Adrenal Medullary Autografts in Anterior Eye Chamber, Lateral Ventricle and Striatum of Adult Rats: A Long Term Study

The neurobiological basis for the short-term recovery in Parkinson's patients and experimental animals grafted with adrenal medulla is not yet clear. Structural details of the grafted chromaffin cells are also not available. In the present study, autografts of adrenal medulla in the anterior eye chamber, lateral ventricle and striatum of adult rats were studied for 360 days. Though a large number of cells degenerated, a few healthy chromaffin cells survived up to 360 days in the anterior eye chamber. In the ventricular and striatal regions, cells degenerated more rapidly, and a few surviving cells were seen only up to 120 and 150 days, respectively. Degeneration of the cells was evident from the alteration of the cytoplasmic granules, appearance of vacuoles and lysosomes, rapid decline in the number of TH and DBH positive cells and diffusion of enzymes in the intercellular region. Only lymphocytes and connective tissue cells were seen in the ventricle after 120 days, while outlines of a few chromaffin cells and background fluorescence were still evident in the striatum up to 150 days. In some of the intrastriatal transplants, morphologically identifiable Schwann cells were present and, in one transplant, there was evidence of myelination of axons by Schwann cells. These axons were obviously derived from the adjacent host tissue. From the findings it has been concluded that autografts of adrenal medulla survive for only a Limited period of time on transplantation to the central nervous system and anterior eye chamber. Survival seemed to be better in the anterior eye chamber than at the sites preferentially chosen for treating Parkinson's patients or experimental animals.


INTRODUCTION
The chromaffin cells of adrenal medulla, known for producing dopamine as an intermediary in the synthetic pathway of epinephrine/19/, are used for grafting in rats and monkeys to reverse the deficits induced by cheInieal denervation of the striatum/3,9,10,12,20,25,26/. Results of these experiments prompted the use of adrenal medulla autografts in the treatment of Parkinson's patients/2,16-18/. Only moderate recovery of short duration is reported so far in a small proportion of patients/15,16/. The neurobiological basis of such recovery is still unexplained. Survival of allografts of ehromaffin cells in the striatum of rats denervated chemically and amelioration of some of the deficits are reported in some studies by using fluorescence histoehemistry and immunolabelling/8,12/. Continued  A recent autopsy report also shows complete degeneration of the adrenal medullary autograft in a Parkinson's patient/23L These reports have brought to light the necessity for a more detailed basic study than hitherto exists, on the transplanted adrenal medullary cells. Transplanted adrenal chromaffin cells grafted to the anterior chamber of the eye were shown to have achieved neuronal characteristics with their processes innervating co-grafted cerebral cortex /21,22/.
Similar observations were also recorded in culture of chromaffin cells in the absence of corticosteroids /27-29/. The present study was undertaken to analyse the morphological details of the autografts of adrenal medulla in the anterior eye chamber, lateral ventricle and striatum.

MATERIALS AND METHODS
Adult Wistar rats of stock bred colony weighing 150 to 200 g were used in this study. The left adrenal gland, after removal from an anaesthetised (ketamine, 10mg/kg body weight) rat, was dissected to free the medulla from the cortex in sterile lactated Ringer's solution under an operation microscope. Standard techniques were used to transplant the medullary grafts into the anterior eye chamber, lateral ventricle and striatum /4,5,11,21/. Behaviour and vascularisation of the intraoeular grafts were observed under an operation microscope. On 30, 60, 90, 120, 150, 180, 300 and 360 post-transplantation days, the rats were sacrificed and the transplants were processed for morphological and histochemical analysis. The number of transplants studed on each experimental day is listed in Table 1.
Tissue preparation for light and electron microscopy After anaesthetising the animals, the intraocular grafts were rapidly dissected free from the host iris and were fixed in a buffered solution containing 4% paraformaldehyde and 2% glutaraldehyde for 24-48 hours. Intracardiac perfusion using the same fixative was done for ventricular and striatal grafts. Following perfusion, brains were sliced to identify the transplants before further processing for routine electron microscopy. Adrenal medulla of adult control rats was also processed simultaneously for comparison. One micron thick semithin sections were cut and stained with 1% toluidine blue for light microscopic analysis. Ultrathin sections of the representative areas were cut and stained with uranyl acetate and lead citrate before examination with a Philips electron microscope 300.

