John T. Povlishock

Axonal Change in Minor Head Injury

Anterograde axonal transport of horseradish peroxidase (HRP) in selected cerebral and cerebellar efferents was studied in cats subjected to minor head injury. After trauma, the animals were allowed to survive from one to 24 hours, when they were perfused with aldehydes and processed for the light and electron microscopic visualization of the peroxidase reaction product. By light microscopy, the brain injury elicited an initial intra-axonal peroxidase pooling. With longer post-traumatic survival, HRP pooling increased in size, demonstrated frequent tabulation, and ultimately formed large ball- or club-like swellings which suggested frank axonal separation from the distal axonal segment. Ultrastructural examination revealed that the initial intra-axonal peroxidase pooling was associated with organelle accumulation which occurred without any other form of axonal change or related parenchymal or vascular damage. This accumulation of organelles increased with time and was associated with conspicuous axonal swelling. Ultimately these organelle-laden swellings lost continuity with the distal axonal segment and the axonal swelling was either completely invested by a thin myelin sheath or protruded without myelin investment into the brain parenchyma. This study suggests that axonal change is a consistent feature of minor head injury. Since these axonal changes occurred without any evidence of focal parenchymal or vascular damage, minor brain injury may ultimately disrupt axons without physically shearing or tearing them.

All roads lead to disconnection? - Traumatic axonal injury revisited

SummaryTraumatic brain injury (TBI) evokes widespread/diffuse axonal injury (TAI) significantly contributing to its morbidity and mortality. While classic theories suggest that traumatically injured axons are mechanically torn at the moment of injury, studies in the last two decades have not supported this premise in the majority of injured axons. Rather, current thought considers TAI a progressive process evoked by the tensile forces of injury, gradually evolving from focal axonal alteration to ultimate disconnection. Recent observations have demonstrated that traumatically induced focal axolemmal permeability leads to local influx of Ca2+ with the subsequent activation of the cysteine proteases, calpain and caspase, that then play a pivotal role in the ensuing pathogenesis of TAI via proteolytic digestion of brain spectrin, a major constituent of the subaxolemmal cytoskeletal network, the “membrane skeleton”. In this pathological progression this local Ca2+ overloading with the activation of calpains also initiates mitochondrial injury that results in the release of cytochrome-c, with the activation of caspase. Both the activated calpain and caspases then participate in the degradation of the local axonal cytoskeleton causing local axonal failure and disconnection. In this review, we summarize contemporary thought on the pathogenesis of TAI, while discussing the potential diversity of pathological processes observed within various injured fiber types. The anterograde and retrograde consequences of TAI are also considered together with a discussion of various experimental therapeutic approaches capable of attenuating TAI.

Engaging neuroscience to advance translational research in brain barrier biology

The delivery of many potentially therapeutic and diagnostic compounds to specific areas of the brain is restricted by brain barriers, of which the most well known are the blood–brain barrier (BBB) and the blood–cerebrospinal fluid (CSF) barrier. Recent studies have shown numerous additional roles of these barriers, including an involvement in neurodevelopment, in the control of cerebral blood flow, and — when barrier integrity is impaired — in the pathology of many common CNS disorders such as Alzheimers disease, Parkinsons disease and stroke.

Functional, morphological, and metabolic abnormalities of the cerebral microcirculation after concussive brain injury in cats.

We induced experimental concussive brain injury by a fluid percussion device in anesthetized cats equipped with a cranial window for the observation of the pial microcirculation of the parietal cortex. Brain injury resulted in transient but pronounced increases in arterial blood pressure and in sustained arteriolar vasodilation associated with reduced or absent responsiveness to the vasoconstrictor effect of arterial hypocapnia and with reduced or absent ability of the vessels to undergo autoregulatory vasodilation in response to reductions in arterial blood pressure. Such vessels had reduced resting oxygen consumption in vitro. Electron microscopic examination of the same vessels that were studied physiologically disclosed the presence of discrete endothelial lesions consisting of either vacuolization or crater formation. Occasionally there was extensive destruction and necrosis of the endothelial cells. There was little or no morphological evidence of vascular smooth muscle damage. There was a close association between the presence of endothelial lesions and vessel dilation and unresponsiveness, suggesting a causal relationship. In cats in which the transient posttraumatic hypertensive episode was prevented, the vessels retained their normal caliber, remained normally responsive, and had no endothelial lesions. The results show that the vascular lesions in the pial microcirculation following this type of brain injury are due to the rise in arterial pressure. Circ Res 46: 37-47, 1980

Increased vulnerability of the midly traumatized rat brain to cerebral ischemia: the use of controlled secondary ischemia as a research tool to identify common or different mechanisms contributing to mechanical and ischemic brain injury

