Axonal cytoskeletal changes after non-disruptive axonal injury

In animal models of human diffuse axonal injury, axonal swellings leading to secondary axotomy occur between 2 and 6 h after injury. But, analysis of cytoskeletal changes associated with secondary axotomy has not been undertaken. We have carried out a quantitative analysis of cytoskeletal changes in a model of diffuse axonal injury 4 h after stretch-injury to adult guinea-pig optic nerves. The major site of axonal damage was the middle portion of the nerve. There was a statistically significant increase in the proportion of small axons with a diameter of 0.5 μm and smaller in which there was compaction of neurofilaments. Axons with a diameter greater than 2.0 μm demonstrated an increased spacing between cytoskeletal elements throughout the length of the nerve. However, in the middle segment of the nerve these larger axons demonstrated two different types of response. Either, where periaxonal spaces occurred, there was a reduction in axonal calibre, compaction of neurofilaments but no change in their number, and a loss of microtubules. Or, where intramyelinic spaces occurred there was an increased spacing between neurofilaments and microtubules with a significant loss in the number of both. Longitudinal sections showed foci of compaction of neurofilaments interspersed between regions where axonal structure was apparently normal. Neurofilament compaction was correlated with disruption of the axolemma at these foci present some hours after injury. We suggest that the time course of these axonal cytoskeletal changes after stretch-injury to central axons is shorter than those changes documented to occur during Wallerian degeneration.     

Temporal Profiles of Cytoskeletal Protein Loss following Traumatic Axonal Injury in Mice

To examine the time course and relative extent of proteolysis of neurofilament and tubulin proteins after traumatic axonal injury (TAI), anesthetized mice were subjected to optic nerve stretch injury. Immunohistochemistry confirmed neurofilament accumulation within axonal swellings at 4, 24, and 72 h postinjury (n = 4 injured and 2 sham per time point). Immunoblotting of optic nerve homogenates (n = 5 injured and 1 sham at 0.5, 4, 24 or 72 h) revealed calpain-mediated spectrin proteolytic fragments after injury. Protein levels for NF68 progressively decreased from 0.5 h to 24 h postinjury, while NF200 and α-tubulin levels decreased acutely (0.5–4 h), with a secondary decline at 72 h postinjury. These data demonstrate that diffusely distributed TAI is associated not only with a localized accumulation of neurofilament proteins, but also significant decreases in total cytoskeletal protein levels which may be mediated, in part, by calpains. Protection of the axonal cytoskeleton represents a potential therapeutic target for axonal damage associated with injury or neurodegenerative diseases. Special issue in honor of Naren Banik.     

Computational modeling of axonal microtubule bundles under tension

Microtubule bundles cross-linked by tau protein serve a variety of neurological functions including maintaining mechanical integrity of the axon, promoting axonal growth, and facilitating cargo transport. It has been observed that axonal damage in traumatic brain injury leads to bundle disorientation, loss of axonal viability, and cognitive impairment. This study investigates the initial mechanical response of axonal microtubule bundles under uniaxial tension using a discrete bead-spring representation. Mechanisms of failure due to traumatic stretch loading and their impact on the mechanical response and stability are also characterized. This study indicates that cross-linked axonal microtubule bundles in tension display stiffening behavior similar to a power-law relationship from nonaffine network deformations. Stretching of cross-links and microtubule bending were the primary deformation modes at low stresses. Microtubule stretch was negligible up to tensile stresses of ～1 MPa. Bundle failure occurred by failure of cross-links leading to pull-out of microtubules and loss of bundle integrity. This may explain the elongation, undulation, and delayed elasticity of axons following traumatic stretch loading. More extensively cross-linked bundles withstood higher tensile stresses before failing. The bundle mechanical behavior uncovered by these computational techniques should guide future experiments on stretch-injured axons.     

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons

Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (delta(psi)m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, delta(psi)m was measured using rhodamine-123, and ATP was quantified with a luciferin-luciferase assay. In astrocytes, delta(psi)m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, delta(psi)m declined at 15 min only in the severely stretched group. At 48 h postinjury, delta(psi)m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, delta(psi)m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24-48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.     