Gtyoxylic acid histochemistry
The method of de la Torre/6/was used to demonstrate catecholamine fluorescence in fresh transplants. Cryostat sections of 10 microns thickness were used in this method. Adrenal medulla of normal adult rats was also processed simultaneously.
7rosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunolabelling After perfusion with buffered 4% paraformaldehyde and 0.1% glutaraldehyde, tissues were further fixed in buffered 4% paraformaldehyde  medulla of normal adult rats was also processed simultaneously for comparison. Control sections were processed without the primary antiserum.
The adjacent vibratome sections of intraventricular and intrastdatal transplants were also processed for routine electron microscopy for the structural details of the transplants used.for immunolabelling.

Morphomec studies
The numerical density per unit volume (Nv) of chromaffin cells in the intraocular autografts on the 60th, 90th, 120th, 180th, 300th and 360th post-transp!antation days was determined using 1 micron thick toluidine blue stained sections using an IBAS Image Analyser (Kontron, FRG). A total of 3 samples was analysed in each group.
The mean values of area, perimeter and diameter of chromaffin cells were determined from 10 frames in each sample. Nv was calculated using the stereology program. The differences between the Nv of the transplanted ehromaffin cells on each experimental day and the control value was statistically analysed using Student's ttest (two-tailed).

Intraocular transplants
Ninety percent of the autografts survived transplantation and developed rich vaseularisation from the host iris within seven days. The size of the graft declined during the first week but remained constant thereafter. By about the 15th post-transplantation day, the transplants had developed a thin connective tissue capsule and were in contact with the iris.
The number of transplants with identifiable ehromaffin cells on different post-transplantation days is shown in Table 1. Based on the structural details, histo-and immunofluorescence staining characteristics and the number of such cells, the viability of ehromaffin cells in different transplants was assessed.

Light microscopic observations
Up to 120 days after transplantation, the arrangement of the cells remained compact in the form of clusters and cords as in the control adrenal medulla (Figs. la & b). Thereafter the cell density gradually decreased and the cells were discretely arranged in the central region in dose proximity to the blood vessels. Cell shape had become polygonal or elongated. halo. The core of the granules was either highly electron dense or moderately electron dense. These characteristics were retained by the chromaff granules up to 120 days after transplantation (Fig. 3b). Chromaffin cells in the transplants at 180-360 days had increasinly fewer granules and other cytoplasmic organelles. The chromaffin granules present were polymorphic and had highly electron dense cores without any halo. The outer membrane was absent in most of the granules. Some of the granules were empty. Clear vacuoles and dense bodies were frequently TH and DBH immunolabelling Cells positive for TH and DBH immunoreactivity were seen in clusters or in isolation in all the transplants. The number of immunopositive cells was reduced as the age of the graft advanced. However, a number of discrete immunopositive cells could be seen at the end of one year (Fig. 7). Background fluorescence was strong in all the transplants studied. Both TH and DBH immunopositive fibers were seen between the host iris and the transplant.
Morphometfic studies Numerical density per unit volume (Nv) of chromaffin cells in the transplants at different post-transplantation days and the control are given in Table 2. The Nv of chromaffin cells showed a steady decline with increasing age of the transplant. The Nv of chromaffin cells in different transplants on all post-transplantation days was significantly different from the control value (Table 2).

