Comparative Medicine
Volume 56, Number 1 (2006)
LABORATORY INVESTIGATIONS
Slenka et al. Effect of Age on Collagen Fibril Diameter in Rabbit Patellar Tendon
Repair, pp. 8-11
Summary: Collagen fibril diameter is not altered with increasing age in the healing rabbit
patellar tendon.
It is generally believed that tissue repair in geriatric patients occurs more slowly and
incompletely than in adolescents and young adults. To test this theory, the authors
simulated a common human orthopedic procedure (cruciate repair) using rabbits. The
authors defined rabbits 4-5.5 years old as geriatric (using a definition of the age at which
a population has reached 50% mortality). One of the key components to re-establishing
tendon strength is the deposition of properly aligned collagen fibers into the repair site.
Previous studies have shown that both in rabbits and humans, tendon strength is
correlated most closely with the size of the type I collagen fibril, with larger fibrils and
better packing providing greater strength. Repairing collagen in young adult rabbits
regain less than 40% of normal uninjured strength and have smaller collagen fibrils.
This study involved resecting the middle 3rd of the patellar tendon in 1 and 4-5.5 year
old rabbits. After surgical recovery, the tendons were evaluated at 3 different locations
along the tendon at 6, 12 and 26 weeks after surgery. Tendons had ultrastructural
analysis by electron microscopy on both the operated and non-operated tendons to
allow each animal to become its own control.
When compared to the un-operated normal patellar tendon, there was a significant
reduction in the mean fibril diameter in the repairing tendons at all time points. No
differences were found in collagen fibril diameter between the young and geriatric
animals in any tendon section at any time point. A single significant difference was seen
in 4 year old rabbits when there was an increase in fibril diameter at one time point and
only at the tibial end of the tendons. This was difficult to explain however the authors felt
may be attributed to a problem in collection technique and inadvertent harvesting of noninjured tissue. This study is further supported by other studies out of this laboratory
which confirmed no changes in biomechanical properties of healing and normal tendons
at 6 and 12 weeks post injury. Perhaps the current dogma is directly correlated to
studies on aging skin which also relies on collagen repair. One study demonstrated that
less force is required to disrupt wounds in the skin of aged people compared with
younger adults. Another study showed that dehiscence occurs more frequently in
geriatric persons compared to young adults. Authors also note that another possibility
for the reduction in repair strength in the elderly may be due to a change in the
organization of the fibrils. No changes were found in this study in the fibril size studies to
look at the packing density or cross-linking between fibrils could play an important role in
differences in tendon strength in repairing tissues.
Questions:
1) Define geriatric.
2) What factors play a role in tendon strength?
Answers:
1) The age at which a population has reached 50% mortality.
2) Fibril diameter, packing density and cross-linking.
Schuler et al. Ovarian Stimulation of Squirrel Monkeys (Saimiri boliviensis
boliviensis) Using Pregnant Mare Serum Gonadotrophin, pp. 12-16
Squirrel monkeys, the most commonly used New World Primates used in biomedical
research are seasonally polyestrous, and thereby limiting the application of Assisted
Reproductive Technologies (ART) to the breeding season. If these animals can be
stimulated to produce oocytes during non-breeding season, ART studies could continue
throughout the year and will be of viable alternative to rhesus monkeys. In rhesus
monkeys, repeated use of pregnant mare serum gonadotrophin (PMSG) has been
associated with induction of refractory state; and further there is no commercial
preparation for ovarian stimulation is available for squirrel monkeys. The objective of the
study is to determine the effectiveness of PMSG and rhFSH for controlled ovarian
stimulation and to determine whether the repeated use of PMSG induces the refractory
state and affects the future reproductive performance of Bolivian squirrel monkeys.
Materials & Methods: Two groups of Bolivian squirrel monkeys were given daily injection
of PMSG (250 IU twice daily 0800 and 1500 SC) and rhFSH (75 IU once 0800 SC) for 4
days during both breeding and non-breeding season. Also, 3 groups of 9 animals were
challenged with PMSG for 1 or 3 cycles of PMSG stimulation. Follicular
recruitment/oocytes production was determined by measuring serum estradiol (E2) and
confirmed by ultrasonography.
