Comparative Medicine
Volume 60, Number 6, December 2010
ORIGINAL RESEARCH
Mouse Models
Vaickus et al. Inbred and Outbred Mice Have Equivalent Variability in a Cockroach
Allergen-Induced Model of Asthma, pp. 420-426
Domain 3: Research; T2 Advise and consult with investigators on matters related to
their research; K3 Animal Models
Primary Species: Mouse (Mus musculus)
SUMMARY: Historically, outbred mice have been thought to have inherent variability that
limits their use to certain studies. It is believed that inbred strains of mice have less
variability due to their homogenous genetic background. This study looked at the validity
of this by comparing the variability of the pulmonary inflammatory response that the
inbred BALB/c strain and the outbred stock HSD-ICR mice have to cockroach allergen
(CRA). The mice were allocated to 4 groups (naive, 0, 1.5, and 24h). The CRA was
delivered intratracheally by direct pharyngeal delivery on day 0 (immunization), and two
challenge doses on day 14 and day 21. Naïve control mice received no CRA
challenges. 0-h mice only received 2 CRA challenges (this group was euthanized prior
to the third challenge). The 1.5-h and 24-h mice received all three challenges and were
euthanized at 1.5 and 24 h after their final challenge respectively. The mice were
placed in a chamber and exposed to 2 minute aeresolization of methacholine at either
25mg/ml or 50 mg/ml in order to acquire respiratory measurements. Respiratory
parameters at the 50mg/ml methacholine were used for comparison. BAL samples were
analyzed for an absolute cell count for total WBC, neutrophils, macrophages,
eosinophils, and lymphocytes. Cytokine and chemokine analysis were done on the BAL
and lung homogenate samples by ELISA technique. The inflammatory response data
points were normalized (z-score normalization relative to each parameter's standard
deviation and population mean). This normalized data was then averaged across
groups and used to construct a heat map.
Analysis of the chemokine Eotaxin-1 in BAL fluid had a similarly shaped curve for both
BALB and ICR. At the measured time points 0, 1.5, and 24h the levels were also
equivalent between the two groups. Other parameters analyzed, such as the level of
the neutrophil chemoattractants MIP2 and KC were elevated in both groups at 1.5h.
This rise in the chemoattractants coincided with the peak neutrophil levels that also
occurred at 1.5h.
MIP2 levels were significantly higher at 1.5 h in BALB/c mice,
but levels of neutrophils at 1.5h were significantly higher than in the ICR
mice. Although the extent that certain parameters varied in the BALB and ICR mice,
analysis of the inflammatory cells showed a similar trend in both groups with a strong
inflammatory response early followed by a partial recovery.
Analysis of the respiratory parameters also showed similarities. In comparison to the
naive animals, both the BALB and the ICR mice had diminished minute ventilation (MV)
at 1.5h and improvement at 24h. The MV of the ICR mice, though, showed a
significantly greater improvement. The second respiratory parameter analyzed was the
respiratory rate. Both the BALB and ICR mice had a diminished rate at 0, 1.5, and 24h
in comparison to the naive animals.
The CRA was demonstrated to induce a strong inflammatory response in both the BALB
and ICR mice. Although the extent of variance of certain parameters of the pulmonary
inflammatory response (ex. chemokines and cytokine profiles) differed between the
groups, the primary goal of this study was to compare the variability of pulmonary
response to the allergen. Both BALB and ICR developed significant pulmonary
inflammatory response to the CRA. Eotaxin, MIP2, and KC were elevated in both
groups, along with inflammatory cells. Measured respiratory parameters (minute
ventilation and respiratory rate) indicative of pulmonary inflammation were similar
between the two groups. A comparison of the coefficients of variance for each of the
data sets within the strain and stock revealed that both the BALB and ICR groups had
virtually equivalent inherent variability. This study concluded that for pulmonary studies,
both BALB and ICR mice are equally suited for targeted studies.
