Fetal Brain Transplantation in Kainic Acid Lesioned Caudate Nucleus of Adult Rats

This study confirmed that bilateral kainie acid (KA) injection at the caudate produces aphagia and adipsia in rats. The reduction in food and water intake was fatal after a higher dosage of the drug. To test the effect of transplantation on. the mortality, rate, KA was first injected in the left caudate, in one set of rats. After a gap of three days, fetal striatal tissue was unilaterally transplanted at this lesioned site, along with a second injection of KA in the right caudate. Successful transplantation, as ascertained morphologically, did not significantly alter the mortality rate. The morphometric study revealed that the neurons of the transplant were larger in size, and their numerical density lower than those of the caudate of normal rats. Only very few neurons of the transplant developed functional connectivity with the host, as demonstrated by electrophysiological studies.


INTRODUCTION
Bilateral KA lesions of the striatum in rats produce severe aphagia and adipsia, ultimately causing death/18/. Tulipan et al. /21,22/ reported that neonatal striatal tissue transplants protected the lesioned animals from the syndrome of aphagia and adipsia, but a recent report showed that animals which received neonatal striatal transplants had increased aphagia and adipsia as compared to the controls /15/. According to Tulipan et al. /21/, the life-saving effect of the transplant was due to graft survival. However, the post-mortem examination of the brains of these rats did not show any surviving transplants /21/. The optimal age of the donor caudate tissue, which shows the maximal survival rate after transplantation, is 15-17 days of gestation /16/.
In the present study, caudate nuclei of rats were lesioned by local injection of KA. Fetal striatal tissue of the optimal age was transplanted within the lesioned caudate of one set of animals. The extent of destruction produced in the caudate nucleus by KA was ascertained morphologically. Survival of the transplant was confirmed on post-mortem examination. In addition, the size and density of the neurons in the transplant were studied and compared with those in normal animals. The changes in food and water intake in the transplanted animals were compared with those of lesioned rats without a transplant. The survival rates of rats with KA lesion alone and those with transplantation (in addition to the lesions) were noted for comparison with the VOL. 3, NO. [2][3]1992 125 earlier reports in the literature. The spontaneous activity of neurons in the transplant was studied electrophysiologically. The changes in the firing rate of these neurons in response to the stimulation of the host substantia nigra (SN) were also investigated to ascertain whether the beneficial effect of the transplant, in terms of survival of the host, was dependent upon the establishment of connectivity.

METHODS
Experiments were conducted on adult male Wistar rats weighing 150 to 250 g. The animals were housed in a room maintained at 26_+2C with constant light from 05.00 to 19.00 h. Food and water were given ad libitum. The basal food and water intake were measured daily (between 9.00 and 10.00 h) for seven days/2/. Fifty of those animals which showed regular food and water intake were selected for this study. The food and water intake were continuously monitored for 15 more days after the first operative procedure. In addition, six normal rats were used for morphometric measurements of neuronal size and density for comparison with the experimental group.
The rats were divided into two groups. One group (controls) ( side. All the animals were given an intraperitoneal injection of 2 ml of normal saline with 5 % glucose on the day following the surgery. The pre-injection food and water intake (at different days) of all the four groups were analysed by using two-way analysis of variance. The food and water intakes of the transplant groups were compared with those of the group which had received an identical dose of KA injections alone by using Wilcoxon's rank sum test. Similarly, the survival rates of transplantation groups of animals were compared with the non-transplanted animals by applying x2-test with correction factors.
Three to seven months after the transplantation, all the surviving rats were again anaesthetised with urethane (1.5 g/kg b.wt. i.p.) for electrophysiological recording. A concentric bipolar stainless steel stimulating electrode was lowered to the SN (A 2.6, L 2, H-2.5). Glass micropipettes, filled with 2% pontamine skyblue dissolved in 0.5 M sodium acetate solution, were guided towards the transplant in the caudate, with the help of a hydraulic microdrive (Narishige Scientific Instruments Laboratories, Japan). Extracellular unit activity picked up by the micropipettes was fed to a P17 unity gain, high input impedance probe (Grass Co., USA) and the signal was amplified 1000 times by passing it through a preamplifier (Tektronix, USA). The amplified signal was fed to a spike analyser (Mentor, USA). The spike analyser was JOURNAL OF NEURAL TRANSPLANTATION & PLAHcrI set to deliver square wave pulses with each spike falling within the set window level. The single cell activity and window discriminator levels were monitored on a storage oscilloscope. Viewing the response on an oscilloscope was extremely important for checking that the spikes did not change their amplitude and the artifacts did not fall within the window level. The window discriminator output was recorded on one of the channels of the polygraph. The normal firing rate of the unit was recorded about five min before the effect of the stimulation of SN was studied. The interspike intervals of prestimulatory records were measured to plot their distribution pattern. Frequency of occurrence of various intervals was plotted. The plotted histograms were visually classified as Poisson and Gaussian types.
The effect of stimulation of the SN, with square wave pulses (of 0.3 msec duration, 80-600 /xA current, and 100 Hz frequency) for 5-10 sec, on caudate unit activity was studied. The stimulus effects on each unit were studied thrice.
The firing rate of caudate units (per sec) over 5 sec periods prior to stimulation, during stimulation, and for 3 sec after stimulus trains, were calculated for statistical analysis. The stimulatory and post-stimulatory rates were compared with the pre-stimulatory rate using Wilcoxon's rank test at the 5% significance level. At the end of the recording session, the last point of recording was stained by ejecting pontamine skyblue (Fig. 1A).
The rats were sacrificed at the end of the experiment and the brains perfused with 10% buffered formalin (formalin diluted in phosphate buffer). Seven /.m thick paraffin sections of brain, cut by microtome, were stained with cresyl violet for verification of the lesion and the transplant. The extent of lesion produced by KA on both sides of the brain was assessed by light microscopy. The histological changes in the caudate in transplanted and non-transplanted lesioned rats were compared. Six normal rats were also sacrificed, and their brains were processed, for comparison of morphometric measurements of the host caudate and transplanted cells. The numerical density of neurons per unit volume, the area, perimeter, maximum diameter and minimum diameter of the cells in three, four, five, six and seven month old transplants, kainic acid lesioned caudates and control caudates were carried out using an IBAS image analyser (Kontron, FRG). The mean values of area, perimeter and diameter of neurons were determined from ten frames in each animal. Numerical density was calculated using a stereology programme. All the values were statistically analysed using Student's t-test (two-tailed). Similarly, values from the transplant and kainic acid lesion sites were compared with the control using the same test.