Changes in cytoskeletal protein synthesis following axon injury and during axon regeneration

Injury to the axons of facial motoneurons stimulates increases in the synthesis of actin, tubulins, and GAP-43, and decreases in the synthesis of neurofilament proteins: mRNA levels change correspondingly. In contrast to this robust response of peripheral neurons to axotomy, injured central nervous system neurons show either an attenuated response that is subsequently aborted (rubrospinal neurons) or overall decreases in cytoskeletal protein mRNA expression (corticospinal and retinal ganglion neurons). There is evidence that these changes in synthesis are regulated by a variety of factors, including loss of endoneurially or target-derived trophic factors, positive signals arising from the site of injury, changes in the intraaxonal turnover of proteins, and substitution of target-derived trophic support by factors produced by glial cells. It is concluded that there is, as yet, no coherent explanation for the upregulation or downregulation of any of the cytoskeletal proteins following axotomy or during regeneration. In considering the relevance of these changes in cytoskeletal protein synthesis to regeneration, it is emphasized that they are unlikely to be involved in the initial outgrowth of the injured axons, both because transit times between cell body and injury site are too long, and because sprouting can occur in isolated axons. Injuryinduced acceleration of the axonal transport of tubulin and actin in the proximal axon is likely to be more important in providing the cytoskeletal protein required for initial axonal outgrowth. Subsequently, the increased synthesis and transport velocity for actin and tubulin increase the delivery of these proteins to support the increased volume of the maturing regenerating axons. Reduction in neurofilament synthesis and changes in neurofilament phosphorylation may permit the increased transport velocity of the other cytoskeletal proteins. There is little direct evidence that alterations in cytoskeletal protein synthesis are necessary for successful regeneration, nor are they sufficient in the absence of a supportive environment. Nevertheless, the correlation that exists between a robust cell body response and successful regeneration suggests that an understanding of the regulation of cytoskeletal protein synthesis following axon injury must be a part of any successful strategy to improve the regenerative capacity of the central nervous system.     

Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions.

The mechanism(s) underlying eccentric damage to skeletal muscle cytoskeleton remain unclear. We examined the role of Ca(2+) influx and subsequent calpain activation in eccentric damage to cytoskeletal proteins. Eccentric muscle damage was induced by stretching isolated mouse muscles by 20% of the optimal length in a series of 10 tetani. Muscle force and immunostaining of the cytoskeletal proteins desmin, dystrophin, and titin were measured at 5, 15, 30, and 60 min after eccentric contractions and compared with the control group that was subjected to 10 isometric contractions. A Ca(2+)-free solution and leupeptin (100 microM), a calpain inhibitor, were applied to explore the role of Ca(2+) and calpain, respectively, in eccentric muscle damage. After eccentric contractions, decreases in desmin and dystrophin immunostaining were apparent after 5 min that accelerated over the next 60 min. Increased titin immunostaining, thought to indicate damage to titin, was evident 10 min after stretch, and fibronectin entry, indicating membrane disruption, was evident 20 min after stretch. These markers of damage also increased in a time-dependent manner. Muscle force was reduced immediately after stretch and continued to fall, reaching 56 +/- 2% after 60 min. Reducing extracellular calcium to zero or applying leupeptin minimized the changes in immunostaining of cytoskeletal proteins, reduced membrane disruption, and improved the tetanic force. These results suggest that the cytoskeletal damage and membrane disruption were mediated primarily by increased Ca(2+) influx into muscle cells and subsequent activation of calpain.     

Changes in Neurofilament and Microtubule Distribution following Focal Axon Compression

Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism.     