Results: The rhFSH did not affect circulating E2 levels on any day of the treatment, but
PMSG produced a robust E2 response. PMSG was effective in stimulating E2
production during both non-breeding season and breeding season. Repeated use of
PMSGH for multiples cycles in squirrel monkeys did not induce refractory state in
squirrel monkeys as reported in rhesus. Further the repeated stimulation of rhFSH did
not affect the reproductive performance in squirrel monkeys.
Summary: The PMSG treated squirrel produce showed robust E2 response during both
breeding and non-breeding season and was had significantly higher E2 response than
with rhFSH challenge. PMSG can also more used for repeated stimulation in same
animal without inducting refractory state or affecting reproductive performance.
Questions:
1. In Bolivian Squirrel monkey, the breeding season occurs during
a. March-April
b. November-January
c. June-August
d. September-November
2. Which of the following statements is “false” regarding the effectiveness of PMSG and
rhFSH in Bolivian squirrel monkeys as reported in this study
a. PMSG is as effective in stimulating serum E2 during the non-breeding season as
during the breeding season
b. Squirrel monkeys do not become refractory when repeatedly challenge with
PMSG
c. Repeated stimulation with rhFSH does not affect future reproductive outcome
d. rhFSH resulted in a robust Estradiol response compared to that of PMSG treated
squirrel monkeys
Answers: 1) b; 2) d
Callicott and Womack.
Telomeres, pp. 17-22
Real-time PCR Assay for Measurement of Mouse
Summary: Measurement of telomeres by PCR was problematic due to the production of
primer dimers during the amplification phase of the process. Recently a new set of
primers was developed for this purpose in humans. The authors used these primers and
modified the assay to study mouse telomere length. The primers were able to amplify
the mouse telomeres without the issues of primer dimer formation. The results obtained
using this real time PCR assay were similar to those obtained by terminal restriction
fragmentation (TRF) analysis by pulse field gel electrophoresis followed by Southern
hybridization. The main difference between the two is that the real time PCR assay was
easier to perform, took less time, and was less expensive than TRF analysis by
Southern blotting and other methods of measuring individual telomere length like
quantitative florescence in situ hybridization. Telomere length is important because it
has been implicated in cell senescence, as a "mitotic clock" for aging, and as a factor in
tumorigenesis. This assay will allow for a more efficient way to study telomere length
and thus aid in the study of telomere function in diseases associated with aging and
neoplasia.
Questions:
1. The problem with PCR assays used to study telomere length in the past was the:
a. High assay costs
b. Primer dimer formation
c. Technical difficulty
d. Slow turnaround time of PCR testing.
2. This real time PCR assay was:
a. More difficult to perform than TRF with southern blotting
b. More technically challenging than quantitative florescence in situ hybridization
c. Produced similar results to TRF with southern blotting but was easier and less
difficult to perform
d. a and c
3. The development of this assay would allow for:
a. More efficient study of telomere length and its role in cancer and age related
diseases.
b. A less expensive way to study mouse telomere length.
c. A faster method of studying mouse telomere length
d. All of the above.
Answers: 1. b., 2. c, and 3. d.
Fletcher et al. Characterization of an Anti-lymphocyte Function-associated
Antigen-1 Antibody in a Simian Immunodeficiency Virus-Pig-tailed Macaque
(Macaca nemestrina) Model, pp. 23-30
Summary:
The simian immunodeficiency virus (SIV)/pig-tailed macaque model of acquired immune
deficiency syndrome (AIDS) is used to study cell adhesion molecules and retroviral
pathogenesis in vivo. This manuscript describes the use of such animal model to
characterize an antibody (MHM.23) specific to human lymphocyte function associated
antigen-1. Besides CD4, lymphocyte function associated antigen-1 (LFA-1) is a key
cellular adhesion molecule that plays a role in HIV-1 induced syncytium formation of T
cells. It initiates the immune response by providing adhesion in the formation of the
immunologic synapse via binding to its ligand, intracellular adhesion molecule (ICAM)-1
on leukocytes. LFA-1 on HIV-1 could potentially target virus particles to sites of immune
activation, allowing HIV-1 to interact with susceptible cells of the immune system and
facilitates HIV transmission.
