Comparative Medicine
Volume 56, Number 6, December 2006
OVERVIEW
Rat Model
Pollard and Suckow. Dietary Prevention of Hormone Refractory Prostate Cancer
in Lobund-Wistar Rats: A Review of Studies in a Relevant Animal Model, pp. 461467
Task: 9 - Collaborate on the Selection and Development of Animal Models
Primary Species: Rat
Summary: Lobund-Wistar (LW) rats, which have high testosterone levels, (1865 pg/ml
vs. 265 for SD and 612 for Wistars) develop spontaneous prostatic tumors in 30% of
animals without treatment and 87% if treated with methylnitrosourea and testosterone.
The spontaneous rate in SD rats is 0.04% and 0.5% in Wistar rats. Tumors that have
progressed to a terminal stage, either in patients with advanced stage tumors or tumors
that have relapsed after failure of treatment are called refractory, and often have
intratumor necrosis and hypoxia. Typical progression of prostatic tumors in LW rats is
0.5 cm palpable tumors at 18 mo. growing to 2-3 cm by 22 mo., with metastasis to lungs
or within the peritoneal cavity. Tumor type is adenocarcinoma. Early tumors are
reversible with testosterone deprivation.
The incidence of prostatic tumors was reduced by moderate dietary restriction and by
dietary supplementation with a soy protein isolate high in isoflavones. This dietary
supplement also reduced the serum testosterone level.
Questions:
1) The rat prostate is composed of _____ distinct paired lobes that are called _______,
________, _______, and _______.
2) The lobund-Wistar (LW) rat has serum concentrations of _______ that are 3-6 times
than for some other rat strains which makes them a model for prostatic tumors.
3) T/F Soy-based diets are possible thought to prevent prostatic tumors in Asian men.
Answers:
1) 4; ventral, lateral, dorsal, & anterior
2) Testosterone
3) True
ORIGINAL RESEARCH
Mouse Models
Krugner-Higby et al. Heritable, Diet-induced Hyperlipidemia in California Mice
(Peromyscus californicus) Is Due to Increased Hepatic Secretion of Very Low
Density Lipoprotein Triacylglycerol, pp. 468-475
Task 9 - Collaborate on the Selection and Development of Animal Models
Species: Tertiary / Peromyscus californicus (rodent)
Summary: California Mice (Peromyscus californicus), fed a high fat diet (25.8% energy
from fat), are a model of hyperlipidemia and type II diabetes. The authors mated 3
couples of high (HR) and low (LR) responders according to their serum TAG
(triacylglycerol), and studied seven progeny mice over a 12 weeks diet. The 4 HR
progeny exhibited, as their parents, higher concentrations in serum of TAG, insulin,
glucose and cholesterol. Lipoproteins profiles in plasma of the 4 HR mice showed
elevated plasma concentrations of high density lipoprotein cholesterol (HDL), and of very
low density lipoprotein TAG compared with the 3 LR mice. No difference was observed
in lipoprotein lipase and hepatic lipase activities, or in food consumption or body weights.
Questions:
1. What is type II diabetes mellitus (T2DM)?
2. What are commonly used rodent models of T2DM and obesity?
3. What is tyloxapol used for in T2DM models?
4. What are fasting guidelines for rodents?
5. What is polygenic inheritance ?
Answers:
1. Type 2 diabetes mellitus (T2DM) is a heterogeneous syndrome resulting from
defects of both insulin secretion and action. T2DM seems to be a polygenic disorder
in the majority of cases, influenced by a variety of environmental factors. T2DM is
characterized by PUPD (polyuria, polydypsia), hyperglycemia and hyperinsulinemia.
Glucose and lipid metabolism in the liver is impaired (elevated fasting glucose..).
T2DM is frequently progressive, chronic and associated with polyphagia, obesity and
complications.
2. Rodents models of obesity and T2DM are numerous and heterogeneous, and some
are widely used (for review see ILAR Journal 47(3) -2006):
mouse:
- leptin deficient 0b/0b mouse (massive obesity)
- leptin resistant db/db mouse (mutation in a hypothalamic receptor for leptin)
- diet induced models (C57BL / 6J mice fed a 60% HF diet for 12 weeks..)
