The Recovery of the Ultrastructure of Hyperchromic Host Neurons Under the Influence of Fetal Neurotransplants

Fetal neocortical transplants placed into a previously prepared cavity in the cortex, of adult animals inhibited destructive processes in host neurons surrounding the place of injury. This was demonstrated by a gradual reduction in the number of hyperchromic and shrunken host neurons and the recovery of the ultrastructure of dystrophic cells which die as a result of neuronophagia or sclerosis in the absence of the transplants.


INTRODUCTION
Nervous tissue regeneration is one of the central problems in neurobiology. It is well known that spontaneous processes leading to normalization of disturbed functions sometimes occur in the damaged brain. For example, as a result of sprouting of neuronal axons, new synaptic connections may be established and result in the recovery of CNS functions. The regenerative processes of nerve cells are, however, restricted/4/. The successful development of neurotransplantation in recent years Reprint address: V.N. Kleshchinov Laboratory of Experimental Neurogenetics N.I. Vavilov Institute of General Genetics, USSR Academy of Sciences, Gubkin St., 3,117809 Moscow, USSR has considerably extended the possibility of regeneration in the damaged brain [5/. Brain damage is often accompanied by the appearance of dystrophic neurons. It is generally accepted that dystrophic nerve cells (shrunken neurons) are dying or dead cells/25/. However, developing embryonic nervous tissue (ENT) can prevent degenerative processes in the host brain /19,22/and promote the recovery of the structure and function of neurons after damage/1,7,17/. In our previous work, it was shown that ENT transplantation into a recently prepared cavity reduced the hyperchromic and shrunken neurons which normally appeared around the wound in the host brain (i.e., prevented the degenerative processes following injury). Moreover, in some nerve cells, partial differentiation /10/, replicative DNA synthesis and mitotic figures were observed/11,12,18/. The purpose of the present work was to study the state of hyperchromic and shrunken neurons appearing around a mechanically-induced brain wound at various times after ENT transplantation.

MATERIALS AND METHODS
Transplantation of 15-day-old embryonic ENT into adult female Wistar rats (160-200 g) was carried out according to the following scheme. An aspirative cavity (5 mm3) was made in the sensorimotor cortex under hexenal-ether anesthesia. The cavity was then covered with gelfoam. After 10 days an embryonic neocortical transplant was placed into the cavity. The animals were then transcardially perfused with 2.5% glutaraldehyde/0.1% cacodylate buffer (pH 7.4) either 10 days following injury or 4, 7, 13, or 30 days after ENT transplantation (n 3 per group). The tissue was post-fixed for 1.5 h in VOL. 3, NO. 1,1992 51 ethanol at increasing concentrations and embedded in araldite. For electron microscopy, the tissue was first examined using semithin sections stained with methylene blue. Ultrathin sections were prepared on an LKB ultratome and viewed under an EM-420 electron microscope.