Abstract Fasted Wistar rats were subjected to either a mild mechanical injury, 6 min of transient forebrain ischemia, or a mild mechanical injury followed 1 h later by 6 min of forebrain ischemia. EEG and evoked potentials were assessed intermittently and morphological analyses were performed after 7 das postinjury survival. In all groups complete qualitative recovery of electrical activity and general behavior was observed with 7-day survival. However, rats subjected to combined concussion and ischemia displayed EEG spike activity and a delayed return of EEG and evoked potentials during acute recovery not evident in other groups. No overt neuronal cells loss was seen in trauma alone and was minimal or absent in ischemia alone. However, extensive bilateral CA1 and subicular pyramidal cell loss was found in the septal and mid-dorsal hippocampi in the combined trauma and ischemia group. In contrast, no overt axonal injury was found in any group. We conclude that even mild mechanical injury can potentiate selective ischemic hippocampal neuronal necrosis in the absence of overt axonal injury. This potentiation also occurs in conjunction with more generalized electrophysiological disturbances such as EEG evidence of postischemic neuronal hyperactivity suggesting that mild concussion may also decrease the threshold for post-ischemic neuronal excitation. These results suggest the potential of this model for examining common or different injury mechanisms in mechanical and ischemic brain injury.

Appearance of superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats.

When increased prostaglandin synthesis was induced in anesthetized cats equipped with cranial windows by topical application of arachidonate (200 μg/ml) or bradykinin (20 μg/ml), there was reduction of nitroblue terrazolium, resulting in deposition of the reduced insoluble form of this dye on the brain surface. The amount of reduced nitroblue terrazolium deposited on the brain surface was measured spectrophotometrically after fixation of the brain by perfusion with aldehydes to eliminate interference from hemoglobin. Topical application of 56 U/ml superoxide dismutase or 20 μg/ml indomethatin inhibited nitroblue terrazolium reduction by 76.5%–82.5% and by 78%–85.5%, respectively. These results show that most of the nitroblue terrazolium reduction was accounted for by superoxide anion radical generated in the course of arachidonate metabolism via the cyclooxygenase pathway. No superoxide production could be detected in the absence of arachidonate or bradykinin. Histological examination showed no evidence of parenchymal cellular damage or vascular damage and no accumulation of leukocytes. Pronounced leukocyte accumulation occurred 24 hours after topical arachidonate in rabbits with chronically implanted cranial windows. Superoxide appearance was reduced severely by 4, 4′-diisothiocyano-2, 2′-stilbene disulfonate and phenylglyoxal, two specific inhibitors of the anion channel. The most likely explanation for these findings is that increased metabolism of exogenous or endogenous arachidonate via cyclooxygenase results in the appearance of superoxide anion radical in cerebral extracellular space. Superoxide crosses the membrane of undamaged cells via the anion channel.

Cytochrome c release and caspase activation in traumatic axonal injury.

Axonal injury is a feature of traumatic brain injury (TBI) contributing to both morbidity and mortality. The traumatic axon injury (TAI) results from focal perturbations of the axolemma, allowing for calcium influx triggering local intraaxonal cytoskeletal and mitochondrial damage. This mitochondrial damage has been posited to cause local bioenergetic failure, leading to axonal failure and disconnection; however, this mitochondrial damage may also lead to the release of cytochrome c (cyto-c), which then activates caspases with significant adverse intraaxonal consequences. In the current communication, we examine this possibility. Rats were subjected to TBI, perfused with aldehydes at 15–360 min after injury, and processed for light microscopic (LM) and electron microscopic (EM) single-labeling immunohistochemistry to detect extramitochondrially localized cytochrome c (cyto-c) and the signature protein of caspase-3 activation (120 kDa breakdown product of α-spectrin) in TAI. Combinations of double-labeling fluorescent immunohistochemistry (D-FIHC) were also used to demonstrate colocalization of calpain activation with cyto-c release and caspase-3-induction. In foci of TAI qualitative–quantitative LM demonstrated a parallel, significant increase in cyto-c release and caspase-3 activation over time after injury. EM analysis demonstrated that cyto-c and caspase-3 immunoreactivity were associated with mitochondrial swelling–disruption in sites of TAI. Furthermore, D-IFHC revealed a colocalization of calpain activation, cyto-c release, and caspase-3 induction in these foci, which also revealed progressive TAI. The results demonstrate that cyto-c and caspase-3 participate in the terminal processes of TAI. This suggests that those factors that play a role in the apoptosis in the neuronal soma are also major contributors to the demise of the axonal appendage.

