Comparative Medicine
Volume 56, Number 5 (2006)
LABORATORY INVESTIGATIONS
Mouse Models
Altholtz et al. Dose-dependent Hypothyroidism in Mice Induced by Commercial Trimethoprim-Sulfamethoxazole Rodent Feed, pp. 395-401
Task 1 - Prevent, Diagnose, Control, and Treat Disease
Primary Species - Mouse
Summary: The main objective of the study was to evaluate the (TMP-SMX) present in two different commercially available diets to produce hypothyroidism in C57BL/6j mice in a 11week study period. These diets are used as prophylactic Pneumocystis in mouse. They had four different group- Brand A TMP-SMX and brand A control, then Brand B TMP-SMX and control. Brands A and B has 1:6 ratio TMP-SMX at a daily dosages of 240 mg/kg and 2400 mg/kg respectively. No change was observed in mice fed with brand A feed, but there was significant reduction in T4 levels in mice fed with brand B diet in two weeks and the levels of TSH increased rapidly in these mice, showing thyroid hyperplasia and hypertrophy. In conclusion, SMX at a daily dosages of 240 mg/kg is recommended in preventing P. murina.
Questions
1. Which is the drug of choice for prophylactic treatment for Pneumocystis carnii (now called P. murina)
2. Recommended dose for Trimethoprin-sulfamethoxazole as prophylactic to Pneumocystis carnii or P. murina
3. Name two mouse models used to study thyroid carcinogenesis
Answers
1. Trimethoprin-sulfamethoxazole
2. 240 mg/kg
3. CB6F1 (wild type) and Tg-rasH2 mice
Rat Models
Toth et al. Sleep, Temperature, Activity, and Prolactin Phenotypes of Genetically Epilepsy-prone Rats, pp. 402-415
Task 9- Collaborate on the selection and development of animal models
Primary species- Rat
Background: Sleep disturbances are common in epileptic patients such as increase sleep latency, sleep fragmentation, and an increase in time spent in stage 1 or 2 of NREM sleep. Genetically epilepsy prone rats (GEPR's) are genetically susceptible to audiogenic seizures. There are two strains. GEPR9-displays clonic tonic seizures, seizure susceptibility 90-99%. GEPR3-wild running followed by clonic seizures, seizure susceptibility 92-97%. The purpose of this study is to characterize sleep, activity and temperature patterns of GEPR9 and GEPR3 and SD rats in the basal state, after a period of sleep deprivation and after exposure to sound induced seizures at light onset and dark onset. Serum prolactin was also assessed.
Materials and Methods: Rats were surgically implanted for monitoring EEG, EMG, locomotor activity, and core temp. The rats were monitored for 24 hrs and then subjected to a 6 hr period of sleep deprivation then they were monitored for 42 hours afterwards. Rats were subjected to seizure induction at light onset and at dark onset. A separate set of rats were used for prolactin measurement
Results: GEPR3 rats spent less time in SWS and REMS than GEPR9 and SD during
the light phase. During the dark phase GEPR3 rats spent less time in SWS than SD rats
GEPR3 had higher core temps. GEPR0 rats showed 2 peaks if locomotor activity during the dark phase. All 3 strains showed inc. in temp and activity during sleep deprivation. Afterwards all 3 strains had an inc. in SWS and REMS. Light onset induced seizures delayed the inset of SWS in GEPR strains. Dark onset induced seizures didn't alter sleep. Genotype-both GEPR's are inbred but they differ from each other at 74 of the 107 loci. Prolactin-Lower in GEPR9 than GEPR3 or SD rats.
Questions:
1. What type of seizures does the GEPR9 rats experience?
2. What are NERM and SWS?
Answers:
1. Clonic tonic seizures
2. Non Rem Sleep, slow wave sleep
Pig Models
Olsen et al. Effect of Hypercapnia on Cerebral Blood Flow and Blood Volumes in Pigs Studied by Positron Emission Tomography, pp. 416-420
Task 2 - Prevent, Alleviate, and Minimize Pain and Distress
Task 9 - Collaborate on the Selection and Development of Animal Models
Primary Species - Pig (Sus scrofa)
Summary: The aim of this study was to determine the effects of a broad range of arterial pCO2 levels on cerebral blood flow (CBF) and cerebral blood volume (CBV) in anesthetized pigs undergoing dynamic H215O and C15O positron emission tomography (PET) scans. Different levels of pCO2 were randomly induced in 4 pigs during the PET scans by decreasing the minute volume of air delivered from the respirator. CBF was estimated from the dynamic H215O PET scan and CBV was estimated from the dynamic C15O PET scan. Results indicated that pCO2 significantly affects CBF and CBV in pigs. Generally, as pCO2 increased, CBF and CBV increased. Accurate control of pCO2 levels when using pigs for PET brain studies is critical.
