Shereen D. Farber

Chapter 13 Neural transplantation of horseradish peroxidase-labeled hippocampal cell suspensions in an experimental model of cerebral ischemia

Publisher Summary One of the major obstacles in assessing the use of neuronal transplants to replace cells that are lost because of ischemia is the inability to identify and distinguish homotypically-transplanted cells from similar cells in the host brain. This chapter discusses the use of horseradish peroxidase (HRP) to label cell suspensions of fetal rodent hippocampus that were injected into the hippocampi of post-ischemic rats to determine whether homotypically transplanted cells could be distinguished from those of the host. When homotypic cells are to be transplanted and are labeled with HRP, a label is required, which might help in distinguishing cells of transplant origin from those of the host. One factor influencing the transplant survival appears to be the placement of the cells within the hippocampus. Whether the minor differences in histocompatibility between the donor tissue and host in combination with the cytotoxicity factors influence cellular survival is an issue that remains to be resolved. The chapter suggests that neural transplantation may be feasible for treatment for focal lesions produced during ischemia. Sufficient cell growth and development that occurred in the four vessel occlusion (VO) animals, justify further transplantation experiments in this model.

Distribution of thyrotropin-releasing hormone (TRH) in the hippocampal formation as determined by radioimmunoassay

The distribution of thyrotropin-releasing hormone (TRH) in the hippocampal formation was determined using a radioimmunoassay (RIA) specific for TRH. RIA of hippocampal subregions revealed that the CA3 region of the hippocampal formation contained the highest amount of TRH, followed by intermediate levels in region CA1 and the dentate gyrus. The hilus and subiculum contained the lowest levels. The issue of whether hippocampal TRH is derived from extrinsic and/or intrinsic sources was evaluated by making lesions of the major subcortical afferent to the hippocampus, the fornix pathway. Analysis of the hippocampal formation by RIA revealed that the ventral hippocampus contains higher levels of TRH than the dorsal hippocampus (6.01 +/- 0.62 pg/mg tissue weight vs 1.11 +/- 0.19 pg/mg tissue weight). Lesions of the fornix produced significant decreases in ventral TRH to 52.9% of its control level and in dorsal TRH to 28.8% of its control level. The results from these studies suggest that (1) there is a differential distribution of TRH in the hippocampal formation, (2) the hippocampal formation might be composed of extrinsic and intrinsic sources of TRH, and (3) extrinsic sources of TRH might enter the hippocampus via the fornix pathway. In addition (4) the greater post-lesion decrement in ventral vs dorsal hippocampal TRH suggests that TRH fibers traversing the fornix innervate the ventral hippocampal formation in preference to its dorsal counterpart.

Evidence for Extrinsic and Intrinsic Sources of Thyrotropin‐Releasing Hormone (TRH) in the Hippocampal Formation as Determined by Radioimmunoassay and Immunocytochemistry

The hippocampal formation is a region of brain that undergoes long-term enhancement of thyrotropin-releasing hormone (TRH) levels after seizure-induced activation.I4 At present, it is not known whether TRH found within the hippocampal formation is derived from intrinsic and/or extrinsic sources. To determine whether there is an extrinsic component to hippocampal TRH, the two major afferent inputs to the hippocampus, the fornix pathway and the perforant path, were aspirated. Hippocampal TRH was quantified by a specific TRH radioimmunoassay (RIA). Immunocytochemical methods were used to determine the location of intrinsic hippocampal TRH.

Oxygen affinity of hemoglobin and peripheral nerve degeneration in experimental diabetes

Peripheral neuropathy remains a major complication of diabetes. Numerous etiological theories of metabolic and/or vascular disturbances have been suggested including decreased endoneurial oxygen tension with presumed tissue hypoxia. Increases in the affinity of hemoglobin for oxygen (Hb-O2 affinity) may also produce tissue hypoxia and such Hb-O2 affinity changes have been implicated in the pathogenesis of diabetic microangiopathy. In order to test whether affinity hypoxia might contribute to the development of diabetic peripheral neuropathy, we have utilized a rat model of high and normal Hb-O2 affinity produced by backcrossing animals with increased and decreased levels of 2,3-diphosphoglycerate (DPG). Diabetes was induced in ten high and ten low DPG animals with a tail vein injection of 55 mg/kg streptozotocin (STZ). Five animals in each group were treated with 2.4 U protamine zinc insulin (PZI)/day while the remaining animals were untreated. All rats were killed after 30 days, sections of tibial and sural nerve were rapidly removed and processed for teased fiber analysis. A minimum of 125 axons were assessed per nerve for E degeneration (myelin ovoids) using the classification developed by Dyck et al. Untreated animals, regardless of DPG levels, demonstrated 0% neuropathy. In contrast, all insulin-treated animals showed degeneration (0.4-17%) that inversely correlated with the DPG level (r = -0.59, P less than 0.04). The results of this study suggest that the level of RBC DPG (and presumably the Hb-O2 affinity) with its attendant effect on tissue oxygen release may play a role in the development of peripheral neuropathy in STZ-induced diabetic rats treated with insulin.