QUESTIONS:
1. Eotaxin1, a potent eosinophil chemotactic agent is produced by both epithelial cells
and macrophages. T or F
2. The coefficient of variance was calculated by dividing the standard deviation by the:
a. Value-average
b. Mean
c. Average
d. Median
3. All the inflammatory response data was concisely displayed by a:
a. Scatter plot
b. Bar graph
c. Heat map
4. The ELISA technique that uses a secondary antibody for detection is:
a. An indirect method
b. A direct method
c. The sandwich ELISA
d. Both a and c
ANSWERS:
1. T
2. b
3. c
4. d
Dole et al. Assessment of rpoB and 16S rRNA Genes as Targets for PCR-Based
Identification of Pasteurella pneumotropica, pp. 427-435
Domain 1, T3. Diagnose disease or condition as appropriate
Primary, Mouse (Mus musculus)
SUMMARY: Pasteurella pneumotropica is considered to be an opportunistic pathogen
that has been associated with respiratory disease and infection of the eye, genital tract,
uterus, skin, mucous membranes and intestinal tract. P. pneumotropica affecting rodents
is classified in to 2 distinct biotypes named as “Heyl” and “Jawetz”. Biotypes are most
commonly identified by using microbiologic and biochemical methods. However, many
discrepancies have been found in the diagnosis of P. pneumotropica by using
commercial kits that are based on various biochemical characteristics, resulting in
different interpretations by different laboratories.
Methods based on DNA sequence divergence potentially offer a more specific method
for classification of P. pneumotropica. Various genes including 16S rRNA, atpD, infB,
gyrB and rpoB from various members of Pasteurellaceae family have been sequenced in
an attempt to characterize Pasteurellaceae with more confidence than biochemical
assays. Gel-based PCR assays, although sensitive, are prone to false positives due to
nonspecific primer binding to related Pasteurellaceae, in comparison, real-time PCR
assays add an additional level of specificity and sensitivity and can be used to determine
copy number. 16S rRNA is highly conserved among many bacterial families, portions of
the gene are unique and can be used to speciate bacteria.
Although rpoB is used frequently for genotyping of bacteria, the usefulness of rpoBbased genotyping is limited due to the availability of far fewer sequences (approximately
19000) in the GenBank compared with 16S rRNA (approximately 1726000) sequences.
The authors of this article have performed a study and demonstrated the sensitivity and
specificity of a 16S rRNA based real-time PCR assay to characterize P. pneumotropica
from isolated bacterial colonies. The study also demonstrates multiple advantages of
targeting the 16S rRNA genes over rpoB for PCR based diagnosis of P. pneumotropica
on a large scale, such as for routine health monitoring.
In conclusion their results demonstrate that the 16S rRNA but not rpoB gene of the
Pasteurellaceae family is a useful target for development of real-time PCR based
diagnostic assays that can provide conclusive differentiation without the need for DNA
sequencing.
QUESTIONS:
1. In microbial screening of mice, which among the following has higher prevalence?
a. Pasteurella multocida
b. Pasteurella pneumotropica
c. Other pasteurellacea species
2. Gel-based PCR assays, although sensitive, are prone to false positives
a. True
b. False
3. Real Time PCR assays are more specific and sensitive when compared to Gelbased PCR assays
a. True
b. False
4. The results of this study demonstrates that the following gene of the Pasteurellacea
family is a useful target for development of real-time PCR based diagnostic assays
that can provide conclusive differentiation without the need for DNA sequencing:
a. rpoB
b. 16S rRNA
c. atpD
d. infB
ANSWERS:
1.
2.
3.
4.
Pasteurella pneumotropica
True
True
16S rRNA
Zhou et al. Pharmacologic Characteristics of Bladder Micturition Function in
Anesthetized Mice, pp. 436-442
SUMMARY: Although mice are potentially useful models of in vivo bladder function,
there is a limited amount of data available regarding bladder function in normal mice.
