Intracephalic transplants of freeze‐Stored rat hippocampal tissue

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Abstract
The survival and cellular and connective organization of intracephalic transplants of developing, freeze‐stored rat hippocampal tissue were examined. Blocks of tissue containing the hippocampus and fascia dentata were obtained from late embryonic (E16‐‐E22) and early postnatal rats (P0‐‐P4) and immersed in a tissue culture medium with 10% of the cryoprotective agent DMSO, frozen at a cooling rate of approximately 1°C/minute, and stored for 1‐‐226 days in liquid nitrogen. After quick thawing and washing out of the DMSO the tissue blocks were transplanted to the brain of adult rats. From 2 weeks to 3 months later the recipient brains were processed histologically. The cellular and connective organization of the transplants and their interaction with the host brains were analyzed after thionin cell staining, Timm's staining for hippocampal and dentate afferents, immunohistochemical staining for enkephalin‐, CCK‐, and somatostatin‐reactive neurons and afferents, AChE staining for cholinergic afferents, and silver stains for fiber architectonics and tracing of connections by anterograde axonal degeneration. Freeze‐storage narrowed the range of donor ages with good transplant survival. The best surviving hippocampal and dentate transplants thus came from 17‐‐21‐day‐old embryos. There was no correlation between the length of storage and survival. Structurally the transplants of stored tissue were more frequently fragmented than the transplants of fresh tissue when located outside the brain parenchyma in the brain ventricles. This was in accordance with the results of a previous study of grafts of freeze‐stored and fresh hippocampal tissue placed in the anterior eye chamber. Despite the decrease in survival and the tendency for fragmentation many well‐structured and organotypically organized hippocampal and dentate transplants were recovered corresponding to the donor ages E19‐‐E21. In addition to the main cell types (granule cells and pyramidal cells) the freeze‐stored transplants also contained peptidergic nerve cells reacting for CCK, somatostatin, and enkephalin. The organization of the intrinsic nerve connections and the exchange of connections with the host brain were similar for transplants of stored and fresh tissue. Besides the consistent innervation of the hippocampal and dentate transplants by host cholinergic afferents monitored by AChE staining, several appropriately located dentate transplants thus sent mossy fibers to the host CA3. Others received host perforant path projection. A CA3‐associated transplant projection to the denervated perforant path zones in the host fascia dentata was also observed. From this and the previous study of intraocular grafting of freeze‐stored immature brain tissue we conclude that cryopreservation is a feasible method that increases both the practical and the experimental potentials of transplantation of CNS tissue.