RESULTS
Analyis of semithin sections of the host sensorimotor cortex 10 days following surgery revealed neurons in various stages of hyperchromia, ranging from cells with poorly pronounced hyperchromia to dark shrunken neurons. This was accompanied by an acute edema, astrogliosis and narrowing of blood vessel lumina. The largest number of dark cells was observed in cortical layers V-VI ( Fig. 1).
Electron microscopic examination revealed that the nuclei of normochromic neurons contained small conglomerations of condensed chromatin and reduced aggregates of interchromatin granules (Fig. 2). In the cytoplasm of these cells, the endoplasmic reticulum cisterns were expanded and did not form parallel aggregates.
The mitochondria exhibited focal zones of clearing and damaged cristae. This pattern of neural damage was primarily evident in small to medium-sized nonpyramidal neurons.
In some cells with a poorly expressed hyperchromia, the nuclei had irregular profiles with deep invaginations and contained large nucleoli, small conglomerations of condensed chromatin and a set of RNA particles characteristic of neuronal nuclei (Fig. 3). No pronounced destructive changes were observed in the cytoplasm. Hyperchromia occurred mainly as a result of the diminution of the cytoplasm and a compacted distribution of free polysomes. These cells were usually surrounded by swollen astrocyte processes.
The dark shrunken neurons had reduced volume compared to normochromic neurons (Fig.   4). The nuclei of these cells were irregularly shaped with deep invaginations together with conglomerations of condensed chromatin, an increased number of interchromatin granules (both free and clustered) and an increased number of perichromatin granules. The nucleoli were small and had a poorly distinguishable ultrastructure. The cytoplasm was densely packed with dissociated polysomes and the lumina of the endoplasmic reticulum cisterns and the perinuclear space were slightly widened. Vacuoles also occurred in the cytoplasm due to the destruction of mitochondrial cristae.
In summary, 10 (Fig. 6). Aggregates of interchromatin and perichromatin granules were sharply reduced. Within the cytoplasm, the cisterns of the endoplasmic reticulum were only slightly expanded and the majority of free ribosomes were assembled into polysomes. Other neurons had normochromic nuclei. A distinctive featJre of these hyperchromic neurons was the presence of vacuoles in the cytoplasm due to swollen mitochondria with damaged cristae and increased cytoplasmic electron density (Fig. 7).
At 4 days post ENT transplant, the majority of neurons were pale with large nuclei and numerous invaginations (Fig. 8). The nuclei contained small blocks of condensed chromatin with few RNA particles and vacuoles of various sizes. In the cytoplasm of these cells were free polysomes, single endoplasmic reticulum cisterns, small mitochondria and occasional lysosomes. Bundles of filaments were observed in the bodies of astroglial cells in the host tissue surrounding the developing transplant (Fig. 9).
Seven days after ENT transplantation, no hy-     Semithin sections of rat brain sensorimotor cortex on the 10th day after nervous tissue aspiration and the 4th day after ENT transplantation. Note the presence of few hyperchromic and several light nerve cells (a). There are several hyperchromic neurons in the injured brain without a graft on the 14th day (b). 400 x.    perchromic cells were found in the surrounding tissue. On th contrary, most neurons had an electron-lucent karyoplasm, a small number of condensed chromatin blocks and a minimal number of RNA particles (Fig. 10). Vacuoles were also found in the nuclei. In the cytoplasm, free polysomes were not as prevalent as they were in pale cells at 4 days post implantation. The length of individual endoplasmic reticulum cisterns and their number were considerably larger, but there were no structural characteristics of neurons including the chromophilic substance. Consistently present in the cytoplasm were lysosomes, small lipid droplets, a large number of multivesicular bodies of different size and single swollen endoplasmic cisterns (Fig. 11). At this time, large numbers of fibrils were also detected in the bodies and processes of the host brain astrocyte cells.
At 10-15 days after ENT transplantation, host neurons exhibited a normal electron density. The nuclear ultrastructure of most nerve cells appeared normal. In the cytoplasm, there were numerous free polysomes, single endoplasmic reticulum cisterns with ribosomes, and randomly distributed mitochondria generally with a dense matrix. There were also normal synaptic contacts within the host tissue and nerve cells containing loci of clearing in which broken membrane remnants and amorphous material could be observed (Fig. 12). Processes and bodies of astrocytes with densely packed fibrillar material were also observed.
Thirty days after ENT transplantation, no hyperchromic neurons were observed in the host tissue. The majority of nerve cells had a normal structure with the exception of a few neurons which contained loci of clearing in the nuclei. These loci included a floccular material, single membranes and an occasional unidentified granular component (Fig. 13).

DISCUSSION
A developing fetal neocortical transplant placed into a previously prepared cavity in the brain of an adult animal inhibited destructive processes in host neurons surrounding the site of iniury. This was expressed primarily as a general reduction in the number of hyperchromic and shrunken host brain neurons and in the recovery of the ultrastructural properties of dystrophic cells which normally die through neurophagia or sclerosis in the absence of a transplant.
The present results on the recovery of the ultrastructure of shrunken neurons are in good agreement with previous reports of the positive effects of ENT transplanted into the CNS. The mechanism by which embryonic tissue exerts its protective effects is unclear but might relate to the production and release of trophic factors by the transplanted tissue. Following brain damage there is a gradient of trophic activity within the damaged region which stimulates the survival and growth of transplanted tissue/3,16/. Moreover, the exogenous administration of trophic factors promotes the survival of neurons and produces functional recovery. Nerve growth factor injected into the brain of aged rats not only minimized the age-related atrophy of cholinergic neurons but promoted recovery of cognitive performance in these same animals/6/. Accordingly, the present results may reflect the ability of specific embryonic trophic factors released by the ENT to influence the regenerative processes of damaged neurons/8,9/.
It is generally believed that shrunken nerve cells are irreversibly dystrophic and will ultimately die. However, the present study indicates that the cellular dystrophy which occurs following neural damage is indeed reversible. While regenerating neurons exhibited various structural abnormalities, including breakage of portions of the endoplasmic reticulum cisterns and mitochondria, and the appearance of lipid inclusion and vacuolization of nuclei, these    neurons were largely indistinguishable from, normal cells within 30 days. It appears, then, that under specific conditions (i.e., presence of developing fetal tissue), adult CNS tissue possesses a robust regenerative capacity.