- KK mouse (several lines of obese mice produced by repeated selective breeding).
- numerous mouse lines with targeted disruption (KO) in metabolic genes or
pancreas genes
rat:
- Zucker fa/fa leptin resistant obese rats (mutation in a hypothalamic receptor for
leptin)
- Goto-Kakizaki (GK) rat - sucrose treated (spontaneous animal model of NIDDM
without obesity, produced by repeated selective breeding of rats with glucose
intolerance starting from a nondiabetic Wistar rat colony. The GK rats exhibit
progressive loss of ß cells in the pancreatic islets with fibrosis, mild hyperglycemia at
fast, an impaired glucose tolerance on glucose load, and complication-like tissue
damage in the peripheral nerves as well as kidneys).
- induced models (Fat-fed streptozotocin-treated rat..)
other rodents:
- Psammomys obesus ("desert gerbil" aka "sand rat"): when fed laboratory chow, the
animals become obese, insulin resistant and hyperglycemic. If a cholesterol-rich diet
is used, hyperlipidaemia and atherosclerosis develop.
- Acomys cahirinus ("spiny mice"): when fed laboratory chow, the animals become
obese and moderately hyperglycemic. With time, animal suffer from ketosis.
3. Tyloxapol is a detergent: tyloxapol, by IV injection (5µl/g of body weight of 10%
solution), coats plasma lipoproteins particles, and therefore inhibits their absorption :
this enables to dose them and to study lipid metabolism ( lipoproteins transport the
lipids secreted by the liver).
4. Fasting is complete withdrawal of food, with continuous access to water. Since
rodents feed all the day long and have a high metabolic rate, stress occurs rapidly:
the duration of fasting should then be kept to a minimum.
- Since rodents do not regurgitate, fasting is not necessary prior to standard surgery
and anesthesia.
- fasting duration for oral treatment or digestive study is based on food transit (gastric
emptying time is < 60 min, colonic transit time in rat is approximately 320 min).
- fasting for behavioral studies should be less than 4 hours
- fasting for nutritional and metabolic studies vary from 4 to 16 hours depending on
the experimental objectives.
5. Polygenic inheritance or quantitative inheritance refers to inheritance of a phenotypic
characteristic (trait) that is attributable to two or more genes and their interaction with
the environment. Unlike monogenic traits, polygenic traits do not follow patterns of
Mendelian inheritance (qualitative traits). Instead, their phenotypes typically vary
along a continuous gradient depicted by a bell curve. Though not necessarily genes
themselves, quantitative trait loci (QTLs) are stretches of DNA that are closely inked
to the genes that underlie the trait in question.
Cho et al. The Circling Mouse (C57BL/6J-cir) Has a 40-kilobase Genomic Deletion
that Includes the Transmembrane Inner Ear (tmie) Gene, pp. 476-481
Task 9 - Collaborate on the Selection and Development of Animal Models
Primary species Mouse
Summary: The circling mouse (C57BL6-cir) shows deafness and circling behavior in
homozygotes. The mutation is transmitted with 100% penetrance by an autosomal
recessive gene on chromosome 9. The circling mouse arouse due to spontaneous
mutation and is an animal model of deafness because of its inner ear abnormalities.
Most notable findings in these mice are their almost completely degenerated cochlea
and remarkably reduced cellularity. Mutation in the cir gene causes deafness, head
tossing and circling behavior in mice. Urocortin- deficient mice show hearing impairment
and increased anxiety-like behavior. The investigators find the outer hair cells of
Urocortin- deficient mice also are dysfunctional.
Questions:
1. Provide some mutant mice model for deafness.
2. Pathophysiological finding include _______________ and ___________ in the
circling mice.
3. Symptoms in the circling mice are _________, _____________ and
__________________.
Answers:
1) cir, sr and Ucn-null are examples for deafness animal model
2) Degenerated cochlea and reduced cellularity.
3) Deafness, head tossing and increases anxiety
Grindle et al. Validation of High-throughput Methods for Measuring Blood Urea
Nitrogen and Urinary Albumin Concentrations in Mice, pp. 482-486
Task 1- Prevent, Diagnose, Control, and Treat Disease
Species-Primary, Mouse
Summary: Chronic kidney disease is an important medical problem in the United States.