Mechanisms of inflammation in spinal cord injury

An increase in protease activity is a hallmark event of the secondary injury cascade following contusion SCI. Elevated levels of protease activity result in the degradation of cytoskeletal components and myelin proteins essential for cellular function and survival. We have shown that a member of the cathepsin protease family is affected by SCI. The excessive release and activity of cathepsin B, a fairly ubiquitous lysosomal cysteine protease, has been implicated in several pathologies including tumor metastasis and progression, arthritis and Alzheimer's disease. Thus, our goal was to characterize any SCI-induced changes in cathepsin B expression. Following a T12 laminectomy and a moderate contusion (NYU device), the gene and protein profiles of cathepsin B in rats were examined using real-time PCR and immunoblots, respectively. Both the contusion injured animals and the time-matched sham controls exhibited elevated pro-enzyme protein levels (37 kDa form) at the lesion site, with significant differences between the two groups at 48 h, 72 h and 7 days post-SCI. Furthermore, there was a surge in the active species of the protein with significant differences at 72 h and 7 days post-SCI for the 30 kDa form and at 48 h. and 7 days for the 25 kDa form. Real-time PCR revealed increases in cathepsin B mRNA levels following contusion SCI as early as 6 h postinjury. These data indicate that SCI causes an up-regulation of cathepsin gene expression and protein levels, and suggest that this protease may be involved in the secondary injury cascade perhaps for as long as 1 week postinjury.     

Poster Sessions CP12: Neurotrauma & Injury. Characterization of cathepsin B expression following contusion spinal cord injury (SCI)

An increase in protease activity is a hallmark event of the secondary injury cascade following contusion SCI. Elevated levels of protease activity result in the degradation of cytoskeletal components and myelin proteins essential for cellular function and survival. We have shown that a member of the cathepsin protease family is affected by SCI. The excessive release and activity of cathepsin B, a fairly ubiquitous lysosomal cysteine protease, has been implicated in several pathologies including tumor metastasis and progression, arthritis and Alzheimer's disease. Thus, our goal was to characterize any SCI‐induced changes in cathepsin B expression. Following a T12 laminectomy and a moderate contusion (NYU device), the gene and protein profiles of cathepsin B in rats were examined using real‐time PCR and immunoblots, respectively. Both the contusion injured animals and the time‐matched sham controls exhibited elevated pro‐enzyme protein levels (37 kDa form) at the lesion site, with significant differences between the two groups at 48 h, 72 h and 7 days post‐SCI. Furthermore, there was a surge in the active species of the protein with significant differences at 72 h and 7 days post‐SCI for the 30 kDa form and at 48 h. and 7 days for the 25 kDa form. Real‐time PCR revealed increases in cathepsin B mRNA levels following contusion SCI as early as 6 h postinjury. These data indicate that SCI causes an up‐regulation of cathepsin gene expression and protein levels, and suggest that this protease may be involved in the secondary injury cascade perhaps for as long as 1 week postinjury.     

Softness, strength and self-repair in intermediate filament networks

One cellular function of intermediate filaments is to provide cells with compliance to small deformations while strengthening them when large stresses are applied. How IFs accomplish this mechanical role is revealed by recent studies of the elastic properties of single IF protein polymers and by viscoelastic characterization of the networks they form. IFs are unique among cytoskeletal filaments in withstanding large deformations. Single filaments can stretch to more than 3 times their initial length before breaking, and gels of IF withstand strains greater than 100% without damage. Even after mechanical disruption of gels formed by crossbridged neurofilaments, the elastic modulus of these gels rapidly recovers under conditions where gels formed by actin filaments are irreversibly ruptured. The polyelectrolyte properties of IFs may enable crossbridging by multivalent counterions, but identifying the mechanisms by which IFs link into bundles and networks in vivo remains a challenge.     

Axonal damage, autophagy and neuronal survival

In recent years autophagy modulation has been shown to reduce or increase neuronal cell death in several models of neurodegeneration. How autophagy exerts these dual effects is currently unknown. Here we review recent evidence from our laboratory demonstrating that autophagy can protect the cell soma after axonal traumatic injury. Damage in the optic nerve induces retinal ganglion cell (RGC) death in glaucoma and other retinal diseases and is often modeled by axotomy of the optic nerve in laboratory animals. Using this well-known model of RGC degeneration we show that autophagy is strongly upregulated following the insult and before cell death. Enhancement of autophagy by pharmacological treatment with rapamycin decreases the number of degenerating neurons. Conversely, axotomy in Atg4B (-/-) mice increases the number of dying cells in the retinal ganglion cell layer. Similar findings were observed in Atg5 (flox/flox) mice following specific downregulation of the autophagy regulator ATG5 in RGCs, by intravitreal injection of a cre-expressing vector. Taken together, these findings point to a cytoprotective role of autophagy following axonal damage in vivo.     