The purpose of this study was to evaluate the effects of various drugs on bladder
micturition function in mice, primarily using cystometry, which is a measure of bladder
pressure in response to filling.
Mice: Adult male KM strain
Drugs tested: Phenylephrine (a1-adrenergic agonist), isoprenaline (B1-agonist),
carbachol (muscarinic cholinergic agonist), doxazosin (a1-antagonist). A range of doses
for each drug were used.
Cystometric parameters that were measured: Vesical micturition pressure (maximal
pressure during micturition), micturition threshold pressure (bladder pressure
immediately prior to micturition), micturition basal pressure (the lowest pressure during
filling), intercontraction interval (the interval between each large amplitude spontaneous
bladder contraction), and micturition time (high frequency oscillations of bladder
pressure associated with urine flow).
Anesthetic protocol: 1.5 g/kg urethane SQ followed by intubation
Experiment 1: Measure the effects of all four drugs on bladder function. Phenylephrine
increased vesical micturition pressure in a dose-dependent fashion and increased
micturition basal pressure at the highest dose. Carbachol increased the intercontraction
interval and micturition time in a dose-dependent fashion and increased the micturition
basal pressure at the highest dose. Isoprenaline increased micturition time and
decreased vesical micturition pressure in a dose-dependent manner. Doxazosin had no
significant effects.
Experiment 2: Measure the effects of doxazosin on phenylephrine-induced increase in
vesical micturition pressure. Doxazosin dose-dependently inhibited the micturition
pressure induced by phenylephrine.
Experiment 3: Measure the effects of all four drugs on blood pressure. Blood pressure
was reduced by carbachol, doxazosin, and isoprenaline, and blood pressure was
increased by phenylephrine. All effects were transient except for those of doxazosin.
QUESTIONS:
1. Name the drug class for the four drugs used in this study: phenylephrine,
isoprenaline, carbachol, and doxazosin
2. What is the term used to describe measurement of bladder pressure in response to
filling?
3. What are the advantages of urethane anesthesia in studies such as this?
ANSWERS:
1. Phenylephrine (a1-adrenergic agonist), isoprenaline (B1-agonist), carbachol
(muscarinic cholinergic agonist), doxazosin (a1-antagonist)
2. Cystometry
3. Induces of long-term anesthesia; less CNS-mediated effects on cardiovascular
function; peripheral stimuli are still able to activate the CNS and produce reflexive
changes in autonomic functions
Preda and Burlacu. Electrocardiography as a Tool for Validating Myocardial
Ischemia-Reperfusion Procedures in Mice, pp. 443-447
Domain 1, T2. Control spontaneous or unintended disease or condition
Primary Species: Mouse (Mus musculus)
SUMMARY: Cardiac ischemia-reperfusion in mice is induced by transient ligation of the
left coronary artery (LCA) and remains one of the most representative models of the
ischemic cell death typical in humans. However, this model is associated with variability
in infarct size of 20% to 90% of the entire left ventricle, thereby requiring a large number
of mice for each study. This study describes a surgical procedure for LCA ligation in
mice that takes advantage of electrocardiography to verify correct ligation and
reperfusion and reduce variability in infarct size.
For LCA ligation, the skin was excised, the layers of the chest muscles were separated,
and a thoracotomy was made in the 4th intercostals space. The pericardium was
removed, and the LCA was ligated 1mm distal from the tip of the left auricle by using 7-0
polypropylene suture. The modifications induced by the LCA ligation were monitored by
electrocardiography. Three-lead electrocardiography was performed and recorded for 2
minutes. Two traditional indicators of acute myocardial ischemia (ST height elevation
and QTc prolongation) confirmed that electrocardiography was an appropriate tool to
validate LCA ligation and reperfusion. Electrocardiography was also used to evaluate
the effect of increasing the duration of the ischemic period on the severity of myocardial
modifications. Whereas 30-min ischemia resulted in reversible changes in the
electrocardiogram, 1-h ischemia induced more severe modifications, which were not
completely abrogated after reperfusion. Thus electrocardiogram patterns indicated
various irreversible changes in the myocardium after 1-h ischemia. The findings indicate
that the electrocardiographic profile corroborates the histological modifications and
predicts the severity of the myocardial modifications induced by ischemia in the mouse
model of LCA ligation and reperfusion.
