Neuronal and glial apoptosis after traumatic spinal cord injury

Abstract

Cell death was examined by studying the spinal cords of rats subjected to traumatic insults of mild to moderate severity. Within minutes after mild weight drop impact (a 10 gm weight falling 6.25 mm), neurons in the immediate impact area showed a loss of cytoplasmic Nissl substances. Over the next 7 d, this lesion area expanded and cavitated. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive neurons were noted primarily restricted to the gross lesion area 4-24 hr after injury, with a maximum presence at 8 hr after injury. TUNEL-positive glia were present at all stages studied between 4 hr and 14 d, with a maximum presence within the lesion area 24 hr after injury. However 7 d after injury, a second wave of TUNEL-positive glial cells was noted in the white matter peripheral to the lesion and extending at least several millimeters away from the lesion center. The suggestion of apoptosis was supported by electron microscopy, as well as by nuclear staining with Hoechst 33342 dye, and by examination of DNA prepared from the lesion site. Furthermore, repeated intraperitoneal injections of cycloheximide, beginning immediately after a 12.5 mm weight drop insult, produced a substantial reduction in histological evidence of cord damage and in motor dysfunction assessed 4 weeks later. Present data support the hypothesis that apoptosis dependent on active protein synthesis contributes to the neuronal and glial cell death, as well as to the neurological dysfunction, induced by mild-to-moderate severity traumatic insults to the rat spinal cord.

Fig. 1.

Schematic drawings showing longitudinal (coronal) sections through the central canal in animals receiving the 6.25 mm impact injury and being perfused at 5 min, 4, 8, and 24 hr, and 3 d (A); 7 d (B); and 14 and 30 d (C). Each contour is the average from four animals. Progressive expansion of the lesion was seen, initially defined by the disappearance of Nissl substance from neurons (5 min–4 hr), later defined by the breaking down of axonal segments and myelin as well as the invasion of blood cells into white matter, and still later defined by gross cavitation (7–30 d,cross-hatched areas). By 14 d, the lesion consisted entirely of a cavity and was somewhat smaller than the lesion defined at 3 d; this cavity was somewhat increased by 30 d.WM, White matter; GM, gray matter; *Site of impact. Scale bar, 1 mm.

Fig. 2.

TUNEL-labeled (A–F) and Hoechst 33342-stained (G–I) cells in the spinal cord after a 6.25 mm injury. A, Longitudinal section of the cord showing numerous TUNEL-positive cells (arrowheads) present within the injury area 8 hr after injury. B, Higher magnification of demarcated region in A showing two TUNEL-labeled neurons with chromatin condensation. C, Section showing TUNEL-labeled neuron) (arrowhead) that can be distinguished from labeled glial cells (thin arrows) by its relatively larger nucleus, its prominent cytoplasm, and the pericellular space created by its shrinkage (thick arrows). The labeled nucleus of this neuron is smaller than the nuclei of neighboring morphologically normal neurons (D). E, Section showing breakdown of the nucleus of a glial cell in the white matter near the injury epicenter into several fragments. F, Section showing TUNEL-labeled glial cell, presumably an oligodendrocyte (thick arrow), in the peripheral white matter away from the injury epicenter. Note the close association of the cell with the space (asterisks) likely created by a degenerated axon. A morphologically normal oligodendrocyte nucleus (thin arrow) and a degenerating axonal segment (arrowhead) are also marked for comparison. G–I, Hoechst 33342-stained sections. These show nuclear fragmentation of a probable neuron (H, arrow) and a glial cell (I, arrow) in the spinal cord 24 hr after injury. The nuclear labeling of a morphologically normal neuron (G, arrow) is presented for comparison.WM, White matter; GM, gray matter. Scale bars: A, 50 μm; B–F, 10 μm; G–I, 10 μm.

Fig. 3.

Double staining of cells in the spinal cord after a 6.25 mm injury. TUNEL-positive cells were also positive for neuronal-specific enolase or rip. A, Section from rat euthanized 8 hr after injury showing two shrunken neurons within the lesion area that were labeled simultaneously by TUNEL (brown) and neuronal-specific enolase immunohistochemistry (blue gray). B, Two other neurons in the same section used in A but outside the lesion area that were labeled for neuronal-specific enolase but were TUNEL negative. C, Section from rat euthanized 7 d after injury showing representative immunofluorescence micrograph showing a spinal cord oligodendrocyte cell body that was located in white matter labeled for TUNEL (yellow) as well as rip (green). Scale bars: A,B, 10 μm; C, 10 μm.