The dynamics of axolemmal disruption in guinea pig spinal cord following compression

Membrane damage has been postulated as a critical factor in mediating axonal degeneration in brain and spinal cord trauma. Despite compelling evidence of membrane disruption as a result of physical insults in both in vivo and in vitro studies, the dynamics of such damage over the time post injury in in vivo studies has not been well documented. Using a well-characterized in vivo guinea pig spinal cord compression model and horseradish peroxidase exclusion assay, we have documented significant membrane disruption at 1 hr, 3 days, and 7 days following injury. Furthermore, the membrane damage was found to spread laterally 10 mm beyond the center of original compression site in both rostral and caudal directions. A second-degree polynomial fit of the measured data predicts a bilateral spread of approximately 20–21 mm of membrane disruption from the epicenter of injury over a period of about 20 days. Thus, this study shows that membrane damage exists days, and possibly weeks, after spinal cord trauma in live guinea pigs. This provides the evidence necessary to investigate the role of membrane damage in triggering axonal deterioration in the future. Furthermore, this study has also revealed a long therapeutical window for membrane repair and functional enhancement following traumatic injury in the central nervous system.     

Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits.

The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury (time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect (P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase (P = 0.015) and glutathione reductase (P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione (P = 0.008) and total glutathione (P = 0.015). Morphological analysis (hematoxylin and eosin staining) showed significant polymorphonuclear cell infiltration of the damaged region at 24 h postinjury. We conclude that acute mechanical muscle stretch injury results in increased free radical production within 24 h after injury. Antioxidant enzyme and glutathione systems also appear to be affected during this early postinjury period.     

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca²⁺) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase II (CaMKII), a target substrate for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon outgrowth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that the activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.     

Molecular regulation of Nogo-A in neural cells: Novel insights into structure and function

Nogo-A is part of the reticulon family of proteins localized to the myelin and oligodendroglial plasma membranes. Nogo-A specifically initiates signal transduction cascades limiting axonal regrowth following injury and disease in the adult mammalian central nervous system (CNS). Recent novel data support the contention that neuronal Nogo-A plays an important role in regulating cytoskeletal re-organization without the requirement of signaling through its cognate receptor (Nogo receptor). These data, along with the recent findings that the N-terminus of Nogo-A can interact with integrins and that NgR1 interacts with the amyloid precursor protein extracellularly, as well as novel findings showing ubiquitin ligases binding with Nogo-A intracellularly add a layer of complexity to its functional role in the CNS.     

Cellular reactions to axotomy in rat superior cervical ganglia includes apoptotic cell death

The cellular response to axonal injury in the superior cervical ganglion was examined by immunofluoresence at intervals from 6 h to 14 days after transection of the internal and external carotid nerves. GAP-43-immunoreactivity (IR) appeared in some neurons in the ganglia 1 day after axotomy, while neurons in control ganglia were GAP-43 negative. In 3 days axotomized ganglia GAP-43-IR structures were increased in number and intensity in nerve fiber bundles, while GAP-43-positive perikarya were restricted to the middle and caudal parts of the ganglia and showed an intensity that was stronger than at 1 day after axotomy. These GAP-43-positive neurons were also galanin positive. In the cranial part of the ganglia, S100-IR in satellite cells was weak at 18 h after axotomy. Peripheral to this area, S100-IR was stronger and co-localized with HSP-72-IR, preferentially located in satellite cells. HSP-72-IR was, however, occasionally observed also in principal neurons at 1 and 3 days after axotomy. In eosin-stained sections, neurons and satellite cells in the cranial part of 1 day axotomized ganglia were reduced in number, and a further loss was noted at 3 days. At 12 h some satellite cells in the cranial part of the ganglia were labelled by the in situ DNA 3'-end labelling method, indicating apoptosis, and at 18 h many cells were labelled. Some neuronal perikarya were also labelled in this region. Labelling was not observed at 1 day or later after axotomy, nor in control ganglia. The results may imply that not only neurons but also satellite cells react to neuronal axonal injury with apoptosis. Neurons in the middle and caudal part of the ganglia survived and showed increased content of GAP-43 and galanin, possibly a sign of regeneration/neuronal plasticity.     

