Cyclin-dependent kinase 5 in axon growth and regeneration.

Injury to the central nervous system often leads to irreversible deficits because of the failure of damaged axons to regrow and restore the functional neural circuitry. Coordinated orchestration of multiple cellular processes including cytoskeletal dynamics and gene expression are essential for both developmental and regenerative axon growth. Recently, mounting evidence suggests that cyclin-dependent kinase 5 (Cdk5), a neuronal kinase implicated in almost all aspects of brain development and function, regulates multiple players required for axon formation and regeneration. Indeed, Cdk5 functions as a "plastic" kinase that maintains the axon growth ability by enabling efficient cytoskeletal reorganization, enhancing protein translation, reducing protein degradation, and promoting injury-induced gene transcription. Here, we summarize the up-to-date information on the mechanisms underlying the axon growth and regeneration after injury.

Neuron-specific enolase and S100BB as outcome predictors in severe diffuse axonal injury.

BACKGROUND: Diffuse axonal injury (DAI) is a common type of traumatic brain injury, mostly associated with mild changes on computed tomography (CT) scan. Serum biomarkers might be used in the diagnosis and prognosis of this injury type. Our purpose was to determine temporal profile and predictive values of serum concentrations of protein S100BB and neuron-specific enolase (NSE) after DAI. METHODS: Twenty-eight isolated severe DAI patients (Glasgow Coma Scale score ≤ 8) with normal CT were enrolled in the study. Serum levels of S100BB and NSE were determined at 6 hours, 24 hours, 48 hours, and 72 hours after injury, using enzyme-linked immunosorbent assay. Clinical outcome variables of DAI comprised survival at discharge and Glasgow Outcome scale (GOS) after 3 months and also 2 years. RESULTS: S100BB concentration was maximum in 6 hours after injury (median = 280.75 ng/L) followed by a quick drop. Its value was significantly higher on third day in patients with unfavorable outcome (GOS score = 1-3) versus favorable outcome (GOS score = 4, 5) (p < 0.0001). The values of NSE had mild changes during 3 days; however, these measured values at 72 hours after trauma manifested higher in unfavorable outcome (p < 0.05). CONCLUSIONS: Increased serum concentrations of NSE and S100BB within first 3 days after DAI are associated with poor outcome despite mild CT findings. S100BB level at 72 hours after injury can predict late outcome in DAI patients. LEVEL OF EVIDENCE: Prognostic study, level III.

Diffuse traumatic axonal injury in the mouse induces atrophy, c-Jun activation, and axonal outgrowth in the axotomized neuronal population.

Traumatic axonal injury (TAI) is a consistent component of traumatic brain injury (TBI) and is associated with much of its morbidity. Little is known regarding the long-term retrograde neuronal consequences of TAI and/or the potential that TAI could lead to anterograde axonal reorganization and repair. To investigate the repertoire of anterograde and retrograde responses triggered by TIA, Thy1-YFP-H mice were subjected to mild central fluid percussion injury and killed at various times between 15 min and 28 d post-injury. Based upon confocal assessment of the endogenous neuronal fluorescence, such injury was found to result in diffuse TAI throughout layer V of the neocortex within yellow fluorescent protein (YFP)-positive axons. When these fluorescent approaches were coupled with various quantitative and immunohistochemical approaches, we found that this TAI did not result in neuronal death over the 28 d period assessed. Rather, it elicited neuronal atrophy. Within these same axotomized neuronal populations, TAI was also found to induce an early and sustained activation of the transcription factors c-Jun and ATF-3 (activating transcription factor 3), known regulators of axon regeneration. Parallel ultrastructural studies confirmed that these reactive changes are consistent with atrophy in the absence of neuronal death. Concurrent with those events ongoing in the neuronal cell bodies, their downstream axonal segments revealed, as early as 1 d post-injury, morphological changes consistent with reactive sprouting that was accompanied by significant axonal elongation over time. Collectively, these TAI-linked events are consistent with sustained neuronal recovery, an activation of a regenerative genetic program, and subsequent axonal reorganization suggestive of some form of regenerative response.