This report stresses the importance of using electrocardiography during the procedure,
as a rapid and inexpensive tool to validate LCA ligation and reperfusion. 3-lead
electrocardiography offers several advantages over 12-lead systems, including reduced
signal noise in small animals, more rapid equipment set up, and easier preparation of
subjects.
QUESTIONS:
1. Two traditional indicators of acute myocardial ischemia include:
a. QRS interval decrease
b. ST height elevation
c. QTc prolongation
d. R amplitude increase
2. T/F: The more detailed 12-lead electrocardiography system is preferred for this
procedure.
ANSWERS:
1. B and C
2. False
Rat Models
Wiedmeyer and Royal. Urinary Biomarkers for Monitoring Disease Progression in
the Han:SPRD-cy Rat Model of Autosomal-Dominant Polycystic Kidney Disease,
pp. 448-454
Primary Species: Rat (Rattus norvegicus)
SUMMARY
Introduction: The Han:SPRD-cy rat is an established model of autosomal-dominant
polycystic kidney disease (ADPKD) as it features many of the human disease
characteristics such as dominant inheritance, slow progression to end-stage renal failure
and males being more severely affected. Progression is commonly assessed using
plasma urea (UN) concentration as an estimate of GFR. Identifying urine biomarkers is
useful because a urine sample may be easier to acquire and may be collected at a
larger volume in live animals. This study was aimed at identifying a urine biomarker for
monitoring progression.
Methods: The biomarkers chosen were N-acetyl-b-D-glucosaminidase (NAG), aglutathione S-transferase (aGST), glutathione S-transferase Yb1(GST-Yb1), collagen IV
and urinary albumin. Each one was chosen for their association with separate portions
of the nephron. Urine was collected from 3,6,9 and 12 wk old heterozygote and wild
type male and female Han:SPRD rats using metabolic cages. Histology was performed
(see photos on p.451)
Results and Discussion: Two urine biomarkers may be useful to measure progressive
renal function: aGST and albumin. Cyst number correlated with the changes in plasma
UN and albuminuria in all male rats. aGST decreased as the disease progressed. This
decrease was thought to be due to 1) loss of functioning nephrons or 2) decreased
expression of the GST gene. The concentrations of GST-Yb1, collagen IV and NAG did
not change.
QUESTIONS:
1. What is the name of the rat that is a well-recognized model of human autosomaldominant polycystic kidney disease?
2. What are the two urine biomarkers found in this study to potentially be useful in
determining progressive renal dysfunction in Han:SPRD-cy rats?
ANSWERS:
1. Han:SPRD-cy
2. Urinary albumin and aGST (a-glutathione S-transferase)
Bardi et al. Fecal Dehydroepiandrosterone (DHEA) Immunoreactivity as a
Noninvasive Index of Circulating DHEA Activity in Young Male Laboratory Rats,
pp. 455-460
Primary Species: Rat
SUMMARY: Evidence suggests that dehydroepiandrosterone (DHEA) plays a key role in
stress and coping responses. DHEA has been shown to be released parallel to cortisol
during physical stress to protect the body against the negative effects of prolonged
exposure to glucocorticoids. DHEA can act centrally to decrease glucocorticoid induced
neuronal death. Fecal sampling permits assessment of hormone behavior interactions
reliably. The objective of this article was to compare circadian or stress-dependent
alterations between DHEA and its fecal metabolites.