Questions:
1. Which statement is false?
A. a rise in pCO2 increases local H+ concentration
B. a rise in pCO2 leads to cerebral vasoconstriction
C. changes in pCO2 directly affect brain arterioles
D. a rise in pCO2 leads to increased CBF and CBV
E. none of the above
2. The minute volume is?
A. tidal volume * respiratory rate
B. the volume of air moved through an animal's lungs in one minute
C. the volume of air an animal should breathe in one minute under ideal circumstances
D. the minimum volume of air needed to keep arterial pCO2 constant
E. both A and B
3. During periods of normocapnia, regulation of cerebral circulation causes cerebral blood flow to ______under varying physiologic conditions
A. vary
B. increase
C. remain constant
D. decrease
E. both A and C
Answers:
1. B. a rise in pCO2 leads to cerebral vasoconstriction
2. E. both A and B
3. C. remain constant
NonhumanPrimate Models
Mills and Tanumihardjo. Vitamin A Toxicity in Wild-caught African Green Vervet Monkeys (Chlorocebus aeithiops) after 2 Years in Captivity, pp. 421-425
Task 1 - Prevent, Diagnose, Control and Treat Disease
Species - Tertiary
Summary: The main objective of this study was to evaluate the levels and effects of excessive dietary vitamin A intake in vervet monkeys and assess the effects of chronically excessive concentrations of vitamin A found in standard laboratory rhesus and marmoset diets. Vervets are primarily vegetarian and obtain most of their vitamin A from provitamin A carotenoid sources that are not absorbed as efficiently as the preformed vitamin A found in laboratory diets. Liver and serum vitamin a concentrations were determined by reversed phase high performance liquid chromatography with analysis performed under fluorescent gold lights using pre-cooled solvents. Evidence of toxicity was diagnosed based on the finding of hypertrophy and hyperplasia of hepatic stellate cells, in conjunction with the finding of elevated hepatic vitamin A concentrations in vervet monkeys held in captivity for approximately 2 years. They estimate that approximately 1.1 µmol excess vitamin A is stored in the liver daily and that reduction in the amount of preformed vitamin A is warranted. Diets should be reformulated to contain more provitamin A carotenoids to achieve sufficient, but nontoxic, vitamin A storage.
Questions
1. Vitamin A is an essential nutrient necessary for which of the following body functions
a. Vision
b. Growth
c. Immune function
d. Reproduction
e. All of the above
2. Which of the following is not linked to vitamin A toxicity?
a. Elevated hepatic vitamin A concentrations
b. Hepatic stellate cell irregularities
c. Low serum retinyl ester concentrations
d. Abnormal extrahepatic vitamin A concentrations
e. All of the above
3. Vervet diets are primarily ______in the wild.
4. True or False???? Vervet monkeys can also store large amounts of vitamin A in the kidney.
Answers
1. e. All of the above
2. c. Low serum retinyl ester concentrations (should be high serum retinyl ester concentrations)
3. vegetarian
4. False. This is true for marmosets.
CLINICAL INVESTIGATIONS
Trammell et al. Fatal Hemorrhagic Diathesis Associated with Mild Factor IX Deficiency in PL/J Mice, pp. 426-434
Summary: The study was planned after the unexpected death of some PL/J mice that had surgery to implant transmitters into the abdomen. The mice recovered well from the surgery but 9 of 10 died 10 days later and gross exam showed large amounts of unclotted blood in the abdomen. This study investigated the cause of the diathesis in the PL/J mice.
In the coagulation process there are 3 general steps: 1) initiation- endothelial injury exposes platelets and plasma factors to tissue factor (TF) and factor VIIIa on TF-bearing cells, with conversion of factor X to Xa, 2) amplification- factor Xa and VIIa interact and trigger formation of thrombin, leading to platelet activation and positive feedback loops and negatively charged phospholipids on activated platelets facilitate assembly of coagulation factors on the membrane of the activated platelet, and 3) propagation- platelet activation and attachment, conversion of fibrinogen to fibrin, and stabilization of the platelet plug. The 2 pathways- intrinsic and extrinsic- converge onto the common pathway, leading to the formation of a clot.
They used 3 strains of mice: C57BL/6J, BALB/cByJ, and PL/J. They divided the mice into groups: ones which had surgery but no abdominal transmitters, some received transmitters, some had magnetic stir bars (the same size as the implant) sterilized by ethylene oxide implanted, some had magnetic stir bars sterilized by high-steam pressure implanted. They collected blood for coagulation studies on the day of surgery and on day 3, 6, and 9 post-op and tested antiphospholipid antibody titers and on day 9 post-op measured activated partial thromboplastin time (aPTT), fibrinogen, and D-dimer levels. They also took samples of tissue for histology on day 9 post-op from euthanized mice. Deficiencies in the intrinsic pathway (factors VIII, IX, XI, & XII; prekallikrein, and high molecular-weight kininogen- HK) were tested in untreated mice in all 3 strains. They analyzed the sequence of factor IX in all 3 strains.
