Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

Male LACA albino mice were used for these experiments. The average weight was 47 grams and the individual weights ranged from 35-55 grams. Mice were classified into two groups; aged 7-8 weeks and 9-12 months. Mice aged 9-12 months represented the old animals. The average life expectancy of mice could be upto 22-24 months. All animals were maintained at 20 °C ±1°C (Celsius) for the duration of the experiment. The animals were housed under a 12 hour : 12 hour light-dark cycle and the normal laboratory chow and water were available ad libitum.

The animals were placed in specialy constructed chambers for measurement of metabolic rate (MR). Oxygen consumption was measured as an index of metabolic rate. Using an open circuit system, 1 litre per min of air was extracted from each chamber. The flow rate was monitored by the flow meters, one for each channel of the system, including the reference channel. Gas removed from the chambers was analysed for oxygen content using an oxygen analyser. Gas from each channel was analysed for one minute at every ten minute interval. The oxygen analyser was on-line to a BBC Model B computer that compared each value to the reference.

Deep body temperature (DBT) was measured by radiotelemetry and was recorded continuously from a radio transmitter implanted inside the peritoneal cavity of the animals. The complete radiotelemetry system consisted of a radio transmitter, a transistor radio receiver, a frequency to voltage integrator and a BBC model B computer. The radio transmitter (Model X, Mini Mitter Inc; Sunriver, U.S.A) consisted of a small plastic capsule which housed the electronics and a 1.4 Volt mercury battery. The minimitter emitted pulses on a broad spectrum between 550 and 1600 kilohertz (kHz), and had a range of approximately 0.5 meters. Before implanting, the transmitter was coated with three layers of molten Paraffin/Elvax to ensure complete coverage. This material prevented any adhesions inside the peritoneal cavity of the animals and also any consequent damage to the minimitter by providing a seal around the electronic circuitry.

Animals were housed in the animal keeping facility for approximately 4 weeks prior to the experiments. During this time surgery to implant the minimitters in peritoneal cavity was performed and recovery was allowed. For three days before the test day, i.e; from Monday at 0900 hours through to Thursday 0900 hours, MR and DBT, were recorded. The MR was recorded every 10 minutes and the DBT was recorded every 2 minutes. Diurnal variation was thus assessed.

Recordings of oxygen consumption of all the animals were made by computer and calculated in both ml O₂/min and ml O₂/kg/min. For each individual animal these recordings were obtained for three days prior to the test day. On Thursday mornings at 0915 hours the animals were removed from these cages and placed in another chamber designed to allow temperature variation. Animals were given time to adjust to the new environment; this was generally achieved within one hour. This one hour reading of DBT in the test cage (between 0930 - 1030 hours), was compared to the mean of the previous three days DBT readings to assess the bias of the environmental change on the readings.

At the end of this one hour recording period a gradually increasing cold challenge was applied. The rate applied was 1°C every 3 minutes dropping down to 4 °C. DBT and MR recordings continued for 2.5 to 3 hours, i.e; till the time the respective animal was taken out of the cage for euthanasia and dissection of the brain.

Animals were killed by cervical dislocation and decapitation. The brain was placed on a glass plate on ice and the hippocampus and hypothalamus were dissected free. Portions of the tissue were used for immunohistochemical analysis using VECTASTAIN® ABC KIT (Vector laboratories). For immunohistochemistry, the blocking and the secondary antibodies were derived from rabbit and the primary antibody was extracted from sheep. The remaining hippocampal and hypothalamic tissue was added to tubes containing ice cold Tris buffer in an ice bath for use in the binding experiments.

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