckland,
New Zealand by the authoring group. It was tested in a rat model and found to provide long term
relief of postsurgical visceral pain. One injection provided at least 48 hours of analgesia, while the
standard drug must be given every 4-8 hours for rats. For other pain such as nerve injury the effect
of the LE product lasted for 7 days. This study looked further at use of the new product in mice, using
splenectomy as the visceral pain model. The authors also sought to evaluate use of running wheels
along with behaviors normally monitored, food intake, and changes in body weight.
Review the figures in the text for summary of the results of each parameter. These show
nicely that that:
Mice receiving analgesics recover faster than saline controls
Voluntary running post op was near the level of mice only anesthetized for the LE group, while
mice receiving buprenorphine (repeated doses) showed decreased running even without surgery at
24 hours.

Body weight dropped after surgery and recovered rapidly (2-3 days) with the LE group, while
mice receiving buprenorphine did not return to baseline weight within 5 days. Their weights were
even below mice that were anesthetized only.
Rats given buprenorphine at clinical doses showed decreased food intake and increased home
cage activity without surgery. In rats, buprenorphine improved all measures of post op recovery,
while in mice the groups given buprenorphine were the slowest to recover even over saline treated
animals and showed decreased activity. This may be due to the dose or dosage interval used in
mice, a differing amount of pain in the model surgeries used, or the ceiling effect of this partial agonist
drug.
Running wheels have not previously been reported for evaluation of surgical recovery, but
have been used in behavioral and physiologic evaluations. Interestingly, at 48 hours using the
standard ethnographic score (moving in cage, grooming, awkward walking, hunching, etc.) all mice
scored as recovered, while when wheel activity was used the animals were not all recovered to their
normal levels.
Comparisons are made to other tests traditionally used in rodents to evaluate analgesic
preparations either in developing analgesics or finding the doses to use for various procedures.
These include withdrawal tests of tail or paw to a heat source (tail flick or hot plate tests). These tests
evaluate high intensity, short duration pain. Surgery is more likely to produce low intensity, long
duration pain. Using a battery of tests can be expensive and time consuming but is needed for
behavioral phenotyping and in assessment of novel compounds. The running wheel used here is in
the author's use a valuable and simple test to use in addition to the more traditional ethogram.
Questions:
1. In this study of mice, what model was used to produce visceral pain? What was previously
used in rats?
2. Buprenorphine is a ____________agonist opioid. Oxymorphone is a ____________opioid.
What effect is seen with Buprenorphine but not Oxymorphone?
3. What test did this group find valuable in monitoring post operative recovery?
Answers:
1. Splenectomy (for mice)
Intestinal resection or bile duct ligation (for rats)
2. Partial/pure agonist/ceiling effect
3. Running wheel activity.
Improved In Vitro Fertilization and Development by Use of Modified Human Tubal Fluid and
Applicability of Pronucleate Embryos for Cryopreservation by Rapid Freezing in Inbred Mice
p. 564-570
Background: Cryopreservation of in vitro fertilized embryos is preferred over in vivo fertilization,
especially in genetically modified strains, because it is more efficient, is less costly, requires less
labor, and requires only a few males to yield a large number of zygotes at a time. The sensitivity of
embryos and gametes to in vitro fertilization (IVF), embryo culture, and cryopreservation
manipulations varies among genetically distinct mice, and limited and varied outcomes of these three
techniques are often encountered in some inbred mice. Inbred strains are more sensitive to
cryopreservation manipulations than outbred strains, and freezing at early embryo stages is less
successful than at later stages. Low success rates for IVF and development are reported in BALB/c
and C3H strains (both frequently used to produce congenic strains), and as observed in other
mammalian species, retarded (or blocked) development in vitro has been reported at the two-cell
stage of outbred and some inbred mouse strains (the “two-cell block”). The authors examined in vitro
fertilizability and development of 10 inbred mouse strains (C57BL/6J, C57BL/10,
C57BL/10.D2/newSn, C57BL/10-Thy1.1, C57BL/10.Br/Sn, C3H/He, RFM/Ms, STS/A, BALB/c-nu, and
C.B-17/Icr), and the viability of frozen-thawed in vitro fertilized (IVF) embryos after embryo transfer
(ET). Given a rate of ~50% for in vivo fertilization of superovulated inbred mice, the authors noted
that an IVF success rate > 50%, and ideally, > 80% will be required for practical use of IVF.
Results:
• In seven of the strains tested, fertilizability was significantly greater in modified human tubal fluid
(mHTF) compared with modified Krebs-Ringer’s bicarbonate solution (TYH medium), with TYH
medium
observed
to
support
virtually
no
fertilization
in
four
strains.
