Comparison of brain cell death and inflammatory reaction in three models of intracerebral hemorrhage in adult rats

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Abstract

Intracerebral hemorrhage (ICH) is associated with stroke and head trauma. Different experimental models are used, but it is unclear to what extent the tissue responses are comparable. The purpose of this study was to compare the temporal responses to brain hemorrhages created by injection of autologous whole blood, collagenase digestion of blood vessels, and avulsion of cerebral blood vessels. Adult rats were subjected to ICH. Rats were perfusion fixed with paraformaldehyde l hour to 28 days later. Hematoxylin and eosin, Fluoro-Jade, immunohistochemical, and TUNEL staining were used to allow quantification of damaged and dying neurons, neutrophils, CD8α immunoreactive lymphocytes, and RCA-1 positive microglia/macrophages, adjacent to the hemorrhagic lesion. In all models, eosinophilic neurons peaked between 2 and 3 days. TUNEL positive cells were observed maximal at 2 days in blood injection model, 3 days in vessel avulsion model, between 1 and 7 days in the collagenase injection model, and were evident in small quantities in 21 to 28 days in 3 models. Neutrophils appeared briefly from 1 to 3 days in all models, but they were substantially lower in the cortical vessel avulsion model, perhaps owing to the devitalized nature of the tissue. Influx of CD8α immunoreactive lymphocytes were maximal at 2 to 3 days in the autologous injection model, 3 to 7 days in other 2 models, and persisted for 21 to 28 days in all models. The microglial/macrophage reaction peaked between 2 and 3 days in the blood injection model and at 3 to 7 days in other 2 models, and persisted for weeks in all groups. These results suggest that different models of ICH are associated with similar temporal patterns of cell death and inflammation. However, the relative magnitude of these changes differs.

Section snippets

Animal preparation

All experimental procedures were done in accordance with the guidelines of the Canadian Council on Animal Care. Protocols were approved by the local experimental ethics committee.

Autologous whole blood-induced ICH

Twenty-five young adult male Sprague-Dawley rats weighing between 175 and 250 grams were used. Each rat was anesthetized intraperitoneally with pentobarbital (50 mg/kg) and placed in a stereotactic frame (David Kopf Instruments, Tujunga, CA). A midline scalp incision was made and a hole was drilled in the skull (3 mm

Results

All rats tolerated the surgical procedure well, and there was no surgical mortality. In all groups, the damaged brain cells surrounding the hematoma appeared pale on H&E stain because of edema and/or necrosis. Following autologus whole blood injection, the irregular hematoma core was located in striatum (Fig 1a), although there was extension into the adjacent white matter in approximately 25% of the rats. Following collagenase injection, the hematoma core was more spherical; it was

Discussion

ICH causes brain damage through multiple mechanisms. Direct tissue destruction by the hemorrhagic event and dissection of blood along tissue planes occurs immediately. This is followed by development of edema and secondary ischemic damage caused by raised intracranial pressure and distortion of the microvasculature. Delayed damage could result through a variety of mechanisms, including local ischemia, the release of toxins by blood breakdown products, thrombin release, or inflammatory cell

Acknowledgements

We thank Dr. Huijin Yan for assistance with the collagenase animal model.

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    Supported by grants from the Heart and Stroke Foundation of Manitoba, Manitoba Health Research Council, and Children’s Hospital Foundation to M.R.D. He also holds the Manitoba Medical Services Foundation Clinical Research Professorship. M.X. has a Doctoral Research Award from the Canadian Institutes of Health Research/Heart and Stroke Foundation of Canada.

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