An Intrathecal Bolus of Cyclosporin a before Injury Preserves Mitochondrial Integrity and Attenuates Axonal Disruption in Traumatic Brain Injury

Traumatic brain injury evokes multiple axonal pathologies that contribute to the ultimate disconnection of injured axons. In severe traumatic brain injury, the axolemma is perturbed focally, presumably allowing for the influx of Ca2+ and initiation of Ca2+-sensitive, proaxotomy processes. Mitochondria in foci of axolemmal failure may act as Ca2+ sinks that sequester Ca2+ to preserve low cytoplasmic calcium concentrations. This Ca2+ load within mitochondria, however, may cause colloid osmotic swelling and loss of function by a Ca2+-induced opening of the permeability transition pore. Local failure of mitochondria, in turn, can decrease production of high-energy phosphates necessary to maintain membrane pumps and restore ionic balance in foci of axolemmal permeability change. The authors evaluated the ability of the permeability transition pore inhibitor cyclosporin A (CsA) to prevent mitochondrial swelling in injured axonal segments demonstrating altered axolemmal permeability after impact acceleration injury in rat. At the electron microscopic level, statistically fewer abnormal mitochondria were seen in traumatically injured axons from CsA-pretreated injured animals. Further, this mitochondrial protection translated into axonal protection in a second group of injured rats, whose brains were reacted with antibodies against amyloid precursor protein, a known marker of injured axons. Pretreatment with CsA significantly reduced the number of axons undergoing delayed axotomy, as evidenced by a decrease in the density of amyloid precursor protein-immunoreactive axons. Collectively, these studies demonstrate that CsA protects both mitochondria and the related axonal shaft, suggesting that this agent may be of therapeutic use in traumatic brain injury.

Characterization of a distinct set of intra-axonal ultrastructural changes associated with traumatically induced alteration in axolemmal permeability

It has recently been demonstrated [Pettus et al., J. Neurotrauma, 11 (1994) 507-522] that moderate traumatic brain injury evokes alterations in axolemmal permeability associated with rapid local compaction of axonal neurofilaments (NF). The current communication fully characterized these local NF changes, while also exploring the possibility of other related cytoskeletal abnormalities. A tracer normally excluded by the intact axolemma (horseradish peroxidase) was administered intrathecally in cats, which were then subjected to moderate/severe fluid percussion brain injury (FPI). After survival times ranging from 5 min to 6 h post-traumatic brain injury (TBI), the animals were perfused and processed for light microscopic (LM) and electron microscopic (EM) visualization of horseradish peroxidase (HRP). HRP-containing axons identified by LM, were investigated by EM in both the sagittal and coronal planes. Electron micrographs were videographically captured, digitized, and analyzed for cytoskeletal distribution. Local alterations in axolemmal permeability to HRP were detected, and consistently linked with distinct cytoskeletal changes. Within 5 min of injury, the injured HRP-containing axons displayed a significant decrease in inter-NF spacing associated with a lack of NF side arm projections. Density analysis proved a significant increase in NF packing in the HRP-containing axons, and further revealed an associated significant decrease in microtubule (MT) density. All ultrastructural changes were seen within 5 min of injury, and persisted unchanged for up to 6 h post-TBI. Collectively, these abnormalities suggest that altered axolemmal permeability triggers a rapid, yet persisting general cytoskeletal change most likely linked to local ionic disregulation. We posit that this local cytoskeletal collapse/alteration marks a site of impaired axonal transport, associated with upstream axoplasmic swelling and eventual axonal detachment.

Pathobiology of traumatically induced axonal injury in animals and man

STUDY OBJECTIVES Although diffuse axonal injury is recognized as a consistent feature of traumatic brain injury, there is confusion regarding its pathogenesis. To provide insight into its pathogenesis, animal models of traumatic brain injury complemented by post mortem human analyses were used. DESIGN In animals, anterograde tracers together with antibodies targeting the neurofilament subunits were used in light and electron microscopic analyses of axonal injury. In humans, antibodies to the neurofilament subunits also were used to follow diffuse axonal injury. Animals were followed from minutes to months after injury, whereas humans were studied from six hours to 59 days after injury. MEASUREMENTS AND MAIN RESULTS In neither animals nor humans did traumatic brain injury cause direct axonal tearing. Instead, the traumatic brain injury triggered focal intra-axonal change in the 68-kd neurofilament subunit, which became disordered in its alignment and resulted in impaired axoplasmic transport. This caused axonal swelling and disconnection. The sequence of axonal change was similar in animals and man; however, its temporal progression was slower in humans. CONCLUSION Traumatically induced axonal damage is triggered first by focal intra-axonal change involving the neurofilament subunits. This neurofilament change is due to either direct mechanical failure of the axonal cytoskeleton or the initiation of a biochemical event that causes neurofilament disassembly. In general, the temporal progression of the intra-axonal changes that lead to ultimate disconnection is influenced by the severity of the traumatic injury and the species evaluated.