Diffuse brain injury elevates tonic glutamate levels and potassium-evoked glutamate release in discrete brain regions at two days post-injury: an enzyme-based microelectrode array study.

Traumatic brain injury (TBI) survivors often suffer from a wide range of post-traumatic deficits, including impairments in behavioral, cognitive, and motor function. Regulation of glutamate signaling is vital for proper neuronal excitation in the central nervous system. Without proper regulation, increases in extracellular glutamate can contribute to the pathophysiology and neurological dysfunction seen in TBI. In the present studies, enzyme-based microelectrode arrays (MEAs) that selectively measure extracellular glutamate at 2 Hz enabled the examination of tonic glutamate levels and potassium chloride (KCl)-evoked glutamate release in the prefrontal cortex, dentate gyrus, and striatum of adult male rats 2 days after mild or moderate midline fluid percussion brain injury. Moderate brain injury significantly increased tonic extracellular glutamate levels by 256% in the dentate gyrus and 178% in the dorsal striatum. In the dorsal striatum, mild brain injury significantly increased tonic glutamate levels by 200%. Tonic glutamate levels were significantly correlated with injury severity in the dentate gyrus and striatum. The amplitudes of KCl-evoked glutamate release were increased significantly only in the striatum after moderate injury, with a 249% increase seen in the dorsal striatum. Thus, with the MEAs, we measured discrete regional changes in both tonic and KCl-evoked glutamate signaling, which were dependent on injury severity. Future studies may reveal the specific mechanisms responsible for glutamate dysregulation in the post-traumatic period, and may provide novel therapeutic means to improve outcomes after TBI.

Proximity of lesioning determines response of facial motoneurons to peripheral axotomy.

We recently found that rubrospinal (RS) neurons, which typify central neurons projecting within the central nervous system (CNS), exhibited different neuronal and glial reactions to axotomy at proximal as opposed to distal sites. To determine whether distance also determines the reaction to axonal injury of central neurons projecting to the periphery, we studied the temporal expression of four free-radical-related enzymes as well as the severity of cell loss, perineuronal astrocytic and microglial reactions, and degeneration of the proximal central axons of facial motoneurons after axotomies performed at various sites on the brainstem surface and in the stylomastoid foramen, respectively. Distal lesions resulted in upregulation of these neurons' expression of nitric oxide synthase (NOS) and persistent downregulation of their expression of the NOS-activating enzyme calcineurin. It also led to transient upregulation of their expression of manganese-dependent superoxide dismutase (Mn-SOD), and resulted in a mild neuronal loss. Proximal axotomy led to an upregulation of NOS but a transient downregulation in the expression of calcineurin and Mn-SOD at 4 weeks after injury. This was accompanied by severe cell loss and swelling of mitochondria at 2-4 weeks postinjury. However, neither proximal nor distal axonal lesioning led to nuclear fragmentation or TUNEL staining of neurons. Proximal as opposed to distal axotomy produced an earlier transformation of glial morphology, including the hypertrophy of astrocytic processes and metamorphosis of ramified microglia to amoeboid cells. We unexpectedly found that unlike RS neurons, whose central axons degenerated slowly and in an anterograde manner only after the severe cell loss induced by proximal axotomy, the central axons of facial motoneurons degenerated rapidly and in a retrograde manner independently of the severity of loss of these neurons after axotomy. However, degeneration began sooner after proximal than after distal axotomy. Since the central axons of both rubrospinal neurons and facial motoneurons lie within the CNS, the differences in whether and how they degenerated after axotomy suggests that central neurons that project within and outside the CNS are inherently different. The significance of these and also the free radical environment regulation differences between these two types of neurons following close and distant axotomies remains to be explored.

Inhibition of cyclooxygenase 2 by nimesulide improves cognitive outcome more than motor outcome following diffuse traumatic brain injury in rats.