Materials and Methods:
Twelve, 21 day old, male Long Evans rats were housed groups of 3. Rats were assigned
to an experimental and (n=6) or control (n=6) group. Each rat in the control group
participated in a 5-min forced swim test in a glass aquarium just before blood was
collected for the second time. Blood collection started, 1800 h, after one week of
habitation, and was repeated at 2000 h, 2200 h, and on day 2 at 0600 h. Blood sampling
started at the time of awakening for the rats, when glucocorticoids would be at their
circadian peak; collected from the submandibular vein after exposure to halothane.
Feces was collected while the rats were in a designated container and started on day 1
at 1800 and continued until the day after for a total of 8 samples per rats, collected at 2,
4, 12, 14, 16, 18, and 24 h after stress exposure in the experimental animals. Enzyme
immunoassay procedures were carried out using a Corticosterone Enzyme
Immunoassay Kit. Patterns of changes of corticosterone and DHEA trends in both serum
and fecal samples were analyzed statistically.
Results:
There was significant diurnal variation in excreted fecal corticosterone and DHEA
metabolites in the control rats. The patterns of variation between fecal corticosterone
and DHEA metabolite levels were parallel and did not differ significantly. The correlation
between levels of excreted corticosterone and DHEA was significant.
The experimental group of rats demonstrated a significant peak in the concentration of
serum DHEA immediately after the stress test. The patterns of variation in the levels of
both metabolites parallel each other after the stress test. The correlation between
plasma levels of DHEA and its metabolites was significant as was that between the
amounts of corticosterone and its metabolites in plasma. After the stress test, fecal
corticosterone and DHEA metabolites were significantly higher in the experimental
(stress) group compared with the appropriate control groups.
The study validated their method of assessing DHEA metabolites levels in feces of
young male rats. Measurements of DHEA metabolites in feces of undisturbed control
rats produced profiles correlating with corticosterone metabolites. The findings confirmed
that observed trends and hourly values were within the expected range of variation.
Animals in the experimental group that underwent a forced swim test, showed a
significant increase in circulating DHEA levels immediately after the test as well as
several peaks in both metabolized DHEA and corticosterone levels. Findings were in
agreement with previous studies on fecal glucocorticoid metabolites. The time needed to
DHEA metabolites to return to baseline was not established.
Collecting fecal samples in rats is simple and cost-effective in comparison with the blood
collection techniques. The use of a noninvasive technique would be of benefit
particularly in behavioral research.
QUESTIONS:
1. The function of dehydroepiandrosterone (DHEA) in the body is?
2. Serum concentrations of glucocorticoids are not always an accurate indicator of?
3. Whereas serum collection has advantages in acute stress scenarios, steroid levels in
feces?
ANSWERS:
1. DHEA is released parallel to cortisol during physical stress to protect the body from
the negative effects of prolonged exposure to glucocorticoids; particularly neuronal.
2. Chronic stress situations
3. Provides a cumulative endocrine profile as several hours’ worth of previous
endocrine and metabolic activation are assessed.
Rabbit Model
Yee et al. Aerosolized Bacillus anthracis Infection in New Zealand White Rabbits:
Natural History and Intravenous Levofloxacin Treatment, pp. 461-468
Domains 1 and 3
Primary Species: Rabbit
SUMMARY: The purpose of the study was to identify potential, early biomarkers of
inhalational anthrax infection (Bacillus anthracis). Early biomarkers are important as
there is a relatively narrow window of opportunity to treat an infection with B. anthracis
before the death of the patient. Inhaled B. anthracis is an important biothreat. Inhalation
of B. anthracis spores is the most lethal route of exposure. In humans, inhalational
anthrax presents initially with mild influenza-like symptoms and culminates with the
onset of high fever, dyspnea, hypotension, shock, and sudden death.
Bacteremia has been the traditional “gold standard” for the diagnosis of anthrax.
However, bacterial culture is a time-consuming process (takes 18-24 h), and there is a
relatively narrow therapeutic window in that patients die rapidly after infection (this
underscores the critical need for rapid diagnosis and, subsequently, timely treatment to
promote a successful outcome).