Intraventricular and intrastriatal transplants
The number of transplants with identifiable chromaffin cells at different post-transplantation days are shown in Table 1. Eighty percent of the transplants survived up to 60 days.    irregularly arranged chromatin (Fig. 10b). Cells with basement membrane were also seen among collagen fibers in three of the intrastriatal grafts at 150 days. In one of the intrastriatal transplants, myelinating axons with loose myelin lamellae and clearly visible basal laminae were seen in dose association with these cells and hence these cells were identified as Schwann cens (Fig. 11).
Post-transplatation days , 60 8.9 +_ 0.55,, After 60 days, the number of identifiable ehromaffi cells declined rapidly. Degenerating mitoehondria, vacuoles, secondary lysosomes, a reduced density of ehrom granules (those present were polymorphic and were without the limiting membrane), and condensation of nuclear ehromatin, were observed in relation to the few cells present in the transplants (Figs. 8a and b). By 150 days, no reeognisable ehromaffin cells were seen in the intraventricular transplants. Instead, lymphoeytes, plasma cells, cell debris and maerophages were seen (Fig. 9). Intrastriatal sites continued to have a few identifiable ehromaffi cells until 150 days. Thereafter only cell outlines without any normal cytology were observed in the transplant (Fig. 10a) in addition to the connective tissue cells. Vacuoles and degenerating mitoehondria were seen in some of the cells. The nucleus was shrunken with dense and Glyoxylic acid histofluorescence Discrete cells and clusters of fluorescing cells were seen in the transplants until 60 days (Fig.   12a). In addition to the fluorescing chromaffin cells, autofluorescing macrophages and debris were seen after 60 days. There was a high background of yellowish green fluorescence in relation to the intrastriatal transplants. By 150 days, only autofluorescing cells and debris were present in the ventricle. In the intrastriatal implant sites a few aminergic cell outlines could be made out up to 180 days (Fig. 12b). After 180 days, intrastriatal implant sites showed only autofluoresting cells and debris. TH and DBH immunolabelling A few i_m_ munopositive cells were seen in the ventricle during the early days. After 60 days only diffuse reactivity was seen in the ventricular transplants. Discrete immunopositive cells were seen at the intrastriatal implantation sites up to 150 days when treated with antibodies against TH and DBH. Cell outlines were not sharp because of the background reactivity (Fig. 13).
After 150 days, only background reactivity was seen at the site of the transplants. Routine light and electron microscopic observations of the vibratome sections of the same 180-day old transplants revealed only cell outlines with cytoplasmic vacuoles and shrunken nuclei (Figs.  10a and b).  Onset and rate of degenerative changes seem to vary according to the site of transplantation. The ventricular transplants had completely degenerated by 150 days followed by the striatal grafts. Intraocular grafts had surviving cells up to 360 days. A reason for such variation could be the delay in the vascularisation of the grafts in the ventricle and striatum compared to the anterior eye chamber. Adrenal medulla being a highly vascular structure may not survive long without the physical presence of blood vessels, even though temporary nutrition could be provided by the cerebrospinal fluid. This assumption is supported by the observation that the intraoeular transplants develop vascularity within the first week after grafting. Early vascularisation and also innervation by the rich nerve plexus from the iris may have preserved the chromaffin cells better and longer in the anterior eye chamber when compared to the other sites.
Despite the alteration in the cell shape as the cell number declined in the anterior eye chamber and other sites, neuronal morphology was not observed by us in the autografts as ha been observed by Olson  Onset and rate of degenerative changes seem to vary according to the site of transplantation. The ventricular transplants had completely degenerated by 150 days followed by the striatal grafts. Intraocular grafts had surviving cells up to 360 days. A reason for such variation could be the delay in the vascularisation of the grafts in the ventricle and striatum compared to the anterior eye chamber. Adrenal medulla being a highly vascular structure may not survive long without the physical presence of blood vessels, even though temporary nutrition could be provided by the cerebrospinal fluid. This assumption is supported by the observation that the intraoeular transplants develop vascularity within the first week after grafting. Early vascularisation and also innervation by the rich nerve plexus from the iris may have preserved the chromaffin cells better and longer in the anterior eye chamber when compared to the other sites.
Despite the alteration in the cell shape as the cell number declined in the anterior eye chamber and other sites, neuronal morphology was not observed by us in the autografts as ha been observed by Olson  /14/and also by us may lead to later pathological changes in the central nervous system. These cells may also have some beneficial influence on ]casing growth factors /1/. Other investigators have reported sprouting of TH-lik immunorcactive fibers at the implantation sites/3,'//. Gash