• More than 80% of IVF embryos developed to the blastocyst stage by 120 hours in potassiumenhanced
simplex
optimization
medium
(KSOM).
• BALB/c-nu mice show low fertilization success in TYH due to spermatozoal deficiencies, and
tweaking the capacitation media did not improve things; mHTF supported BALB/c-nu spermatozoa
penetration
into
the
zona
pellucida
irrespective
of
the
capacitation
media.
• In vitro fertilized embryos frozen-thawed rapidly were transferred to surrogate mothers at the twocell stage, and compared to unfrozen controls, rapid freezing had no significant effect on fetus
development
in
nine
of
the
ten
strains
tested.
Conclusions:
• mHTF medium is superior to TYH with respect to IVF of inbred mice.
• KSOM adequately supports in vitro fertilized embryo development in inbred mice.
• rapid freezing of pronucleate embryos following IVF is suitable for cryopreservation and embryo
banking of inbred mice and for the production of genetically modified mice.
Questions:
1. Can you list four reasons why cryopreservation of in vitro fertilized embryos is preferred over in
vivo
fertilization,
especially
in
genetically
modified
strains?
2. What are three types of manipulations to which embryos and gametes of genetically distinct mice
show
varied
sensitivity?
3. Which type of mice are more sensitive to cryopreservation manipulations: inbred or outbred?
4. Is freezing at early embryo stages more, or less, successful than freezing at later stages?
5. What is the common term for the retarded (or blocked) development in vitro reported at the two-cell
stage
of
mice
and
other
mammalian
species?
6. Which of the following media used for mouse IVF did the authors demonstrate suitable for the 10
inbred
strains
tested:
a.
modified
Krebs-Ringer’s
bicarbonate
solution
b.
modified
Tyrode’s
solution
c. tissue culture medium with Earl’s balanced salt solution supplemented with amino acids and
vitamins
d.
mHTF,
a
medium
based
on
human
tubal
fluid
Answers:
1. Cryopreservation of in vitro fertilized embryos is it is more efficient, less costly, requires less labor,
and requires only a few males to yield a large number of zygotes at a time.
2.
in
vitro
fertilization
(IVF),
embryo
culture,
and
cryopreservation
3. inbred strains are more sensitive to cryopreservation manipulations than outbred mice
4. freezing at early embryo stages is less successful than freezing at later stages
5.
the
“two-cell
block”
6. d. mHTF, a medium based on human tubal fluid
Helicobacter bilis-Associated Hepatitis in Outbred Mice p. 571-577
Summary
Helicobacter spp. are established pathogens in many species. Enterohepatic Helicobacter spp.
(EHS) is of interest regarding hepatic disease and Inflammatory Bowel Disease (IBD). H. bilis is a
confirmed pathogen causing IBD in immunodeficient mice. This article seeks to examine the
enterohepatic disease potential of naturally occurring infections of H. bilis in outbred mice. The
outbred
strain
used
was
the
Swiss
Webster
(SW)
mouse.
H. bilis is characterized as being fusiform to curved in shape, the presence of periplasmic fibers and
having 3-14 bipolar sheathed flagella. It colonizes the gastrointestinal tract and the liver.
The study confirmed that as in inbred mice H. bilis efficiently colonizes the gastrointestinal tract and
the liver. H. bilis liver infection was associated with hepatitis and the lesions were more severe in
aged mice. The lesions observed in the liver varied from no lesions to disseminated focal aggregates
of leukocytes found in close proximity to portal triads. The cell infiltrates were predominantly
comprised
of
lymphocytes
and
macrophages.
It was noted that in some of the control mice (no H. spp. isolated) a focal idiopathic necrotic hepatitis
was observed. This is thought to be a vascular disease created by microvascular thrombosis and
subsequent infarction. H. bilis has (distinct from idiopathic hepatitis) a portal triad associated
inflammation.
A small population study based on sex suggested that females have more portal inflammation than
do the males. This is in contrast to H. hepaticus where the males have significantly more liver
inflammation.
H. bilis colonizes the gastrointestinal tract and liver of outbred SW mice. It is linked to varying
degrees of hepatitis generally associated with portal triads. H. bilis has the potential to confound
experimental
results.
Questions
1.
H.
bilis
is
colonized
from
which
organs?
2. In what area of the liver are lesions associated with H. bilis generally found?
3. T/F Idiopathic necrotic hepatitis observed in control mice is difficult to differentiate from the
hepatitis
associated
with
H.
bilis.
Answers
1.
H.
bilis
efficiently
colonizes
the
gastrointestinal
tract
and
the
liver.
