Comparative Medicine
Volume 60, Number 4, August 2010
OVERVIEW
Karmarkar et al. Considerations for the Use of Anesthetics in Neurotoxicity
Studies, pp. 256-262
Domain 3 – Research; T2 – Advise and consult with investigators on matters related to
their research
SUMMARY: Anesthetic agents are widely used in laboratory settings, however they may
sometimes alter the outcome of the experiment, and there are several studies showing
that anesthetics alter the processes being studied in neuroprotection and neurotoxicity
experiments. This article reviews the effects of isoflurane, dexmedetomidine, propofol,
ketamine, barbiturates, halothane, xenon, carbon dioxide, and nitrous oxide. They all
showed to have substantial effects on neuronal survival and death. These findings make
it necessary to take anesthetics into account when designing a study that especially
involves in vivo cerebral ischemia. Sham controls with anesthesia but no ischemia
should be used in the study design, as well as control animals without anesthesia or
ischemia. Measuring out endpoints over an extended period as the animal recovers
since the effects of most anesthetics lessen over time. Another option for animals that
will be euthanized is to use non-pharmacologic methods whenever appropriate.
QUESTIONS
1. Which are some acceptable non-pharmacological methods of euthanasia for rats and
mice?
a. Cervical dislocation
b. Decapitation
c. Microwave irradiation
d. A and B
e. All of the above
2. How can studies be planned to minimize the effects of anesthetic agents on
neuroprotection and neurotoxicity studies?
a. Use sham controls treated with anesthesia but no ischemia
b. Never use anesthesia when designing cerebral edema studies
c. No changes need to be made to take in account anesthesia
d. None of the above
3. True or False: Cervical dislocation and microwave irradiation can be used as an
acceptable means of euthanasia for all types of animals.
ANSWERS
1. E
2. A
3. False
ORIGINAL RESEARCH
Mouse Models
Cray et al. Quantification of Acute Phase Proteins and Protein Electrophoresis in
Monitoring the Acute Inflammatory Process in Experimentally and Naturally
Infected Mice, pp. 263-271
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and
Conditions; T3. Diagnose disease or condition as appropriate
SUMMARY: Acute phase proteins (APP) are part of the innate immune system. Some
(C reactive protein, serum amyloid A, and serum amyloid P) are indicators of acute
inflammation while others (haptoglobin) may be more indicative of chronic inflammation.
This project looked to study the changes in APP in laboratory mice after experimental
infection with Sendai virus and MPV. LPS and CFA were used as controls. Changes in
APP were mild, if at all evident, which was an unexpected outcome in this study as
others have shown more sensitivity in these assays in infection and inflammation.
Protein electrophoresis may provide a preliminary test for sentinels before the more
labor-intensive ELISA testing. Other conditions may cause an increase in APP, thus the
electrophoresis cannot be a substitute for ELISA testing entirely.
QUESTIONS
1. True/False: Protein electrophoresis can quantitate single proteins.
2. Short answer: Which conditions can cause a change in acute phase proteins?
3. Short answer: ELISA quantifies proteins at the ng/mL level, while electrophoresis
quantifies at the _____ level
ANSWERS
1. False. Groups of proteins may be quantitated by electrophoresis.
2. Infection, trauma, stress, neoplasia, inflammation
3. mg/Ml
Ding et al. Lack of Association of a Spontaneous Mutation of the Chrm2 Gene
with Behavioral and Physiologic Phenotypic Differences in Inbred Mice, pp. 272281
Domain 3: K6 (Research: characterization of animal models)
Primary Species: Mouse (Mus musculus)
SUMMARY: Type 2 muscarinic receptors (M2R) are present in many peripheral and
central sites of the nervous system and peripheral target organs. The M2R is encoded
by the gene Chrm2, which is relatively conserved across several species. However, a
nucleotide substitution (C797T) has been identified in several strains of inbred mice that
results in an amino acid substitution (P266L) in the protein. Single-nucleotide
polymorphisms in the human Chrm2 gene have been implicated in several abnormal
conditions. Mouse strains that bear the Chrm2 mutation potentially provide a unique
model for exploring mechanisms by which Chrm2 variants may affect cholinergic
mechanisms and associated physiologic processes in both the brain and periphery. This
study attempted to determine whether the C797T Chrm2 mutation confers a detectable
phenotypic difference in M2R-related processes in mice.