RESULTS
The survival rate of the rats which received bilateral injections of (Figs. 1C, D). The transplants were well delineated and could be clearly distinguished from the host tissue on the basis of cellular organisation. There was no difference in the numerical density and cell size of the transplant in the different rats which had been sacrificed from three to seven months after transplantation (Fig. 2). The average cell size of the neurons in the transplant was larger ( Table  2) than the normal caudate neuron.  There was no significant variation in the quantity of food and water consumed by the different animals during the pre-injection period. The food and water intake showed a statistically significant fall on the day following the injection of 2 g of KA but it showed significant recovery by the second day after the injection (Fig. 3). The reduction was more marked and prolonged after injection of 3.5/zg of KA. Similar changes in food and water intake were produced in transplanted animals also after KA injection..  (Fig. 4).

DISCUSSION
In this study the lethal dose of KA was found to be 3.5 /zg. The survival rate of rats which received a striatal transplant in addition to KA was not significantly different from those which did not receive a transplant. KA injection at the caudate produced a dose dependent decrease in food and water intake in all animals. Aphagia and adipsia were persistent in all animals which died after KA injection. The changes in food and water intake in the transplant group were not different from those of the non-transplant group. A well-formed transplant was found in the brains of all the rats which received foetal transplants. The average size of the neurons in the transplant was larger and their density lower than the normal caudate. There were neurons in the transplant which had spontaneous electrical activity. These neurons generally had a Poisson distribution of firing. On stimulation of the SN of the host, two neurons of the transplant showed alteration in their firing rates.
The amount of KA which was required to produce lethal changes was found to be 3.5 as compared to 2 g reported earlier /21,22/.   There are some differences in the experimental approach of our study as compared to those of Nash  The cells of the transplant developed normal spontaneous electrical activity, as is evident from the recording of spontaneous firing from 25 units in this study. Their firing rates were comparable to those found in the striaturn of normal urethane anaesthetised rats /19/. The Poissonian distribution of the firing may indicate the development of functional connections by the transplanted neurons/17,24/. Striatal transplants were reported to develop afferent and efferent anatomical connections with various regions of the host brain including SN/23/. As stimulation of the host SN, which normally forms an important input to the eaudate/1,3,4,5,9,10,12,13/, did not affect most of the neurons studied, it is possible to conclude that the major functional connections of the transplant are not from the host. It is possible that the transplanted cells developed connections between themselves. This was observed in our earlier morphological studies/20/. The percentage (9%) of neurons in the transplant showing response to stimulation of SN was much lower than that reported (63%) in normal animals/3-5/. Even this small number of neurons, which responded to SN stimulation, forms an important landmark in the understanding of the functional integration of the transplant with the host brain.

ACKNOWLEDGEMENT
The financial support Department of Science and gratefully acknowledged.
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