2. disseminated focal aggregates of leukocytes located in close proximity to portal triads.
3. F H. bilis has (distinct from idiopathic hepatitis) a portal triad associated inflammation.
Outbreak of Mycobacterium bovis in a Conditioned Colony of Rhesus (Macaca mulatto) and
Cynomolgus (Macaca fascicularis) Macaques p. 578-584
I would strongly urge the readers to go through the gross and histopathology slides in
this article.
This is an article about outbreak of tuberculosis in rhesus and cynomolgus monkeys in
a animal facility. Simian tuberculosis is caused by two species of aerobic facultative
intracellular bacilli, Mycobacterium tuberculosis and M. bovis. The out break happened when a
new shipment of monkeys were received and later cleared by a quarantine period of 30 days
by TST (tuberculin skin test). Most prominent lesions were enlarged lymph nodes(mainly
tracheobroncheal) with grey, green, soft, cheesy, caseous necrosis. Palpably firm nodules,
areas of parenchymal consolidation, and/ or gross bronchopneumonia with airway exudates
were typical of the lung lesions. Histologically, affected lymph nodes are often effaced by large
areas of amorphous , eosinophilic, granular caseous necrosis surrounded by dense infiltrate of
epitheliod macrophages, called Langhans' giant cells. But the definite cause of the outbreak
was not determined unequivocally. It has been found that cynomolgus have less severe clinical
symptoms than the rhesus. Moreover, the rhesus monkeys produced twice the IFN* than the
cynomolgus.
Questions:
1. Name the organism(s) that cause simian tuberculosis.
2. What will be a ideal quaratine period in an animal facility according to the authors.
3. Name alternative screening method that can be used in an animal
facility for TB
Answers
1.Mycobacterium tuberculosis and M. bovis
2.60 days
3.PRIMAGAM test and ESAT-6 ELISA.
Legg-Calve-Perthes Disease in a Rhesus Macaque (Macaca mulatto) p. 585-588
This article describes a case of LCP in a juvenile Rhesus Macaque. This is thought to be the first time
this disease has been describe in the Rhesus Macaque.
Osteonecrosis of the femoral head and neck of young growing animals and humans is referred
to as Legg-Calve-Perthes disease. It has formerly been described in dogs, spontaneously
hypertensive rats, the red panda, and the lowland gorilla. A similar disease has been seen in
chickens.
Common synonyms for LCP are avascular necrosis of the femoral neck, osteonecrosis, coxa
plana, and osteochondritis deformans juvenilis.
The macaque is this case was a 27-month old female exhibiting an unusual amount of
prominent sex skin for a monkey of this age. The monkey's previous medical history was
unremarkable. The monkey presented with atrophy of 50-75% of the musculature of the left leg and a
subtle gait abnormality due to decreased weight bearing on the left leg. Attempts at pain reduction
with NSAIDS and opioids had little or no effect. A femoral head ostectomy (FHO) was elected and the
surgery was successful. The monkey is regaining musculature of the left leg and has a better use and
range of motion.
The article included photographs and radiographs of classic lesions associated with LCP that
should be seen by those studying for the boards.
The cause of LCP is unknown. A disruption in blood flow to the femoral head is thought to be
the inciting factor. Potential causes include: abnormal limb position, trauma, synovitis, corticosteroid
administration, endotoxins, immune reactions, and endocrine abnormalities. The authors speculate
that a high level of estrogen in this young animal may have led the prominent sex skin and
contributed to the LCP. The authors go on to describe treatment plans including conservative
bandaging, splitting and treatment with anti-inflammatory drug if the disease in detected early before
severe joint disease has occurred and FHO the accepted treatment in dogs with LCP. They feel that
the monkey is doing very well after FHO despite their initial reluctance to try the procedure in a more
bipedal species.
Questions:
1. What species is the Rhesus monkey?
2. At what age is a female Rhesus considered to be sexually mature?
3. What is Legg-Calve-Perthes disease?
4. In what species has Legg-Calve-Perthes been described?
5. What are the common clinical signs and radiographic finding in Legg-calve-Perthes disease?
Answers:
1. Macaca mulatta
2. 34-43 months of age. (LAM 2nd edition)
3. osteonecrosis of the femoral head and neck of young growing animals and
4. humans, dogs, spontaneously hypertensive rats, the red panda, and the lowland gorilla
5. Clinical signs:
Lost of musculature in the affected limb, gait abnormalities, crepitus and reduced range of motion of
the coxofemoral joint.
Radiographic findings:
Widening of the acetabulum, loss of sclerotic margin of the acetabular rim, femoral head small and
irregular.