High-throughput phenotyping of renal function and damage is facilitated by clinical
chemistry auto analyzers to quantify glomerular filtration rate estimated by plasma and
urine creatinine concentrations, BUN (blood urea nitrogen) concentration, and the ratio
of albumin to creatinine concentration in the urine. Recent studies have confirmed that
plasma chromagens in mice interfere with the Jaffe method commonly used in auto
analyzers to measure creatinine concentrations. The authors validated methods to
determine plasma BUN (blood urea nitrogen) and urinary albumin concentrations using
auto analyzer methods and validated these results using high-performance liquid
chromatography (HPLC) to validate BUN concentrations. Albumin level accuracy was
determined by using mouse albumin standards to compare to auto analyzer values over
a wide range of albumin concentrations.
Blood and urine samples were collected from 7-10 wk old, male DBA2/J (D2), A/J, and
C57BL/6J (B6) mice and from separate groups of 10-14 month old and 20-24 month old
D2 mice. Additional groups of A/J and D2 mice underwent 5/6-nephrectomy as a model
of kidney disease and to confirm that BUN is an effective marker for diminished kidney
function. The 5/6-nephectomized mice had significantly elevated BUN concentrations at
both 3 and 10 days post surgery. They observed a significant linear correlation between
BUN concentrations measured on the auto analyzer and by HPLC on nephrectomized
mice. These findings show that plasma constituents from mice with reduced renal
function do not interfere with auto analyzer methods of measuring BUN concentration.
Plasma and urine albumin concentrations were underestimated by 3.5-4 fold, so
although it exhibited good precision, the auto analyzer method was not accurate based
on calibration with human albumin. However, accurate quantification of mouse albumin
is possible by calculating the albumin concentrations of experimental samples by linear
regression of a standard curve generated with mouse albumin standards that are
commercially available.
Authors also note that although HPLC can be used to accurately measure plasma
creatinine concentrations, it is expensive and less amenable to high-throughput analysis
of large scale chronic kidney disease studies using mice.=20
Questions:
1. Urinary albumin concentration tends to increase significantly between 10 wk-2yr old
mice with chronic kidney disease. T F
2. Plasma creatinine levels can be measured accurately using auto analyzer methods
in mice. T F
3. In the 5/6-nephrectomy method of kidney disease, the following is a true statement:
a.
b.
c.
d.
e.
Right kidney is removed, and =BD of left kidney is removed
Left kidney is removed and 2/3 of right kidney is removed
Left kidney is removed and =BE of right kidney is removed
Both kidneys are partially removed
None of the above
Answers:
1. T
2. F
3. b
Rat Models
Smith et al. A Single Dose of Liposome-encapsulated Hydromorphone Provides
Extended Analgesia in a Rat Model of Neuropathic Pain, pp. 487-492
Task 2 - Prevent, Alleviate, and Minimize Pain and Distress
Primary species - Rats
Summary: A chronic constriction injury (CCI) thermal hyperalgesia model of neuropathic
pain was used to test the analgesia effect of liposome-encapsulated hydromorphone
(LEH), liposome-encapsulated vehicle alone, or hydromorphone. Rats were sham
operated or CCI by sciatic nerve ligation. Rats receiving liposome-encapsulated vehicle
or hydromorphone at surgery developed full thermal hyperalgesia by day 3 post surgery.
In contrast, thermal withdrawal latency was not different at d 3 post surgery when
compare to baseline in rats receiving LEH at surgery, and thermal hyperalgesia was
reversed in CCI rats receiving LEH at d 3 post surgery.