The purpose of this study was to characterize in vitro and in vivo effects of humanspecific MHM.23 on peripheral blood mononuclear cells (PBMC) of SIVmac239 infected
pig-tailed macaques. In vitro studies revealed that at concentration of 20ug/ml, MHM.23
blocked LFA-1-mediated adhesion and T-cell activation (> 90%) of \ macaque PBMC.
SIVmac239 infection of macaque cells was inhibited in a dose-dependant manner by
MHM.23; at concentration of 20 ug/ml, macaque PBMCs were completely saturated. In
vivo studies revealed that MHM.23 inhibited LFA-1-ICAM-1-mediated activity and
maintained binding on macaque cells for 4 days; intravenous 5 mg/kg MHM.23 given
every 24 hours was required to maintain saturating levels and inhibit LFA-1-ICAM-1
function in this model.
Authors concluded that anti-LFA-1 mAb (MHM.23) alters (neutralizes) infectivity or
replication of SIV in vitro. It can bind and block function of pig-tailed macaques LFA-1 in
vivo. MHM.23 is the best choice for future in vivo macaque studies, and blockade of
LFA-1 may represent a novel therapeutic approach against primate lentivirus infection.
Questions:
1. What key cellular adhesion molecule plays a direct role in HIV-1-induced syncytium
formation in primary T cells?
a) CD8
b) LFA-1
c) IL-2
d) MHM.23
e) ICAM-1
2. SIV infection progresses to cause an AIDS-like disease when inoculated into which
non-human primate?
a) Cercopithecus aethiops
b) Papio anubis
c) Macaca nemestrina
d) Aotus trivirgatus
e) Cercocebus torquatus atys
Answers:
1. B
2. C
Sugawara et al. Current Status of Chromosomal Abnormalities in Mouse
Embryonic Stem Cell Lines Used in Japan, pp. 31-34
BLUF: Mouse embryonic stem cells (ES) are totipotent cells derived from the inner cell
mass of a pre-implantation mouse embryo. Abnormalities of chromosome number and
karyotype gained during passage in culture are reported to influence the totipotent state
and the ability to contribute to the germ line. However ES cell lines with 40% normal
karyotype leading to cystic embryoid body formation within 7 days of suspension were
capable of germ line transmission.
Of the 540 cell lines examined in Japan, only 66.5% had a normal chromosome
numbers and only 60% of the 88 cell lines randomly karyotyped were normal with the
majority displaying XX sex chromosome constitution. In light of the findings, ES cell lines
should be checked for chromosomal number and karyotype as a means of quality
control.
Questions
1. What is true according to the article regarding ES cells?
A) Karyotype abnormalities do not affect totipotent state
B) Abnormal chromosome number affects germ line contribution
C) Both
D) none
2. An XO karyotype represents:
A) Klinefelter's syndrome
B) Turner's syndrome
C) Any sex chromosome abnormality
D) none
3. Chromosomal analysis is evaluated in what stage?
A) Anaphase
B) Metaphase
C) Telophase
D) None
4. Q banding technique is used:
A) for karyotype analysis
B) to identify ES cells
C) to identify unfertilized eggs
D) none
5. Which diploid chromosome number will not affect ES cell germ line?
transmission in the mouse?
A) 39
B) 40
C) 41
D) 42
Answers
1. B
2. B
3. B [materials and methods]
4. A [materials and methods]
5. B [the normal diploid number]
Dyson et al. Components of Metabolic Syndrome and Coronary Artery Disease in
Female Ossabaw Swine Fed Excess Atherogenic Diet, pp. 35-45
Summary
The authors define metabolic syndrome as a cluster of risk factors that include central
(Intra abdominal) obesity, insulin resistance, impaired glucose tolerance, dislipidemia
(Low HDL and increased triglycerides). The purpose of the study is to show the effect of
feeding excess atherogenic diet on ossabaw swine with eventual goal of optimizing the
animal model for the study of coronary artery disease. These particular swine are known
to have a thrifty genotype that enables them to survive seasonal food shortage in their
native environment.
The study was conducted at the University of Missouri. Swine were fed either a lean diet
(Total number 9) or excess high fat high cholesterol (Total number 8) diet for nine
weeks. Blood pressure measurement, Intravenous glucose tolerance test (IVGTT),
Plasma lipids, body composition, intravascular ultrasonography and histopathology were
conducted at different time point during the nine week study.
