Experimental brain injury induces regionally distinct apoptosis during the acute and delayed post-traumatic period

Abstract

The temporal pattern of apoptosis in the adult rat brain after lateral fluid-percussion (FP) brain injury was characterized using terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labeling (TUNEL) histochemistry and agarose gel electrophoresis. Male Sprague Dawley rats were subjected to brain injury and killed for histological analysis at intervals from 12 hr to 2 months after injury (n = 3/time point). Sham (uninjured) controls were subjected to anesthesia with (n = 3) or without (n = 3) surgery. Apoptotic TUNEL-positive cells were defined using stringent morphological criteria including nuclear shrinkage and fragmentation and condensation of chromatin and cytoplasm. Double-labeled immunocytochemistry was performed to identify TUNEL-positive neurons (anti-neurofilament monoclonal antibody RM044), astrocytes (anti-glial fibrillary acidic protein polyclonal antibody), and oligodendrocytes (anti-cyclic nucleotide phosphohydrolase polyclonal antibody). Compared with that seen with sham controls, in the injured cortex, significant apoptosis occurred at 24 hr (65 +/- 19 cells; p < 0.05) with a second, more pronounced response at 1 week after injury (91 +/- 24 cells; p < 0.05). The number of apoptotic cells in the white matter was increased as early as 12 hr after injury and peaked by 1 week (33 +/- 6 cells; p < 0.05). An increase in apoptotic cells was observed in the hippocampus at 48 hr (13 +/- 8), whereas in the thalamus, the apoptotic response was delayed, peaking at 2 weeks after injury (151 +/- 71 cells; p < 0.05). By 2 months, the number of apoptotic cells in most regions had returned to uninjured levels. At 24 hr after injury, TUNEL-labeled neurons and oligodendrocytes were localized primarily to injured cortex. By 1 week after injury, populations of TUNEL-labeled astrocytes and oligodendrocytes were present in the injured cortex, while double-labeled neurons were present predominantly in injured cortex and thalamus, with a few scattered in the hippocampus. DNA agarose gels confirmed morphological identification of apoptosis. These data suggest that the apoptotic response to trauma is regionally distinct and may be involved in both acute and delayed patterns of cell death.

Fig. 1.

Photomicrographs illustrating type II apoptotic cells (A, B) and type I nonapoptotic cells (C, D) in the rat brain after lateral FP brain injury of moderate severity. Sections were counterstained with hematoxylin. A, B, Apoptotic cells were identified based on the rounded, shrunken nature of the cytoplasm and nucleus and on the intense staining of the nucleus. Note the similar intense TUNEL reactivity and morphological characteristics in apoptotic cells in the injured cortex at 24 hr after injury (A) and in injured thalamus at 2 weeks after injury (B). C,D, Type I nonapoptotic cells were characterized by diffuse TUNEL reactivity, uneven staining in the cytoplasm, and a lack of shrunken cytoplasm. C illustrates a nonapoptotic cell in the cortex at 12 hr after injury, whereas Dillustrates a typical type I TUNEL-stained cell (white arrow) in the thalamus at 1 week after injury. Note the neuron-like appearance and cytoplasmic TUNEL stain of cells inD and a relatively normal-appearing neuron stained with hematoxylin (D, black arrow). Scale bar, 50 μm.

Fig. 2.

Schematic of TUNEL-stained sections of the rat brain 24 hr, 1 week, and 2 weeks after injury illustrating the regional distribution of apoptotic cells. Initially, apoptotic cells were observed primarily in the injured cortex at 24 hr after injury and over time were detected in internal structures, such as the thalamus and hippocampus. Each filled circle (•) represents five TUNEL-positive cells exhibiting apoptotic morphology.

Fig. 3.

Quantitation of TUNEL-positive cells exhibiting apoptotic morphology in the rat brain after lateral FP injury of moderate severity (2.4–2.6 atm) or sham surgery (n= 3/time point). A, Cortex. Average number of apoptotic cells in the cortex peaked at 24 hr, with a second, delayed peak at 1 week after injury. B, White matter. Average number of apoptotic cells in the subcortical white matter, comprising the internal and external capsule, fimbria, corpus callosum, and cingulum, peaked at 1 week after injury. C, Hippocampus. Average number of apoptotic cells in the hippocampus, comprising the CA1–CA3, dentate hilus, and gyrus regions, peaked at 48 hr after injury.D, Thalamus. Average number of apoptotic cells in the thalamus peaked at 2 weeks after injury. By 2 months, apoptotic cells in all regions was similar to that found in sham control brains.Open bars represent the ipsilateral hemisphere;shaded bars represent the contralateral hemisphere; *p < 0.05 when compared with sham. Average number of cells was determined as described in Materials and Methods. Error bars represent SEM.

Fig. 4.

Representative photomicrographs illustrating TUNEL-labeled neurons (A, B), astrocytes (C), and oligodendrocytes (D) in the injured cortex. Neurons were observed at 24 hr (A) and 1 week (B) after injury, whereas GFAP-positive astrocytes were observed predominantly at 1 week (C) after injury. CNPase-labeled oligodendrocytes (D) were observed at 24 hr after injury. Arrows indicate TUNEL-positive nuclei, and arrowheads indicate staining in cell bodies. Tissue sections were subjected to the TUNEL procedure followed by immunohistochemistry as described in Materials and Methods. Scale bar, 50 μm.

Fig. 5.

Representation of internucleosomal DNA fragmentation at 30 min (lane 3), 2 hr (lane 4), 15 hr (lane 5), 24 hr (lane 6), and 1 week (lane 7) after injury in the cortex (A) and hippocampus (B). DNA fragments in 180–200 bp intervals were detected in injured tissue but not in sham tissue (lane 2). Molecular weight standards are shown in lane 1 of each gel with molecular weight markers illustrated.

Fig. 6.

Quantitation of TUNEL-positive, nonapoptotic cells in the rat brain after lateral FP injury of moderate severity. Nonapoptotic cells were identified based on diffuse, uneven TUNEL staining in the nucleus and cytoplasm with little to no cellular shrinkage. A, Cortex. Average number of nonapoptotic cells peaked at 24 hr after injury. B, White matter. Average number of nonapoptotic cells peaked between 24 and 48 hr after injury. C, Hippocampus. Average number of TUNEL-positive, nonapoptotic cells in the injured hippocampus increased slightly (nonsignificantly) at 12 and 48 hr after injury.D, Thalamus. Average number of TUNEL-positive, nonapoptotic cells in the injured thalamus revealed a biphasic response, with a significant peak at 12 hr and a second broader, nonsignificant peak between 48 hr and 1 month after injury. By 2 months after injury, the number of nonapoptotic TUNEL-positive cells had reached sham levels in all regions. * p < 0.05 when compared with sham. Average number of cells was determined as described in Materials and Methods. Error bars represent SEM.