Questions:
1. In rats with CCI, what drug was able to prevent full thermal hyperalgesia?
a. Buprenorphine
b. Liposome-encapsulated vehicle
c. Hydromorphone
d. Liposome-encapsulated hydromorphone
2. Hydromorphone and liposome-encapsulated hydromorphone were able to reverse
thermal hyperalgesia in CCI rats receiving the drug at d 3 post surgery. True or
False
3. Hydromorphone at d 3 post surgery was able to reverse thermal hyperalgesia for only
90 minutes, and LEH was able to reverse the effect for several days. True of False
Answers:
1. d
2. True
3. True
Burdzinska et al. Sodium Ascorbate and Basic Fibroblast Growth Factor Protect
Muscle-derived Cells from H2O2-induced Oxidative Stress, pp. 494-501
TASK: 9 - Collaborate on the Selection and Development of Animal Models
SPECIES: Rat
SUMMARY: Urinary incontinence is a common problem in both humans and animals
(especially older women and spayed bitches). Many non-surgical, peri-urethral injectable
treatments have been tried in the past with varying success. There is typically an
inflammatory response (with varying severity) that results in oxidative damage to
transplanted cells. In vitro, muscle-derived cells which have been pre-incubated with
sodium ascorbate or basic fibroblast growth factor are more resistant to oxidative stress.
If this property holds true in vitro, this could be a useful treatment for urinary
incontinence.
QUESTIONS:
1. True/False: MDCs can form myotubes, contract spontaneously and form
associations with neural endings.
2. Which of the following are strategies that have been attempted to enhance cell
survival post-transplantation:
a. Selecting more resistant sub-populations of satellite cells.
b. Inhibiting injection site inflammation.
c. Transfecting transplanted cells with growth factors.
d. Preconditioning cells prior to implantation.
e. All of the above.
3. True/False: Treating MDCs with both bFGF and ASC provided a synergistic effect on
cell survival, thus suggesting separate molecular pathways.
ANSWERS:
1. True
2. e. all of the above.
3. False. There was an absence of a synergistic effect which would suggest the same
molecular pathway.
Hotchkiss et al. Oral Treatment with Retinoic Acid Decreases Bone Mass in Rats,
pp. 502-511
Tasks 1,2,3,10
Primary species - Rat
Administration of retinoids for the treatment of acne in humans risks pathologic effect on
bone, including hyperostosis and calcification of the tendon and ligaments in adults, and
hypermaturation of growth plates, loss of bone mineral, and decreased radiodensity in
juvenile guinea pigs and humans. Specific isomers of retinoids results in different
effects, but in general, retinoids alter cortical bone modeling and inhibit cortical
expansion. All trans RA is also used in humans to treat acute promyelocytic leukemia.
These adverse effects have not previously been quantified by bone densitometric
measurement techniques in the animal model. Investigators used a DXA scanner to
produce a 2 dimensional projection of 3 dimension bone for quantification of bone
mineral content, density, and area.
SD rats were gavaged for 15 weeks in two separate experiments beginning at either day
59 or 82 of life in 5 treatment groups designed to mimic human clinical dosing with
different doses and isomers of retinoic acid. In experiment one, rats were treated
beginning at day 82 with high and low dose groups of 13-cis-RA and all trans RA. In
experiment two, treatment with low and high dose 13-cis-RA was performed starting at
day 59 of life. Calcium, ALT, AST, AP, and serum pyridinoline levels were obtained at
sacrifice from animals treated beginning at day 82. Histomorphometry of the left femur
from the same rats from experiment one was performed. Bone densitometry of the right
femur was performed in both experiments. The lumbar spine was examined in the rats
dosed beginning on day 59 (experiment two). Open field activity and forced swim testing
was also performed
In experiment one, clinically silent spontaneous fractures occurred in animals treated
with all trans RA. AP was increased in all trans RA rats. High 13-cis RA male rats had
higher calcium levels than low 13-cis-RA male rats, but this was not statistically
significant. All trans RA male rats had decreased bone area, mineral density and
mineral content, and both doses of 13-cis RA male rats had decreased bone mineral
content, but only low 13-cis RA decreased bone mineral density as well. In female rats,
low all trans Ra decreased bone mineral content and density. Bone mineral content was
decreased in the high 13 cis RA group. Bone mineral density and content was
significantly more decreased in the low all trans RA groups compared to the 13-cis RA
groups, but in the high all trans RA group was decreased in comparison to low 13 cis RA
and controls. Specific morphologic changes noted in all trans RA rats included
decreased bone diameter, total area, marrow diameter and area and cortical bone area
and in some of the 13-cis RA animals. Sex differences were present in morphologic
changes in high 13 cis RA groups - males had decreased bone diameter, area, marrow
diameter and cortical bone area, but in females only marrow area was reduced.