Compared with lean animals, obese swine showed two fold greater product of the
plasma insulin to normalize blood glucose concentration. 4.1 fold greater total
cholesterol, 1.6 fold greater post prandial triglycerides, 4.6 fold greater low to high
density lipoprotein cholesterol ratio, hypertension, and neointimal hyperplasia of
coronary arteries. The 1.5 fold greater body weight in obese swine was largely
accounted for by the 3 fold greater carcass fat mass. High correlation of CT, anatomical
measurements, and ultrasonography with direct chemical measures of subcutaneous,
retroperitoneal, and visceral fat indicates high validity of all indirect measures.
The authors conclude that relatively brief feeding of excess atherogenic diet produces
striking features of metabolic syndrome and coronary artery disease in female ossabaw
swine.
Question
1. What other large animal models of metabolic syndrome available?
Answer
1. Gottingen swine, Fat-fed dogs, Macaques and baboons. Alpacas and Llamas and
Alpacas also can be used.
Hukkanen et al. Comparison of Commercially Available and Novel West Nile Virus
Immunoassays for Detection of Seroconversion in Pig-tailed Macaques (Macaca
nemestrina), pp. 46-54
Summary: West Nile Virus (WNV), an encephalitic virus, belongs to the genus Flavivirus,
family Flaviviridae, and Japanese encephalitis (JE) serogroup. WNV was first detected
in North America in 1999, along the East coast, and has since been detected in every
continental state, all but 1 Canadian province, and in parts of northern Mexico. Vertical
transmission of WNV is via mosquito vectors (especially Culex spp), and birds.
Resistant avian species amplify the virus and serve as carriers, migratory patterns of
birds allow reintroduction of WNV to regions on a seasonal basis. Susceptible avian
species are in essence sentinels for disease emergence in a region. Mammals and
reptiles are not considered part of the viral transmission cycle, as infectious titers in the
peripheral blood are rare.
In humans, infection may present in two different forms. WNV fever is associated with
self-limiting and mild pyrexia and malaise in up to 30% of infected people. WNV
neuroinvasive disease manifests as a mild to severe meningitis or encephalitis affecting
up to 1.5% of infected people, but mortality in immunosuppressed individuals may reach
40%.
Current diagnosis of WNV infection in NHPs is through the plaque reduction
neutralization test (PRNT) or hemagglutination inhibition (HAI) assays. Limitations of
these tests include the need of a BSL-3 lab, expense, and/or technical difficulty. The
Washington National Primate Research Center (WaNPRC) evaluated seven
commercially available human immunoassays for WNV. Assays were evaluated for
sensitivity, specificity, positive and negative predictive values, as well as precision,
robustness and sample storage conditions. The test of choice for detection of WNV in
pig-tail macaques was the PanBio KgG ELISA.
Questions:
1) Most commercial assays for WNV are based on which WNV protein?
a) E (envelope)
b) preM (membrane
c) NS1
2) Which viral proteins of WNV are the most immunogenic?
a) E & NS1
b) NS1 & preM
c) preM & E
3) T or F: The NS1 protein is expressed only during viral replication
4) T or F: The PRNT assay does not use live virus
5) What are heterophilic antibodies?
Answers:
1) a
2) a
3) T
4) F
5) Antibodies that cross-react with proteins from multiple species.
Browning et al. Role of Reactive Nitrogen Species in Development of Hepatic
Injury in a C57BL/6 Mouse Model of Human Granulocytic Anaplasmosis, pp. 55-62
Human granulocytic anaplasmosis (HGA), caused by the granulocytic rickettsia-like
organism Anaplasma phagocytophilum, is the 3rd most frequent vector-borne infection in
North America. To understand the disease mechanisms of HGA, we developed a murine
model that lacks clinical disease yet exhibits characteristic histopathologic and
immunologic changes. Because the degree of hepatic histopathology is unrelated to high
bacterial numbers, tissue injury in HGA is thought to occur due to products of innate
immunity, such as nitric oxide (NO) and reactive nitrogen species (RNS) from cytokine-
activated macrophages. To test the hypothesis that RNS cause hepatic tissue damage,
mice received either water treated with a nonspecific inhibitor of inducible nitric oxide
synthase, L-NAME, or untreated water for 7 to 10 d before infection and continuing
thereafter. Mice were euthanized for tissue harvest at 0, 7, 14, or 21 d after infection to
assess differences in histopathology, hepatic bacterial load, RNS quantity in urine and
liver, and serum chemistry values. Overall, L-NAME treatment had a beneficial effect,
resulting in lower histopathology scores and RNS levels compared with those of
untreated mice. There were no significant differences in hepatic bacterial load among
treatment groups of infected mice. The observed increases in serum glucose and
alanine aminotransferase levels on day 14 appear to be unexpected side effects of LNAME administration. HGA is best characterized as an immunopathologic disease
rather than one caused by direct bacterial injury to the host. Therefore, human and
animal patients with HGA likely would benefit from therapy targeting reduced
inflammation to supplement anti-infective modalities.
