Research reportApoptotic morphology of dentate gyrus granule cells following experimental cortical impact injury in rats: possible role in spatial memory deficits
Introduction
Traumatic brain injury (TBI) in humans produces both retrograde (memory of events preceding the trauma) and anterograde (memory of posttraumatic events) amnesia 4, 47. Retrograde amnesia is graded such that recent memories are lost more easily than remote memories. Anterograde amnesia can sometimes be ungraded and extensive depending on the severity of the injury. Unfortunately, no effective therapies for human head injury are available. Animal models for TBI have been developed in an attempt to unravel the cellular and biochemical mechanisms underlying memory deficits in human head injury 17, 24, 39. Cortical impact injury, a widely employed model of TBI, produces spatial learning and memory deficits in rats, similar to those seen in human traumatic brain injury (TBI) 14, 16, 26, 29, 35, 40, 48. It is thought that hippocampal cell death may contribute to these deficits 26, 32, 37. Neuronal loss following TBI can be broadly divided into three categories: (1) cell death due to physical damage as a result of trauma, (2) necrotic cell death by release of excessive excitatory neurotransmitter such as glutamate, and (3) delayed cell death. Using a modified silver impregnation technique, we have identified delayed cell loss in the dentate gyrus, CA1, CA3 and hilar regions of the hippocampus in injured animals [12]. Preliminary light microscope examination suggested that these dystrophic cells may be dying by apoptosis.
Apoptosis is a form of active cell death process occurring during nervous system maturation 28, 30. It is a physiological process of cell elimination, during which, in contrast to necrosis, there is no induction of an inflammatory response 18, 21. The morphological determinants of apoptosis begin with cell shrinkage, condensation of the chromatin, segmentation of the nucleus, and fragmentation of the chromosomal DNA 2, 19, 20, 31, 54. The cell plasma membrane then becomes convoluted, cytoplasmic vacuolization occurs, and the cellular fragments are packaged into membrane enclosed vesicles. These vesicles, known as apoptotic bodies, express surface markers that cause them to be phagocytosed by neighboring cells 21, 54. During apoptosis, intracellular organelles such as mitochondria and rough endoplasmic reticulum (RER) remain intact 1, 13, 41, 50, 51. In comparison to necrosis, apoptosis appears to be a slow process [5]and often depends on gene expression and protein synthesis 15, 44. Recent in vitro studies have shown that neurons can be induced to undergo apoptosis by treatment with the β amyloid peptide as well as with intracellular calcium altering treatments such as glutamate 5, 15, 36. Although controversial, the role of apoptosis in neuropathological conditions is beginning to emerge. For example, DNA fragmentation has been reported to accompany neuronal death in models of ischemia 9, 10, 34, 38, 42, 43. However, neither apoptotic cell death nor its role in memory dysfunction has been examined following TBI.
Apoptosis and necrosis appear to be a continuum of cell death processes 1, 11. Many treatments that induce apoptosis at low levels can cause necrosis at higher concentrations 5, 11. For example, exposure of cortical neurons to low concentrations of NMDA induces apoptotic cell death 1, 5. In contrast, intense expose to high concentrations of NMDA induces necrotic cell damage 5, 11. These data suggest that the intensity and duration of insult may direct the ensuing pathways towards either necrotic or apoptotic neuronal death [11]. In addition, there are findings that suggest that intermediate levels of insults can produce both necrotic and apoptotic features 9, 27.
In this report, we have investigated the morphology of dentate gyrus granule cells following controlled cortical impact injury in rats. These neurons possess the morphological features of apoptosis. DNA laddering and in situ terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL-staining) were also consistent with apoptosis. However, some of these dystrophic cells showed the presence of swollen mitochondria and a lack of well defined rough endoplasmic reticulum, morphological features usually associated with necrosis.
Section snippets
Production of cortical impact brain injury
Lateral controlled cortical impact injury in rats was produced essentially as described previously 14, 40. All protocols were in compliance with NIH's Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. Cortically impacted rats received a single impact at 6 m/s, 2.5 mm deformation. Sham rats underwent identical surgical procedures but were not impacted. Core body temperature was monitored continuously by a rectal thermistor probe and
Light microscope analysis of dentate gyrus granule cell neurons
To look for delayed cell death in the hippocampus, tissue from 24 h (n = 6) postinjury animals, as well as from naive (n = 4) and sham (n = 4) injured animals, were first examined using several staining techniques. Tissue sections 40 μm thick were examined spanning the entire hippocampus. Fig. 1A shows a cresyl violet-stained section obtained from a sham animal and Fig. 1B from an injured animal. At this 24 h time point, no overt loss of hippocampal neuronal cells can be detected. Moreover, no
Discussion
In this study, we have examined the morphological features of dentate gyrus granule cells following controlled cortical impact brain injury. The dystrophic cells, as identified by silver impregnation, cresyl violet, and Hoechst staining, appear shrunken, and have condensed nuclei and chromatin. EM microscope examination showed apoptotic morphology including the association of condensed chromatin to the nuclear membrane and segmentation of nuclei. DNA fragmentation was demonstrated by the
Acknowledgements
The authors would like to thank A. Moore, Dr. R. Hayes and Dr. C.E. Dixon for their comments on the manuscript, Dr. D. Marshak for his help with the interpretation of the EM figures, and Dr. S. Liu and Dr. J. Bao for performing the animal injury. This work was funded by an ARP grant from the Texas Higher Education Board, a fellowship from the Klingenstein Foundation and MH49962.
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