Since some transmitters from the original mice that experienced unexpected death had been sterilized with ethylene oxide, there was a question about whether the diathesis could have been caused by ethylene glycol toxicity, as ethylene glycol is produced as a byproduct of ethylene oxide sterilization and can deplete clotting factors and induce hemorrhage in mice. They develop characteristic bilateral hemothorax associated with deficiencies in the extrinsic pathway and prominent myocardial fibrosis and necrosis. The other differentials included disseminated intravascular coagulation (DIC), coagulation factor(s) deficiency, presence of an inhibitor to (a) coagulation factor(s), or a platelet defect.
All PL/J mice that had implants (transmitters or stir bars sterilized by either method) had abdominal hemorrhage, while the sham surgery mice did not. Histology showed chronic active peritonitis and extramedullary hematopoiesis in the spleen of all mice with abdominal hemorrhage. D-dimer, a specific test for thrombotic conditions such as DIC was normal in the PL/J mice. PL/J mice had normal fibrinogen levels and prothrombin time, but a markedly elevated aPTT. Prolonged aPTT with normal prothrombin time indicates a deficiency in the intrinsic pathway, or the presence of a specific (antifactor VIII or IX) or nonspecific (antiphospholipid antibodies) inhibitor. But there was no increase in antiphospholipid antibodies in the PL/J mice. The functional activity of HK in the PL/J mice was 442% compared to that of normal BALB/cByJ mice. HK is involved in inflammation, fibrinolysis, and thrombosis- it has antithrombotic properties such as inhibition of platelet adhesion and aggregation. The functional activity of factor IX was 16% in the PL/J mice compared to that of normal BALB/cByJ mice. In humans, factor IX deficiency is called hemophilia B, and it is less prevalent than the classic hemophilia A (factor VIII deficiency). The sequence analysis of factor IX in the PL/J mice did not detect functional mutations in the regions examined, which were the regions most relevant for the expression of factor IX. The nature of the genetic mutation in the factor IX determines the level of residual activity of the factor- clinical severity is determined by the plasma level of factor IX and severe disease is associated with <1%. Human patients with mild factor IX deficiency (5-40%) do not have spontaneous bleeding and may be diagnosed when bleeding occurs in association with trauma, surgery, or use of drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs). PL/J mice may provide a model for the study of hemorrhagic diathesis related to mild factor IX deficiency.
Questions:
1. Factor IX deficiency in humans is called
a. Hemophilia VIII
b. Hemophilia IX
c. Hemophilia A
d. Hemophilia B
2. T/F Humans with mild factor IX deficiency have spontaneous bleeding.
3. The functional activity of ______was reduced and the functional activity of ______was increased in PL/J mice.
a. factor VIII, HK
b. factor IX, HK
c. HK, factor VIII
d. HK factor IX
4. Factor IX is in the ______coagulation pathway
a. common
b. extrinsic
c. intrinsic
d. initiation
Answers:
1. d.
2. F
3. b.
4. c.
CASE REPORT
Baze et al. Karyomegaly and Intranuclear Inclusions in the Renal Tubules of Sentinel ICR Mice (Mus musculus), pp. 435-438
Summary: During routine sentinel testing, unusual microscopic lesions were noted in the kidney. The lesions included karyomegaly and intra-nuclear inclusion body and marginated chromatin. These cells were present randomly in cortex and medulla and more prominently near the cortico-medullary junction. Occasionally mild interstitial infiltrates of lymphocytes and plasma cells were also seen near the abnormal cells. Serological screening and PCR testing didn’t detect any viral infection, including polyoma and cytomegalovirus. No viral particles were identified by electron microscopy. Due to the lack of evidence for an infectious agent, the observed tubular lesions were considered as degenerative changes, possibly due to toxic insult. The cause and significance of this finding cannot be explained.
Questions:
1. The Karyomegaly and Intra-nuclear lesions seen in tubular epithelial cells of ICR mice is attributed to?
a. Polyoma virus
b. Toxic insult
c. Herpes virus
d. Unknown
2. List the murine pathogens capable of producing intranuclear inclusion bodies in the kidneys
3. What are the possible causes of observed tubular damage in the Sentinel ICR mice?
a. Toxic insult
b. Hereditary
c. Viral insult
d. All of the above
Answers
1. d
2. Adenovirus, Cytomegalovirus and Polyoma virus
3. d