Questions:
1) Granulocytic anaplasmosis is a severe, potentially fatal, tick-borne disease caused
by Anaplasma phagocytophilum, a_______________________organism of
neutrophils that can infect humans and other mammals.
2) C57BL/6 Mouse infected with Anaplasma phagocytophilum showed clinical
symptoms (T/F)
3) C57BL/6 Mouse infected with Anaplasma phagocytophilum elicited tissue injury via
generation of_____________ and______________
4) Nitric oxide (NO) can be inhibited by ___________.
Answers:
1) Rickettsia-like
2) F
3) Nitric oxide and reactive nitrogen species
4) L-NAME (L-NAME, N=F9-nitro-L-arginine methyl ester hydrochloride)
CLINICAL INVESTIGATIONS
Turner et al. Pharmacokinetics of Meloxicam in Rabbits After Single and Repeat
Oral Dosing, pp. 63-67
This study uses the SIV/pig tailed macaque model of AIDS to investigate the function of
lymphocyte function-associated antigen-1 (LFA1) in the creation of the virological
synapse. A human LFA-1 monoclonal antibody (MHM.23) was used in vivo to attempt to
block the adhesion of pigtail macaque LFA-1 to leukocytes and prevention of T-cell
activation. In vitro 80-100% inhibition of binding was shown by MHM.23 at 20-40
micrograms/ml doses. In vivo a dose of 5 mg/kg IV SID of MHM.23 inhibited LFA-ICAM1 function and was well tolerated by the macaques. Leukocytosis, mostly due to
neutrophillia was seen after 24 hours until day 4.
Questions:
1. What is the scientific name of the Pig tailed macaque?
2. What is the function of LFA-1?
3. Which chemokine receptor is the major coreceptor for HIV and SIV?
Answers:
1. Macaca nemestrina
2. Initiates the immune response by providing adhesion in the formation of the
immunologic synapse via binding to its ligand, intracellular adhesion molecule
(ICAM) on leukocytes.
3. CCR5
Doherty et al. Comparative Anatomy of Rabbit and Human Achilles tendons with
Magnetic Resonance and Ultrasound Imaging, pp. 68-74
Summary: Animal models including rabbits, dogs, cats and rats have been used for
research into Achilles tendon injuries. This paper compares the anatomy of the rabbit
and human Achilles tendons using MRI, ultrasound, and macroscopic observations.
Gastrocnemius - soleus - Achilles tendon – calcaneus complexes were collected from 18
euthanized NZ White rabbits and from one human cadaver. The medial and lateral
heads of the gastrocnemius tendons fused more distally in rabbits than in humans,
forming the Achilles tendon. In rabbits, the medial gastrocnemius muscle was rotated
laterally to lie posterior to the lateral gastrocnemius tendon. This is hypothesized to
allow more elastic recoil to store energy in the Achilles tendon in rabbits. Additional
anatomical differences were found between the anatomy of the flexor digitorum
superficialis muscle in rabbits, which corresponds to the flexor digitorum longus muscle
in humans. These differences must be considered when using the rabbit as a research
model for human Achilles tendon studies.
Questions:
1. Compared to humans, the fusion of the medial and lateral gastrocnemius tendons to
form the Achilles tendon in rabbits occurs:
a. more proximally
b. at the same relative location
c. more distally
d. more rostrally
e. more caudally
2. T/F? Differences in anatomy between the gastrocnemius - soleus - Achilles tendon calcaneus complexes of rabbits and humans should be considered when using rabbits
as a model for research into human Achilles tendon injuries.
Answers:
1. c. more distally
2. True