Fig. 4.

Schematic drawing of TUNEL-stained longitudinal (coronal) sections of the spinal cord through the ventral horns, after a 6.25 mm weight drop insult and euthanization of the rats at the indicated times after injury. Five minutes after insult, no TUNEL-labeled cells were observed. By 8 hr, many TUNEL-positive neurons (large dots) and glial cells (small dots) were observed mostly within the lesion area (defined by loss of Nissl substance from neurons). By 24 hr, TUNEL-positive neurons were no longer found, but many TUNEL-positive glial cells were seen within the injury area. By 7 d, a second wave of TUNEL-positive glial cells was observed mostly outside of the lesion area in the lateral funiculus extending the entire length of the section (1.5 cm). By 14 d, fewer TUNEL-labeled glia were found. WM, White matter;GM, gray matter; *Site of impact. Scale bar, 1 mm.

Fig. 5.

Electron micrograph showing a representative apoptotic cell and a representative necrotic cell in the gray matter of the cord 8 hr after injury. A, Apoptosis. The nucleus has fragmented into several membrane-bounded, highly condensed bodies (thick arrow), and the cell body has shrunk with an intact, infolded cell membrane (arrowheads).B, Necrosis. The nucleus (Nu) is swollen (arrowhead) with scattered granular aggregations of chromatin (thick arrows), and the cell is swollen with dilation of rough endoplasmic reticulum (thin arrows) and mitochondria (asterisks). RBC, Red blood cell. Scale bars, 1 μm.

Fig. 6.

Ethidium bromide-stained agarose gel showing a DNA ladder from a rat spinal cord after a weight drop insult. Data are from rats euthanized: in Lane 1, after no injury (normal control); in lane 2, 4 hr after injury; inlane 3, 8 hr after injury; and in lane 4, 24 hr after injury. Molecular weight markers are shown to theleft of lane 1. DNA laddering is apparent in lane 4 (arrows).

Fig. 7.

Enrichment of mononucleosomes and oligonucleosomes in the cytoplasmic fraction after a 6.25 mm injury (enrichment factor, absorbance of the injured tissue/absorbance of the normal control tissue). An ELISA kit (see Materials and Methods) was used to detect mononucleosomes and oligonucleosomes in the cytoplasmic fraction of spinal cord tissue at the indicated times after injury. Evidence of internucleosomal DNA fragmentation peaked 24 hr after injury and was reduced by cycloheximide treatment. Error bars indicate SEM; *p < 0.05; n = 3 animals per group.

Fig. 8.

Hematoxylin- and eosin-stained horizontal cross-sections taken 4 weeks after injury in rats treated with either vehicle (left) or cycloheximide (right).B, E, Sections through the lesion epicenter (0). A, D, Sections 750 μm rostral to the epicenter (−750 μm). C,F, Sections 750 μm caudal to the epicenter (+750 μm). Scale bar, 100 μm.

Fig. 9.

Quantitation of histological sparing produced by cycloheximide in animals 4 weeks after a 12.5 mm weight drop insult. The rim of nearly normal tissue (some vacuoles can be seen) was measured in both vehicle- and cycloheximide-treated animals. The hypercellular core in cycloheximide-treated animals was considered part of the lesion area and thus not counted as spared tissue. A beneficial effect of cycloheximide on tissue sparing was seen at all levels examined. Error bars indicate SEM; *p < 0.05;n = 12 animals per group.

Fig. 10.

Hematoxylin- and eosin-stained transverse right hemisection at the lesion epicenter in a rat treated with cycloheximide. A, Section showing a wide rim of spared cord tissue (arrow). In the central region, hypercelluar, vascularized tissue with many macrophage-like cells is seen. B, Higher magnification of demarcated region inA showing a border between the peripheral rim of spared cord tissue and this central hypercellular tissue (dotted line). BV, Blood vessel.Arrowheads indicate probable macrophages. Scale bars, 100 μm.

Fig. 11.

Long-term beneficial effect of cycloheximide on the hindlimb neurological function of rats after a 12.5 mm weight drop injury, assessed by the BBB Locomotor Rating Scale. Error bars indicate SEM; *p < 0.05; n = 12 animals per group.