The capsule of B. anthracis and 2 exotoxins, lethal toxin and edema toxin, are primarily
responsible for the symptoms and pathogenesis. Lethal toxin and edema toxin each
possess a unique enzymatic active domain – lethal factor and edema factor, respectively
but share a nontoxic cell binding domain known as protective antigen (PA). Detection of
antigenemia (e.g. detection of PA) was conducted via electrochemiluminescence (ECL)
immunoassay which only takes 1 h to conduct (compared to the 18-24 h for culture).
Rabbits appeared clinically normal until shortly before succumbing to anthrax approx 47
h after challenge (approx 22 h after antigenemia). Please note the rabbits became
antigenemic and bacteremic at roughly the same time (24.5 +/- 1.4 h after infection for
antigenemia and 23.1 +/- 0.2 h for bacteremia), but the ability to detect antigenemia
takes 1 h of sample processing whereas the ability to detect bacteremia takes 18-24 h
of culture time. The results indicate that antigenemia (PA) is a viable, early biomarker for
B anthracis infection that can be used as a treatment trigger to allow for timely
intervention.
Study design as follows: 12 NZW rabbits were exposed to 150 LD50 aerosolized spores
in a head-only chamber for approx 10 min. within the confines of a class III BSC. Body
temp recorded every 15 min by radiotelemetry. Venous access port surgically implanted
to allow for frequent blood collections. Whole body plethysmography was performed to
determine respiratory capacity before challenge. The development of antigenemia and
bacteremia coincided and preceded both pyrexia and inversion of the heterophil:
lymphocyte ratio (the latter is also an indicator of infection).
Rabbits were treated (after determination of antigenemia) IV with levofloxacin
(fluoroquinolone antibiotic) for 5 d at a total daily dose of 25 or 12.5 mg/kg. Results
demonstrated an approximate survival rate of 90% and 70%, respectively, to the study
end (28 d after challenge) which suggests that IV levofloxacin is an effective therapeutic
against inhalational anthrax.
QUESTIONS:
1. The results indicate that antigenemia (PA) is a viable, early biomarker for B anthracis
infection that can be used as a treatment trigger to allow for timely intervention. T/F
2. What has traditionally been the gold standard of diagnosis?
3. Why is the use of PA as a biomarker favorable as compared to bacterial culture?
4. How was the presence of PA in the blood detected?
ANSWERS:
1. True
2. Bacterial culture
3. Detection of antigenemia (PA) allows for a much faster diagnosis which promotes a
better opportunity for a successful outcome.
4. electrochemiluminescence (ECL) immunoassay
Ruminant Model
Inglis et al. Catheterization of Intestinal Loops in Ruminants Does Not Adversely
Affect Loop Function, pp. 469-478
Domain 1: Management of Spontaneous and Experimentally Induced Diseases and
Conditions
Secondary Species: Sheep
SUMMARY: One reason that the intestines are studied is that they play a pivotal role in
immunosurveillance and protection from enteric pathogens. Methods of intestinal study
include xenografts and intestinal loop models. Xenograft models use the fetal intestinal
segments of one species that are transplanted into an immunodeficient animal of
another species. Intestinal loop models had been used in non-survival surgeries to
measure short-term (<24 hours) intestinal changes. The authors created a recovery
intestinal loop model so that longer term changes could be studied. The goals of this
study were to investigate the effect of catheterization on the animal’s health, the
deposition and localization of green fluorescent protein (GFP) E. coli in loops, the effect
of the catheter on the responses of the intestinal tissue and the effect of broad-spectrum
antibiotics on the intestinal flora in the loops. The group investigated these issues on 5
female Arcott sheep.
