This manuscript provides a review of those factors involved in the pathogenesis of traumatically induced axonal injury in both animals and man. The review comments on the issue of primary versus secondary, or delayed, axotomy, pointing to the fact that in cases of experimental traumatic brain injury, secondary, or delayed, axotomy predominates. This review links the process of secondary axotomy to an impairment of axoplasmic transport which is initiated, depending upon the severity of the injury, by either focal cytoskeletal. misalignment or axolemmal permeability change with concomitant cytoskeletal. collapse. Data are provided to show that these focal axonal changes are related to the focal impairment of axoplasmic transport which, in turn, triggers the progression of reactive axonal change, leading to disconnection. In the context of experimental studies, evidence is also provided to explain the damaging consequences of diffuse axonal injury. The implications of diffuse axonal injury and its attendant deafferentation are considered by noting that with mild injury such deafferentation may lead to an adaptive neuroplastic recovery, whereas in more severe injury a disordered and/or maladaptive neuroplastic re-organization occurs, consistent with the enduring morbidity associated with severe injury. In closing, the review focuses on the implications of the findings made in experimental animals for our understanding of those events ongoing in traumatically brain-injured humans. It is noted that the findings made in experimental animals have been confirmed, in large part, in humans, suggesting the relevance of animal models for continued study of human traumatically induced axonal injury.

Recent studies have suggested that severe forms of traumatic brain injury (TBI) can be associated with direct alterations of the axolemma. The present study evaluated whether injuries of mild to moderate severity are associated with comparable change. To this end, we used extracellular horseradish peroxidase (HRP) to determine if altered axolemmal permeability occurred following the traumatic event. Adult cats received intrathecal infusions of peroxidase and then were prepared for mild to moderate fluid percussion injury. At intervals ranging from 5 min to 3 h, animals were perfused with aldehydes and prepared for the histochemical visualization of the peroxidase, in addition to the immunocytochemical visualization of the neurofilament 68 kD subunit, a long recognized marker of reactive axonal change. The histochemically and immunocytochemically prepared tissue was examined at both the light and electron microscopic level. With mild TBI, the injured animals displayed a repertoire of neurofilament misalignment and axonal swelling consistent with that previously described in our laboratories, yet these changes were not associated with the passage of peroxidase from the extracellular to the intraaxonal compartment. With moderate injury, on the other hand, focal axolemmal permeability change to the extracellularly confined peroxidase was recognized. This peroxidase passage was associated with local mitochondrial abnormalities in addition to an increased packing of the neurofilaments. Over a 3 h course, these neurofilaments began to disassemble, showing a delayed progression of reactive axonal change. Collectively, the results of this investigation suggest that traumatically induced axonal injury involves complex subsets of pathobiology, one evoking rapid primary neurofilamentous change and misalignment, the other eliciting altered membrane permeability concomitant with rapid neurofilament compaction, leading to a delayed progression of reactive axonal change.

Diffuse axonal injury (DAI) is observed commonly in traumatically brain injured humans. However, traditional histologic methods have proven of limited use in identifying reactive axonal change early (< 12 h) in the posttraumatic course. Recently, we have reported, in both humans and animals, that antibodies targeting neurofilament subunits are useful in the light microscopic recognition of early reactive change. In the present study, we extend our previous efforts in humans by analyzing the progression of traumatic brain injury (TBI)-induced axonal change at the ultrastructural level. This effort was initiated to follow the subcellular progression of reactive axonal change in humans and to determine whether this progression parallels that described in animals. Two commercially prepared antibodies were used to recognize reactive axonal change in patients surviving from 6 to 88 h. The NR4 antibody was used to target the light neurofilament subunit (NF-L), and the SMI32 antibody was used to target the heavy neurofilament subunit (NF-H). Plastic-embedded tissue sections were screened for evidence of reactive axonal change, and once identified, this reactive change was analyzed at the ultrastructural level. At 6 h survival, focally enlarged, immunoreactive axons with axolemmal infolding or disordered neurofilaments were seen within fields of axons exhibiting no apparent abnormality. By 12 h, some axons exhibited continued neurofilamentous misalignment, pronounced immunoreactivity, vacuolization, and, occasionally, disconnection. At later stages, specifically 30 and 60 h survival, further accumulation of neurofilaments and organelles had led to the further expansion of the axis cylinder, and clearly disconnected reactive swellings were recognized. These contained a dense core of disordered immunoreactive neurofilaments partially encompassed by a cap of less densely aggregated organelles. At 88 h, the reactive axons were larger and elongated, consistent with the continued delivery of organelles by axoplasmic transport. At the later time points, considerable heterogeneity was observed, with focally enlarged disconnected axons being observed in relation to axons showing less advanced reactive change. Our findings suggest that neurofilamentous disruption is a pivotal event in axonal injury.