Trabecular bone was decreased in specific ways in different treatment groups.
In experiment two, increased 13-cis RA dose resulted in no lumbar bone changes.
However, in the male rats, bone mineral density, content and bone area of the femur
was decreased in high dose 13 cis RA. In females mineral content decreased in high
and low dose groups, but mineral density and bone area were only decreased in the
high dose groups.
Based on these results, investigators conclude that all trans RA will inhibit cortical
expansion and reduce bone strength and size. They note that 13 cis RA had similar
specific effects that were not always statistically significant, and that there was a lack of
effect on lumbar vertebrae, making it a potentially safer choice for clinical use. It had
been posited that bone resorption would increase with RA treatment, but serum
pyridinoline levels did not support this theory. Increased AP was not correlated to
increases in liver enzymes.
The authors discuss relative dosing, and the effective serum levels of RA in humans that
correlate with treatment groups in their studies and the pathologic effects observed on
bone, and conclude that further study is needed on the effect of retinoids on long bone
growth.
Questions:
1. What methodology is used for measurement of bone density and mineral content?
2. What were the predominant pathologic effects of treatment with all trans RA and high
dose 13 cis RA?
3. What serum marker was used to evaluate bone resorption?=20
4. Were significant gender effects observed?
Answers:
1. Dual energy x ray absorptiometry - DXA
2. Decrease in bone mineral density, content, bone diameter and cortical thickness of
the femur
3. Pyridinoline
4. Yes - effects were more variable in female rats
CLINICAL INVESTIGATION
Doane et al. Electrocardiogram Abnormalities in Captive Chimpanzees (Pan
troglodytes), pp. 512-518
Task 1 = Prevent, Diagnose, Control and Treat Disease
Species = Tertiary, Other nonhuman primates
Summary: Cardiovascular disease is the leading cause of death in captive chimpanzee
populations. This study set out to use electrocardiogram (ECG) to describe cardiac
arrhythmias over time in a large captive chimpanzee population. The animals were all
research veterans coming from a variety of programs. They had a mixed virus status
including; hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV)
infection, HCV-HIV co-infection, hepatitis B virus (HBV) infection, and HBV-HIV coinfection. Of the 142 male and 123 female chimps there was a broad range of
arrhythmias observed. Fifty-four of the chimps had echocardiograms based on
suspected cardiovascular disease and 36 had echocardiograms to establish normal
values. Only a few of the animals had systemic hypertension, or hyperlipidemia, or had
any other underlying disease. During the course of the study, six animals died of sudden
cardiac disease that had been previously diagnosed with ventricular arrhythmias. There
was a statistical significance between animals having a ventricular arrhythmia and a high
incidence of sudden cardiac death (SCD) compared to animals without ventricular
arrhythmia.
The most commonly observed cardiac arrhythmia was ventricular premature
contractions (VPCs). Of the 13 animals diagnosed with multiform VPC, 5 died of SCD.
The mortality incidence of these chimps was significantly higher than in chimps with
VPCs of another form. An increased risk of cardiac arrhythmias was associated with
animals 30-50 years old and of a male gender. Unlike humans dyslipidemia and
atherosclerosis do not appear to play a role in heart disease in chimps. In chimpanzees,
cardiac fibrosis is reported to be the leading cause of death. Concurrent infection with
HCV or HIV did not appear to be a predisposing factor to cardiovascular disease. As
cardiovascular disease is the leading cause of death in chimps and arrhythmias can be a
predictor of cardiovascular disease, then ECG can be considered a useful modality to
identify high risk chimps.
Questions
1. T/F Chimpanzees have a much lower incidence of disease than do humans?
2. T/F The primary cause of death in chimpanzees is heart disease?
3. The most accurate description of risk factors for CVD in chimps is:
a) Age, dyslipidemia, underlying disease
b) Systemic hypertension, gender, arrhythmia
c) Systemic hypertension, age, gender
d) Arrhythmia, age, gender
Answers
1. T
2. T
3. d