Although all sheep had ileus after surgery, none had evidence of infection. After 4-7
days, all sheep were eating and didn’t have signs of adverse affects from the
catheterization. At necropsy, adhesions were found, but no evidence of peritonitis. The
loops were created from functional ileum (defined by presence of continuous Peyer
patches). Loops remained patent for more than 40 days, and the bacteria stayed in the
loops. None of the sheep had behavior or health effects from the surgery. No changes
in intestinal tissue morphology or differences in gene expression of cytokines were seen
that may indicated changes in intestinal inflammation.
The loops and interspaces between loops were not made sterile by the administration of
broad-spectrum antibiotics. This is in contrast to previous studies. The authors
postulate that this is due to the increased sensitivity of the molecular diagnostics to
ascertain that bacteria were still present. The antibiotics did not affect the composition
of the flora but did selectively favor some taxa.
QUESTIONS:
1. T/F: One benefit of this model is that sheep are able to recover from surgery, and
clear all pharmaceuticals before treatments are started, thus eliminating the
confounding factors of anesthetics and other perioperative medications.
2. T/F: Administration of broad-spectrum antibiotics changed the relative abundance
of microbiota constituents, but did not substantively change the composition of the
microflora.
3. T/F:
This study found that the loops could be sterilized to study microbiota
interactions, as in a gnotobiotic model.
ANSWERS:
1.
T
2.
T
3.
F
Nonhuman Primate Models
Graham et al. Refinement of Vascular Access Port Placement in Nonhuman
Primates: Complication Rates and Outcomes, pp. 479-485
Domain 3: Research; T2 Advise and consult with investigators on matters related to their
research; K8 Experimental surgical techniques and instrumentation
Primary Species: Macaques
Secondary Species: Baboons
SUMMARY: Various methods of implanting vascular access ports (VAP) in nonhuman
primates have been described. The authors recently developed a technique described
as the single-incision, peripheral insertion (SIPI) method. The technique, described in a
previous paper, involves accessing the inferior vena cava through cannulation of the
saphenous vein, allowing for subcutaneous port placement on the lateral aspect of the
thigh. In this study, the SIPI technique was compared to the traditional VAP method of
femoral vein cannulation with subcutaneous tunneling of the catheter, and port
placement in the posterior chest wall. Use of the SIPI technique was evaluated in
cynomolgus macaques, rhesus macaques, and baboons of various ages and weights.
Animals can be trained to cooperatively extend the hind limb to the handler allowing
access to the VAPs placed with the SIPI technique. Because this can be accomplished
in the animal’s familiar home cage, the stress associated with manual or chemical
restraint is avoided. In addition to this refinement, the authors determined that VAPs
placed with the SIPI technique were less prone to other common complications such as
infection and catheter occlusion. The authors ascribed the reduced infection rates to a
lower bacterial density at the leg compared to the groin and to improved acceptance by
the NHP. Excellent patency was demonstrated in the SIPI cohorts. This was attributed to
the absence of fixation between the catheter and vessel. This arrangement
accommodates free movement of the tip in the inferior vena cava, thereby decreasing
the probability of prolonged contact between the tip and the vessel wall that might
disturb normal blood flow. When complications were encountered, removal of the SIPI –
placed VAPs was reported to be easier than VAPs implanted in the posterior chest wall.
The authors concluded that SIPI VAP provides safe and effective long-term intravenous
access for successful drawing of blood samples and delivery of drugs, antibiotics, blood
products, fluids, and nutrition. This refined method of VAP implantation has the
substantial advantage of reduced complications and increased patency and supports
cooperative handling, thereby improving overall animal wellbeing and reducing
experimental stress.