No abstract

The appearance of superoxide anion radical in cerebral extracellular space during and after acute hypertension induced by intravenous norepinephrine was investigated in anesthetized cats equipped with cranial windows. Superoxide was detected by demonstrating the presence of superoxide dismutase-inhibitable reduction of nitroblue tetrazolium. The superoxide dismutase-inhibitable rate of reduction of nitroblue tetrazolium was 4.1 +/- 1.61 nM/min per cm2 during hypertension and 4.55 +/- 0.62 nM/min per cm2 one hour after hypertension had subsided. During norepinephrine administration in the absence of hypertension, the superoxide dismutase-inhibitable rate of reduction of nitroblue tetrazolium was 0.44 +/- 0.17 nM/min per cm2. The reduction of nitroblue tetrazolium during hypertension was also inhibited by prior treatment of the brain surface with phenylglyoxal at pH 10, to induce irreversible inhibition of the anion channel. The results show that acute hypertension is associated with the generation of superoxide which enters the extracellular space of the brain via the anion channel. Following hypertension, the sustained vasodilation caused by acute hypertension was inhibited significantly by topical application of superoxide dismutase and catalase, showing that it was due in part to superoxide and other radicals derived from it. The vasodilator response of cerebral arterioles to topical acetylcholine was converted to vasoconstriction following acute hypertension, and restored to vasodilation following topical application of superoxide dismutase and catalase. The results show that superoxide and other radicals generated after acute hypertension interfere with acetylcholine-induced endothelium-dependent vasodilation, probably because they destroy the endothelium-derived relaxant factor.

SUMMARY. Topical application of sodium arachidonate (50-200 /ig/ml) or bradykinin (0.1-10 fjg/ml) on the brain surface of anesthetized cats caused dose-dependent cerebral arteriolar dilation. This dilation was blocked by 67-100% in the presence of superoxide dismutase and catalase. These enzymes did not affect the changes in arteriolar diameter caused by alterations in arterial blood Pco 2/ or the arteriolar dilation from topical acetylcholine. Enzymes inactivated by heat had no effect on the vasodilation from arachidonate or bradykinin. Superoxide dismutase alone or catalase alone reduced the dilation during application of 200 Mg/ml of arachidonate for 15 minutes; they also completely prevented the residual dilation seen 1 hour after washout, as well as the reduction in the vasoconstrictive effects of arterial hypocapnia observed at this time. The results show that superoxide anion radical and hydrogen peroxide, or radicals derived from them, such as the hydroxyl radical, are mediators of the cerebral arteriolar dilation from sodium arachidonate or bradykinin. These radicals are not the endothelium-derived relaxant factor released by acetylcholine. The presence of both superoxide anionradical and hydrogen peroxide is required for the production of the vascular damage seen during prolonged application of high concentrations of sodium arachidonate. (Circ Res 55: 295-303, 1984)

Traumatic brain injury in animals and humans is well known to cause axonal damage diffusely scattered throughout the brain without evidence of other brain parenchymal change. This observation has prompted some to posit that such damaged axons are well positioned to mount a regenerative attempt. The present study uses an immunocytochemical marker specific for regenerating neurites to explore this issue. Further, in an attempt to expedite and enhance any potential regenerative effort, this study evaluates the efficacy of intrathecally applied nerve growth factor. Three sets of experiments were performed in adult cats. One group of animals was subjected to moderate fluid percussion brain injury and followed for 7 or 14 days post injury, with the continuous intraventricular infusion of nerve growth factor delivered by implanted osmotic pumps. These animals were compared to a second group of time-matched, sham-operated animals receiving artificial cerebrospinal fluid infusion. To assess axonal damage immunohistochemical staining for the low molecular weight neurofilament subunit (NF-L) was carried out using an NR4 monoclonal antibody. To localize axons exhibiting a regenerative response immunohistochemical staining for the growth associated protein GAP43 was employed. In sham controls, at the light microscopic level NF-L-immunoreactive axonal swellings were numerous at 7 days, but by 14 days post injury their frequency declined markedly. In contrast, GAP43-immunoreactive, disconnected reactive axonal swellings were rarely observed at 7 days but were numerous at 14 days. Ultrastructural analysis at 14 days post injury of carefully matched sections revealed reactive axons demonstrating sprouting consistent with a regenerative effort. Analysis of tissue from animals of 14 days of survival indicated that supplementation with nerve growth factor did not appear to enhance the capacity of damaged brain axons to mount a regenerative attempt. Rather, it appears that regenerative efforts seen reflect a spontaneous response. A third group of adult cats, subjected to the same injury but not subjected to osmotic pump implantation, was allowed to survive for 22-28 days. Animals in this group also demonstrated GAP43 immunoreactivity in reactive axonal swellings in the brain stem. This study demonstrates that diffusely injured axons can mount a sustained regenerative attempt that is associated with a reorganization of their cytoskeleton and accompanied by an up-regulation of growth-associated proteins.

Xanthine oxidase and xanthine, a combination that produces hydrogen peroxide and superoxide anion radical, applied topically in anesthetized cats equipped with cranial windows caused arteriolar dilation during application, sustained dilation 1 h after washout, and reduced reactivity to the vasoconstrictive effects of arterial hypocapnia, discrete lesions of the endothelium, and morphological abnormalities of the vascular smooth muscle by electron microscopy. Similar effects were seen in small, but not in large, arterioles during topical application of hydrogen peroxide or hydrogen peroxide plus ferrous sulfate, a combination that produces free hydroxyl radical. The functional changes caused by xanthine oxidase plus xanthine were inhibited completely by superoxide dismutase plus catalase. Superoxide dismutase or catalase, each by itself, eliminated the residual effects seen after washout and reduced the dilation during application of xanthine oxidase. The results show that superoxide anion radical and hydrogen peroxide produce reversible arteriolar dilation and that consistent vascular damage is produced in the presence of both superoxide anion radical and hydrogen peroxide.