Prostanoid synthesis is regulated by the enzyme cyclo-oxygenase (COX) that is present in at least two isoforms: COX-1, the constitutive form, and COX-2, the inducible form. Expression of COX-2 has recently been shown to be an important determinant of the cytotoxicity connected with inflammation following ischemic injury to the brain. The present study examines the temporal and spatial profiles of COX-2 expression following diffuse traumatic brain injury (TBI) in rats, and the effects of the COX-2 inhibitor nimesulide on cognitive and motor outcomes. Adult, male Sprague-Dawley rats were injured using the 2-meter impact acceleration model of diffuse TBI. At preselected time points after injury, animals were killed and the expression of COX-2 was measured in the hippocampus and parietal cortex by immunohistochemistry and Western blotting techniques. Effects of nimesulide (6 mg/kg daily over ten days) on cognitive and motor outcome was assessed in a separate group of animals using the Barnes circular maze and rotarod test, respectively. A highly significant up-regulation of COX-2 expression was found in the hippocampus as early as 3 h post-trauma and persisting for at least 12 days after TBI. In contrast, a slight but significant upregulation of COX-2 expression occurred in the cortex only at 3 days after trauma. Administration of the COX-2 inhibitor nimesulide resulted in a significant and substantial improvement in cognitive function compared to vehicle-treated controls, while motor deficits after injury was only improved at 24 h after injury. We conclude that COX-2 is involved in the development of functional deficits following diffuse TBI, particularly cognitive deficits, and that these can be improved by administration of COX-2 inhibitors.

Apoptotic change and NOS activity in the experimental animal diffuse axonal injury model.

Although nitric oxide (NO) plays an important role in the pathophysiological process of cerebral ischemia or severe traumatic brain injury, its contribution to the pathogenesis of moderate diffuse axonal injury (mDAI) remains to be clarified. The alterations in nitric oxide synthase (NOS) activity and the histopathological response after mDAI was investigated. Forty anesthetized Sprague-Dawley adult rats were injured with a Marmarou's weight-drop device through a Plexiglas guide tube. These rats were divided into 8 groups (control, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr, 24 hr, 48 hr after trauma). The temporal pattern of apoptosis in the adult rat brain after mDAI was characterized using TUNEL histochemistry. In addition, the cDNA for NOS activity was amplified using RT-PCR. The PCR products were electrophoresed on a 2% agarose gel. eNOS activity was not detected, but nNOS activity was expressed after 3 hr and continuously 48 hr after impact, which was approximately double that of the control group at 12 and 24 hr. Subsequently, there was a decrease in activity after 48 hr. The iNOS activity increased dramatically after 12 hr and was constant for a further 12 hr followed by a dramatic decrease below the level of the control group. Significant apoptotic changes occurred 12 and 24 hr. after insult. nNOS and iNOS activity were affected after moderate diffuse axonal injury in a time-dependent manner and there was a close relation between the apoptotic changes and NOS activity. Although the nNOS activity was expressed early, its activity was not stronger than iNOS, which was expressed later.

Apoptotic Change and NOS Activity in the Experimental Animal Dif fuse Axonal Injury Model

Although nitric oxide (NO) plays an important role in the pathophysiological process of cerebral ischemia or severe traumatic brain injury, its contribution to the pathogenesis of moderate diffuse axonal injury (mDAI) remains to be clarified. The alterations in nitric oxide synthase (NOS) activity and the histopathological response after mDAI was investigated. Forty anesthetized Sprague-Dawley adult rats were injured with a Marmarou's weight-drop device through a Plexiglas guide tube. These rats were divided into 8 groups (control, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr, 24 hr, 48 hr after trauma). The temporal pattern of apoptosis in the adult rat brain after mDAI was characterized using TUNEL histochemistry. In addition, the cDNA for NOS activity was amplified using RT-PCR. The PCR products were electrophoresed on a 2% agarose gel. eNOS activity was not detected, but nNOS activity was expressed after 3 hr and continuously 48 hr after impact, which was approximately double that of the control group at 12 and 24 hr. Subsequently, there was a decrease in activity after 48 hr. The iNOS activity increased dramatically after 12 hr and was constant for a further 12 hr followed by a dramatic decrease below the level of the control group. Significant apoptotic changes occurred 12 and 24 hr. after insult. nNOS and iNOS activity were affected after moderate diffuse axonal injury in a time-dependent manner and there was a close relation between the apoptotic changes and NOS activity. Although the nNOS activity was expressed early, its activity was not stronger th an iNOS, which was expressed later.

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 alpha-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.