QUESTIONS:
1. VAP placement using the SIPI technique involves cannulation of which vessel?
a. Saphenous vein
b. Femoral vein
c. Jugular vein
d. Cephalic vein
2. Compared to traditional VAP methods, SIPI is reported to be a significant refinement
because:
a. Obviates the need for general anesthesia
b. Eliminates stress of chemical or manual restraints
c. Fewer complications (i.e., infection and patency)
d. All of the above
3. In this study, SIPI placement of VAPs was evaluated in which species?
a. Macaca fasicularis
b. Macaca mulatta
c. Papio anubis
d. All of the above
ANSWERS:
1. a
2. d
3. d
Tetrick et al. Blood D-(-)-3-Hydroxybutyrate Concentrations after Oral
Administration of Trioctanoin, Trinonanion, or Tridecanoin to Newborn Rhesus
Monkeys (Macaca mulatta), pp. 486-490
Domain 3: Research; T2 - Advise and consult with investigators on matters related to
their research; K3 - Animal Models, K6 - Characterization of Animal Models
Primary Species: Macaques
SUMMARY: Newborn mammals must rely on endogenous reserves of glycogen, lipid
and amino acids until feeding is established after birth. In preterm human infants,
glucose is frequently supplied by infusion, but this source of energy is limited by insulin
resistance and osmotic constraints. Lipids are energy-dense, and medium-chain fatty
acids (MCFA) are more water-soluble and their triglycerides may be more rapidly
absorbed than longer chain fatty acids. MCFA supplementation in newborns results in
metabolism to ketone bodies. Ketone bodies can provide energy to neural tissue and
support myelination as well as lung surfactant production but can have adverse effects
at higher levels.
Based on previous studies in piglets, the authors proposed that supplementation with
even-chain medium-chain fatty acids increased ketone body production to pathologic
concentrations and subsequent toxicity (narcolepsy, mortality). They hypothesized that
supplementation of newborns with odd-chain fatty acids might result in catabolism
products that would reduce the level of ketone body production, thus avoiding toxicity
while providing a source of metabolic energy. This study was conducted to evaluate the
effect of medium-chain fatty acid chain length and even- vs. odd-chain fatty acids on
levels of plasma fatty acid and levels of whole-blood ketone body concentrations. As a
preliminary step in evaluating newborn rhesus macaques as a model for newborn
preterm human infants, the potential for acute toxicity of a single oral dose of mediumchain triglyceride was also assessed.
Newborn rhesus macaques were separated from mothers within 15 hours of birth.
Groups were treated with a single oral octanoate (C8 triglyceride), nonanoate (C9),
decanoate (C10) or water control. Plasma fatty acid metabolites (C8, 9, 10 MCFA) and
the ketone body D-(-)-3-hydroxybutyrate (3HB) in whole blood were measured prior to
and 1 and 3 hours after gavage.
The authors concluded that newborn rhesus macaques are a viable model for studying
medium-chain triglycerides (MCT) and 3HB metabolism in premature human infants.
Monkeys were observed for clinical signs of toxicity, and none were noted.
Concentrations of plasma fatty acids and ketones increased with time after the dose,
indicating that MCFA are absorbed and metabolized from their respective triglycerides in
newborn rhesus macaques. Both chain length and even- versus odd-chain effect
accounted for differences in ketone body concentrations after MCT dose. The authors
proposed that the odd-chain MCT catabolism product propionyl-CoA suppressed
production of 3HB but acknowledged that further studies are needed to elucidate the
effects of different rates of digestion, absorption and metabolism of different MCT and
MCFA released.
QUESTIONS:
1. What is the correct order of lipid metabolism?
a. Triglyceride => ketone body => fatty acid
b. Fatty acid => triglyceride => ketone body
c. Triglyceride => fatty acid => ketone body
d. Ketone body => triglyceride => fatty acid
2. According to this article, newborn piglets and rhesus monkeys have 1-2% body fat.
Which of the following are the primary endogenous fuels consumed after birth?
a. Glycogen and body fat
b. Body fat and body protein
c. Body fat only
d. Glycogen and body protein
3. What was a major concern regarding the need to separate newborn rhesus
macaques from their mothers?
a. Starvation
b. Maternal rejection
c. Cold stress
d. Poor motor development
ANSWERS:
1. c
2. d
3. b