Intraocular grafts of fresh and freeze‐stored rat hippocampal tissue: A comparison of survivability and histological and connective organization

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Abstract
Intraocular grafts of rat hippocampal tissue, grafted either directly from the immature donor brains (fresh) or after storage in liquid nitrogen at –196°C (freeze‐stored), were compared with regard to survivability and histological and connective organization. For direct grafting, pieces of hippocampal tissue from rat embryos (embryonic day 19, E21) and newborn rats (PO) were placed in the anterior eye chamber of adult rats immediately after dissection. For grafting after deep‐freeze storage, pieces of hippocampal tissue were taken from rat embryos (E16–E21) and newborn rats (PO), frozen at a cooling rate of 1°C/min in CO2 or N2 vapours after addition of the cryoprotective agent dimethylsolfoxide (DMSO), and stored in liquid nitrogen for 1 to 33 days before thawing and intraocular grafting. From 20 to 68 days after grafting, the recipient rats were sacrificed, their eyes sectioned, and the sections stained with thionine for cell bodies, Timm's sulphide silver method for hippocampal fiber systems and terminal fields, and acetylcholinesterase (AChE) for cholinergic fibers and AChE‐positive neurons. When examining the 101 grafted eyes (34 grafted with fresh and 67 with freeze‐stored tissue) a significantly lower survival rate of the freeze‐stored tissue was found (28 vs. 88%). The survivability of the freeze‐stored tissue was age‐dependent with no survival at donor ages E16 and PO, while tissue from E18–E21 had a 50% survival rate. The grafts of the freeze‐stored tissue were also smaller and showed an increased tendency for fragmentation. When evaluating the structure of the grafts, the deep‐freeze storage was found primarily to have been harmful to the dentate granule cells and their precursors. The organization of intrinsic fiber connections followed the pattern known from lesion and intracerebral transplant studies. While demonstrating that immature brain tissue can survive deep‐freeze storage and subsequent intraocular grafting, the study also indicates that different schemes may have to be used to get optimal survival of different neuronal populations.