Adult male C57BL/6J, BALB/cByJ, and CXB recombinant inbred mice were utilized for
this study. Methods of assessment included the following:
 Acoustic startle, acoustic gap detection, and prepulse inhibition (PPI) of startle
 Physiologic responses to the muscarinic agonist oxotremorine (OXO)
o Tested for: heart rate, body temperature, tremor, salivation, and response to painful
stimuli
 RT-PCR analysis in hypothalamus and basal forebrain (Chrm2 transcription was
measured in basal forebrain and hypothalamus of healthy mice and mice infected with
the influenza virus)
 Chrm2 sequence and genotype analysis (using cDNA synthesized from the lungs)
 Receptor binding (competition curves for OXO were performed by using heart
homogenates )
The data in this study confirmed the presence of a single-nucleotide polymorphism and
an associated amino acid substitution in A/J and DBA/2J mice as compared with
C57BL/6J mice and further reports the presence of this mutation in BALB/c-based
strains, AKR/J and some CXB RI strains. However, the authors detected no difference
between C57BL/6J and BALB/cByJ mice in several types of assessments that are
relevant to M2R function:
 No relationship was demonstrated between the Chrm2 mutation and acoustic startle
and PPI
o While less PPI and a greater startle amplitude in BALB/cByJ mice was observed
when compared with C57BL/6J mice, this was attributed to a higher basal state of
arousal in BALB/cByJ mice and the significant age-related hearing loss in this strain.
 The 2 strains of mice responded similarly in terms of hypothermia, tremor, salivation,
and analgesia to in vivo administration of OXO
o These responses to OXO are impaired in mice that lack Chrm2 but not in mice that
lack genes for other muscarinic receptors
 Chrm2 transcription in basal forebrain and hypothalamus was not influenced by
genotype and/or health status (influenza infection)
 No differences were noted in competition of OXO at binding sites in heart homogenate
or in a shift in the OXO competition curve
Chrm2 mutation with the phenotypes of receptor binding, Chrm2 mRNA in acetylcholinerich brain regions, behavioral responses to OXO, and measures of arousal and
sensorimotor gating. However, the mutation could potentially influence receptor binding
or signal transduction properties in ways that were not tested by this study.
QUESTIONS
1. How would a single-nucleotide polymorphism in the Chrm2 gene cause functional
consequences of the M2 receptor?
2. T/F - Polymorphisms in the human Chrm2 gene have been associated with major
depression in women in some studies.
3. Acoustic startle, acoustic gap detection, and prepulse inhibition (PPI) of startle
provide sensitive measures of:
a. Hearing function
b. Sensory gating processes
c. Both hearing function and sensory gating processes
4. Heart homogenates were used to determine competition curves for OXO because:
a. M1 is the major muscarinic receptor in the heart
b. M2 is the major muscarinic receptor in the heart
c. M1 and M2 receptors are in equal proportion in the heart
ANSWERS
1. Proline is the only amino acid that contains a secondary amino group and forms
tertiary peptide bonds. Because of this attribute, substitution of leucine for proline
could cause allosteric alterations in proteins, with potential structural or functional
consequences. Furthermore, allosteric modulation is a recognized regulatory
mechanism of muscarinic receptors.
2. True
3. c.
4. b.
Rat Models
Ozaki et al. Insulin-Induced Hypoglycemic Peripheral Motor Neuropathy in
Spontaneously Diabetic WBN/Kob Rats, pp. 282-287
SUMMARY: This study attempted to compare the neuropathy caused by hypoglycemia
caused by intensive insulin therapy to the peripheral motor neuropathy seen in
spontaneously diabetic Wistar Bonn Kobori (WBN/Kob) rats. This study was different
from other experimental studies on type 1 diabetic peripheral neuropathy as it included
morphologic analysis along with motor nerve conduction velocities. Male rats with
ongoing hyperglycemia and glucosuria were divided into three groups for a 40 day
treatment study; D group consisted of untreated animals (blood glucose BG, 350420mg/dL), H group consisted of insulin treated animals (BG, 50-200mg/dL), N group
consisted of insulin treated animals (BG, 150-250mg/dL).
