Elsevier

Brain Research

Volume 917, Issue 1, 26 October 2001, Pages 45-54
Brain Research

Research report
Neuronal injury and loss after traumatic brain injury: time course and regional variability

https://doi.org/10.1016/S0006-8993(01)02905-5Get rights and content

Abstract

We have examined regional neuronal injury after traumatic brain injury using Fluoro-Jade, an acidic dye that exhibits a marked affinity for both the degenerating neuronal cell body and its processes and have determined the extent to which early injury corresponds to regional patterns of neuronal loss. Rats (n=45) were subjected to lateral fluid percussion brain injury and euthanized at 3 h to 28 days post injury. Complementary Fluoro-Jade, silver impregnation methods and TUNEL were used to assess neuronal injury. Neuronal loss was evaluated in sections immunostained for NeuN, a neuronal specific nuclear protein. Overt neuronal cell loss was evident by 7 days post injury in the cortex, hippocampus and thalamus. Injured neurons were apparent in the ipsilateral cortex bordering the impact site, hippocampus (CA1 and dentate), thalamus, and vermis of the cerebellum as early as 3 h post injury. Degenerating neurons were maximal by 1 and 3 days in the cortex and hippocampus, by 3 and 7 days in the cerebellum, and by 7 days in the thalamus. The regional distribution of Fluoro-Jade-labeled neurons corresponded to a similar pattern of silver and TUNEL staining. Together, these findings demonstrate a regionally specific temporal pattern of neuronal injury that results in overt neuronal cell loss within both cortical and subcortical regions.

Introduction

Fluid percussion injury has been shown to reproduce the behavioral and pathophysiological effects of traumatic brain injury observed in human closed head injury. The development of neurological changes accompanying traumatic brain injury is related to a number of pathophysiological events including direct mechanical damage, intraparenchymal and subarachnoid hemorrhage, breakdown of the blood–brain barrier, excitotoxicity, ischemia, and target deprivation.

Recent evidence demonstrates that both acute and delayed neuronal injury occurs after traumatic brain injury [2], [3], [4], [7]. As early as 10 min after injury, extensive neuronal injury, as defined with TUNEL (terminal deoxynucleotidyl transferase nick-end labeling) staining, has been observed in the cortex at the site of injury and in the underlying hippocampus. After several days, injured neurons are apparent in the lateral dorsal thalamus and cerebellum. The present study extends these observations by determining the extent to which progressive neuronal injury results in regionally distinct patterns of neuronal cell loss and by characterizing the degenerative events that occur both in the neuronal cell body as well as in processes.

Over the past several years a number of elegant anatomical approaches have been used to demonstrate apoptotic and necrotic neuronal cell death and to define cytoskeletal derangements in neuronal somata, dendrites and axons after traumatic brain injury [4], [15], [20], [21], [22], [28]. What remains unclear is the extent to which these pathological events collectively contribute to regional patterns of neuronal cell loss.

In this study, we have evaluated regional neuronal vulnerability, using Fluoro-Jade, an anionic fluorochrome that has been shown to be an effective marker of neuronal injury regardless of the nature of the insult [6], [8], [24], [25], [26]. Moreover, unlike TUNEL, which labels fragmented DNA [10], Fluoro-Jade delineates both the degenerating neuronal cell body as well as its processes. Thus, at any given time point, Fluoro-Jade offers a comprehensive approach to evaluating regional neuronal vulnerability by offering a ‘snapshot’ of cortical and subcortical neuronal injury and defining the patterns of degeneration within both the cell body and processes of a given neuron. In this study we have examined both neuronal cell injury and neuronal loss in order to determine the extent to which neuronal cell injury delineates regional patterns of cortical and subcortical neuronal loss.

Section snippets

Surgical procedures

Adult, male Sprague–Dawley rats (n=45, 350–450 g; Simonsen Laboratories, Gilroy, CA) were provided with ad libitum food and water and maintained on a 12-h light/dark cycle. Each animal was anesthetized with 4% chloral hydrate (9 ml/kg, intraperitoneally) and prepared for fluid percussion injury. A catheter was inserted into the right femoral artery to monitor blood pressure and analyze blood gases. After incision of the temporal muscle, a circular craniectomy, 4 mm in diameter, was performed

Physiological parameters

Physiological measures were determined immediately before injury. The mean arterial blood pressure (MABP) was 74±10 mmHg, the PaO2 was 78±12 mmHg, and the PaCO2 was 52±3 mmHg. There were no significant differences in blood gases before injury as compared to post injury values (PaO2=81±12, PaCO2=50±2 mmHg). There was a significant increase MABP (122±20 mmHg) immediately after injury as compared to preinjury values (P<0.05). This transient elevation of MABP was accompanied by apnea lasting

Discussion

A time course was established for regional neuronal degeneration following lateral fluid percussion injury. The most robust labeling of injured cells in the cortex and hippocampus occurred acutely within the first 3 days post injury. By 7 days post-injury the number of injured cells was markedly decreased, and was virtually undetectable by 28 days post injury. In contrast, a large delayed peak in the number of injured cells in the thalamus was observed at 7 days post injury. Purkinje cell

Acknowledgements

This research supported by NS14543 and the Lucille Packard Foundation for Children’s Health.

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