Sample Responses:

C. Lay Description:

Paraplegia (paralysis of the legs) is a major and devastating complication following surgery on the aorta (the major artery of the body). All patients undergoing such operations are at risk for becoming paralyzed. Operations for repair of the aorta necessitate temporary interruption of blood flow through the artery and this can lead to inadequate supply to the spinal cord (which controls leg movements). The mechanism of the injury to the spinal cord is not clearly understood, although there are several treatments that seem to protect (with limited success) the spinal cord from this injury.

Recent evidence has shown that glutamate (normally found in small amounts in the spinal cord) is released in large quantities during interruption of the blood supply and leads to damage and death of cells within the spinal cord. Glutamate causes damage by attaching to receptors on the nerve cells. New drugs called "Glutamate Antagonists" can be used to block the receptors before the glutamate is released, so it cannot attach to the cells. These drugs also prevent the release of glutamate by an unknown mechanism. Although these drugs have shown activity against glutamate in cultured cells, they have never been tested to see if they can prevent the damage to the spinal cord seen with surgery on the aorta.

We propose to study the effectiveness of one of these drugs, dextrorphan, on the protecting the spinal cord in a rabbit model. The rabbit is an excellent model for simulating aortic operations and the paraplegia associated with these operations. Some groups of animals will undergo surgery without giving dextrorphan and some will undergo surgery and receive dextrorphan during surgery. After the operation, each rabbit will receive pain medications for at least two days. The rabbits will be evaluated on their ability to stand and hop for the next 4 days. Differences between the treated and untreated groups will be measured. After the observation period, the rabbits will be euthanized and the spinal cords removed for other measurements.

D. Rationale:

i. What is the scientific background that underlies the approach taken (including literature citations)?

The goal of this project is to determine if Mouse Hepatitis Virus (MHV) can be given intranasally to produce lesions causing dysosmia in Swiss-Webster mice without causing systemic disease. Ideally, this dysosmia will be measurable by the Olfactory Confusion Matrix (OCM) testing system developed in our laboratory (Youngentob et al., 1990). In order for animals to be suitable for OCM system, they must recover from the infection and have no other impairments except for dysosmia. Therefore, the hepatitis strain selected should not cause acute or chronic liver disease from which there is no recovery. There is a great deal of information on MHV and its effect on different genotypes of mice (Barhold, et al., 1986). Unfortunately, the information is then where the older Swiss-Webster mouse is concerned. However, these are the only mice suitable for the OCM system due to their size (see "specific study" section C). To determine the efficacy of the virus to produce the desired results, we will assay several parameters: 1) clinical observation -after intranasal infection, animals will be observed for 30 days. During the observation period, animals will be weighed and monitored for signs of illness daily. Any animal which becomes moribund will be euthanized and have a histopathological examination to determine extent of disease. 2) pathologic changes - after intranasal inoculation, animals will be euthanized at various timepoints and the brains and nasal tracts studied for pathological changes using biochemical techniques, light microscopy and electron microscopy (Schwob, JE et al.,1992). In addition, a recording of odorant-induced mucosal activity patterns will be made (Kent, PF et al., 1992).

Barthold, SW, et al: Mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype. Arch Virol 1986; 91:247-256.

Kent, PF and Mozell, MM: The recording of odorant-induced mucosal activity patterns with a voltage-sensitive dye. J of Neurophysiol 1992; 68:184-1919.

Schwob, JE, et al: Olfactory sensory neurons are tropically dependent on the olfactory bulb for their prolonged survival. The Journal of Neuroscience 1992; 12: 3896-3919.

Youngentob, SL et al: A method for establishing a five odorant identification confusion matrix task in rats. Physiology and Behavior 1990; 47:1053-1059.

ii. What value or potential contributions to biology or medicine may come from this work?

One out of every 11 women will be diagnosed with breast cancer at some point during her lifetime and the incidence appears to be increasing. Despite these alarming statistics, few attempts have been made to treat breast cancer patients with adoptive immunotherapy. Since I have found that significantly higher levels of TIL cells accumulate in mouse mammary tumors than in corresponding normal mammary tissue subsequent to adoptive transfer, the TIL may be the appropriate cell for immunotherapy of breast cancer. If altered TIL cells can be shown to selectively kill mouse mammary adenocarcinoma cells, this may lead the way to clinical trials with human TIL cells in breast cancer patients.

iii. Please give detailed reasons why animals must be used.

Although the induction and inhibition of somatostatin release can be measured in cultured cells its effect on the gastric phase of digestion can only be evaluated on a living system. Somatostatin effects multiple organ systems with feedbacks to gastrointestinal function. The complex biological interactions involved cannot currently be duplicated in a computer model or cell culture system.

iv. Please give detailed reasons why this species must be used?

The pig is currently the standard model for simulating human endoscopic abdominal surgery. The organs are of very similar size and structure to humans, allowing the learning surgeon to train in an environment most similar to what will be encountered in actual human surgery. Smaller species would not allow room for the multiple instruments and access points required for these operations. Pigs are readily available since they are raised in very large numbers for commercial food production.

v. Are your using the fewest number of animals possible? Please explain.* Include the number of groups with the "n" value for animals clearly outlined.

· A guideline that is sometimes appropriate is that the sample size should be adequate to detect reliably a 10% difference in treatment means. If not statistically justifiable, please justify the number of animals to be used or indicate that this is a pilot study designed to demonstrate the amount of variability expected in the data.

Previous work in this area has demonstrated that an n=8 mice per group is necessary to demonstrate a statistical difference (p=0.05) between treatment groups. We propose to study 4 different drugs at both a high and low dose. Saline will be used as the control. One group will be needed for each timepoint (5 timepoints), therefore we need the following:

All of the experiments proposed in this protocol are performed in vitro using primary cultured hepatocytes harvested from rats without prior manipulation. All of the experiments listed in section G will be undertaken in the next 3 years. Each experiment requires variable amounts of hepatic cells which are taken from a central "stock culture" maintained at all times within the laboratory. The stock culture must be renewed weekly (with any remaining cells discarded) due to senescence of the cells causing changes in biochemical properties. It takes 3 livers from neonatal rats to make up the stock culture and the rats can be used any time in the first two weeks after birth. Sprague-Dawley rats average about 8 pups per litter. This means we should be able (with some flexibility in when the cultures are prepared) get two weeks of stock culture from each litter. Therefore, we will need 1 pregnant female ever two weeks for the 3 years of this protocol (1 X 26 X 3 = 78 pregnant rats). Contamination of the stock culture occurs occasionally (5-10%) and some litters are too small to get two cultures (5%), so we are requesting an additional 10% of the required pregnant females (8) to ensure adequate numbers to maintain our stock culture. Therefore, we require a total of 86 pregnant female SD rats*.

*Examples note: The total number of animals should always be consistent throughout the protocol and should be equivalent to the total number of animals requested in section B.