RESULTS: Conduction velocity was not significantly different in N, D, and H groups.
Morphologic analysis of the sciatic nerves of H rats showed severe changes, including
axonal degeneration, myelin distention, and endoneurial fibrosis, that tended to occur in
large, myelinated fibers. Only relatively mild changes were noted within the N and D rats.
The degree and distribution of degenerated nerve fibers in H rats were significantly
higher than in N and D rats. The authors suggest that hypoglycemia of less than
50mg/dL induced severe peripheral neuropathy, and also stated that hypoglycemic
lesions differed from the hyperglycemic lesions in diabetic WBN/Kob rats.
QUESTIONS:
1. T/F Diabetic WBN/Kob rats spontaneously develop diabetic peripheral motor
neuropathy characterized by segmental demyelination and secondary axonal
degeneration.
2. WBN/Kob rats generally acquire hyperglycemia and glucosuria by what age?
3. In this study, how often was blood glucose samples obtained?
ANSWERS:
1. T
2. 40-45 weeks
3. Once weekly for H and N groups, at the beginning and end of study for group D
Shin et al. Spatiotemporal Expression of tmie in the Inner Ear of Rats during
Postnatal Development, pp. 288-294
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and
Conditions; K1. anatomy with emphasis on features which have significance with regard
to clinical medicine or experimental medicine
Domain 3: Research
Primary Species: Rat (Rattus norvegicus)
SUMMARY: Inner ear defects which affect the vestibular system are thought to be a
potential cause of circling in the mouse and rat, especially in deafness mutants. The
circling mouse (cir/cir) is a model of human nonsyndromic deafness DFNB6. Circling
mice exhibit almost completely degenerated cochlea and remarkably reduced cellularity
in spiral ganglion neurons. The phenotype is linked to a deletion of the transmembrane
inner ear (tmie) whereas an additional mutation of the same gene is reported in the
spinner (sr/sr) mouse. Neither of these models have any noteworthy problems in any
tissues except those of the inner ear systems. The purpose of this study was to track
the spatiotemporal expression of tmie in the rat at the time of hearing development
(approximately postnatal day 12). This time coincides with the formation of the tunnel of
Corti and the establishment or retraction of neuronal connections. At approximately day
P1, the stereocilia of the organ of Corti begin to thicken and elongate into the staircase
pattern of gradually increasing heights. (Recall that these cilia “sway” within the
endolymph of the cochlea to transform the mechanical energy of sound pressure into
electrical signals which can then be transmitted down the vestibulocochlear nerve,
CNVIII). Immunohistochemical analysis of tmie expression in the inner ear during
postnatal development (postnatal day 0-19) was measured. Tmie expression increased
and expanded as the organ of Corti matured and expression was stronger in the
stereocilia of hair cells than in the cell body region. Conversely, expression in vestibular
systems showed no obvious change over this timeframe. These results imply that tmie
may have a key role in the maturation and structure of stereocilia bundles in developing
hair cells. Then after hair cell maturation, the authors hypothesize that tmie is involved
in the maintenance of organ of Corti cells.
QUESTIONS
1. Mutations in the transmembrane inner ear gene, tmie, are believed to be responsible
for the phenotypic changes seen in which two mouse models used to study human
deafness?
2. Tmie expression is thought to have a key role in the maturation and maintenance of
which cells?
a. Stereocilia of the organ of Corti
b. Retinal ganglion
c. Vibrissal cells
d. Vestibular ganglion
ANSWERS
1. The circling mouse (cir/cir) & the spinner mouse (sr/sr)
2. a. Stereocilia of the organ of Corti
Chicken Model
Xu et al. Blood Supply to the Chicken Femoral Head, pp. 295-299
Task 3 - Provide Research Support, Information, and Services; K2 - Normative biology
Secondary Species: Chicken (Gallus domestica)
SUMMARY: This article describes the normal vascular anatomy found in the femoral
head of chickens in further detail than has been previously described. Previous papers
have described the terminal vascular supply to the femoral head, but the origin of these
terminal arteries in respect to the ischiatic and femoralis arteries has not been
described. The team examined twelve 2-kg White Leghorn Chickens by perfusing them
with a casting agent injected into the aorta. The tissues were refrigerated overnight and
then the hind limbs were harvested and fixed in formalin for 72 hours. The bones were
decalcified and then the limbs stored in a glycerin solution.
The article contains many high-quality photographs that significantly aid in the
discussion of these anatomical findings. The team found previously described
vasculature in all 24 hind limbs and also identified three previously undescribed
branches of the femoralis artery providing blood to the acetabulum and femoral head (2
separate vessels). The team named the undescribed vessels supplying the femoral
head arising from the femoralis artery as the “lateral retinacular artery” and the
“acetabular branch of femoralis artery.” Although there have been previous descriptions
of the terminal vasculature, this team confirmed that the femoral head is perfused by two
different major arteries: the femoralis and ischiatic arteries. This creates an anastamotic
ring of blood supply in contrast to the human femoral head which is supplied by superior
retinacular vessels of the deep branch of the medial femoral circumflex artery and an
anastamosis between the deep medial femoral circumflex and inferior gluteal arteries.
The chicken can be an excellent model for human femoral head osteonecrosis because
it is readily available, active, and bipedal. Many previous studies have utilized
quadrapedal animals as models and also used nonphysiologic means to produce
osteonecrosis (e.g. corticosteroids.) Ideally, vascular disruption could produce clinically
relevant and reproducible degrees of osteonecrosis. This team suggests using a
surgical method to disrupt the trochanteric, acetabular branch of femoralis, lateral
retinacular, and middle femoral nutrient arteries to reliably produce osteonecrosis and
eventual end-stage mechanical collapse as a research model in chickens.
QUESTIONS
1. T or F: Chickens are suboptimal for use in osteonecrosis studies due to significant
vascular differences to humans.
2. T or F: The femoral head of chickens is primarily perfused by two major arteries, the
femoralis and iliac arteries.
3. T or F: The vascular supply of chicken femoral heads is almost identical to humans.
4. T or F. Many studies of osteonecrosis have used nonphysiologic means to induce
osteonecrosis in research animals, including drugs such as corticosteroids.
ANSWERS
1. F. Chickens can be an excellent model. Although the vascular is different, the
bipedal and active nature of the birds makes them an excellent option.
2. F. The femoral head of chickens is primarily perfused by the femoralis and ischiatic
arteries.
3. F. There are significant differences in the vasculature in both species; however this
does not prevent the chicken from being a good model for the human disease.
4. T. This is true.
Swine Model
Neeb et al. Metabolic Syndrome and Coronary Artery Disease in Ossabaw
Compared with Yucatan Swine, pp. 300-315
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and
Conditions
Domain 3: Research
Primary Species: Pig - Sus scrofa
SUMMARY: Metabolic syndrome is diagnosed by the presence of 3 or more of the
following conditions: obesity, insulin resistance, glucose intolerance, dyslipidemia, and
hypertension. Having metabolic syndrome increases the risk of type 2 diabetes and
coronary artery disease. Pigs are often used as a model for this disease in humans.
The authors state that Ossabaw pigs are better than other breeds of swine as models of
this disease condition in humans. Ossabaw pigs have a “thrifty” genotype, which causes
them to become obese and develop metabolic syndrome traits through diet
manipulation. In this study, Ossabaw pigs become more obese, had more
characteristics of metabolic syndrome, and had more lesions in the coronary artery stent
injury than Yucatan pigs. These features make them an ideal large animal model for
human metabolic abnormalities and coronary artery disease.
QUESTIONS
1. Despite advances in the creation of mouse models for diabetes and metabolic
syndrome, only large animal models can provide a way to study _____.
2. Characteristics of metabolic syndrome include (choose all that apply):
a. Obesity
b. Glucose intolerance
c. Renal failure
d. Alopecia
3. What are the disadvantages of using standard-size domestic pigs for animal models
of metabolic syndrome or heart disease?
ANSWERS
1. Vascular interventions, like stents, that are human-sized
2. a and b
3. Standard-size pigs are very large (250 kg) and 2 years old before they develop
clinical signs of disease. This is too large for most human-size equipment and
devices, and they are more difficult to handle.