Mechanical Effects of Dynamic Binding between Tau Proteins on Microtubules during Axonal Injury

The viscoelastic nature of axons plays a key role in their selective vulnerability to damage in traumatic brain injury (TBI). Experimental studies have shown that although axons can tolerate 100% strain under slow loading rates, even strain as small as 5% can rupture microtubules (MTs) during the fast loading velocities relevant to TBI. Here, we developed a computational model to examine rate-dependent behavior related to dynamic interactions between MTs and the MT-associated protein tau under varying strains and strain rates. In the model, inverted pairs of tau proteins can dynamically cross-link parallel MTs via the respective MT-binding domain of each tau. The model also incorporates realistic thermodynamic breaking and reformation of the bonds between the connected tau proteins as they respond to mechanical stretch. With simulated stretch of the axon, the model shows that despite the highly dynamic nature of binding and unbinding events, under fast loading rates relevant to TBI, large tensile forces can be transmitted to the MTs that can lead to mechanical rupture of the MT cylinder, in agreement with experimental observations and as inferred in human TBI. In contrast, at slow loading rates, the progressive breaking and reformation of the bonds between the tau proteins facilitate the extension of axons up to ∼100% strain without any microstructural damage. The model also predicts that under fast loading rates, individual MTs detach from MT bundles via sequential breaking of the tau-tau bonds. Finally, the model demonstrates that longer MTs are more susceptible to mechanical rupture, whereas short MTs are more prone to detachment from the MT bundle, leading to disintegration of the axonal MT ultrastructure. Notably, the predictions from the model are in excellent agreement with the findings of the recent in vitro mechanical testing of micropatterned neuronal cultures.     

Axotomy of developing rat spinal motoneurons: Cell survival,soma size,muscle recovery,and the influence of testosterone

During the period of synapse elimination, motoneurons are impaired in their ability to generate or regenerate axonal branches: following partial denervation of their target muscle, young motoneurons do not sprout to nearby denervated fibers and after axonal injury, they fail to reinnervate the muscle. In the rat levator ani (LA) muscle, which is innervated by motoneurons in the spinal nucleus of the bulbocavernosus (SNB), synapse elemination ends relatively late in development and can be regulated by testosterone. We took advantage of this system to determine if the end of synapse elimination and the development of regenerative capabilities by motoneurons share a common mechanism, or, alternatively, if these two events can be dissociated in time. Axotomy on or before postnatal day 14 (P14) caused the death of SNB motoneurons. By P21, toward the end of synapse elimination in the LA muscle, SNB motoneurons had developed the ability to survive axonal injury. Altering testosterone levels by castration on P7 followed by 4 weeks of either testosterone propionate or control injections did not change the ability of SNB motoneurons to survive axonal injury during development, although these same treatments alter the time course of synapse elimination in the LA muscle. Thus, we dissociated the inability of SNB motoneurons to recover from axonal injury from their developmental elimination of synaptic terminals. We also measured the effect of early axotomy on motoneuronal soma size and on target muscle weight. Axotomy on P14 caused a long-lasting decrease in the soma size of surviving SNB motoneurons, whereas motoneurons axotomized on P28 recovered their normal soma size. Axotomy on or before P7 caused severe atrophy of the target muscles, matching the extensive loss of motoneurons. However, target muscle recovery after axotomy on P14 was as good as recovery after axotomy at later ages, despite greater motoneuronal death after axotomy on P14. This result may reflect an increase in motor unit size, a decrease in polyneuronal innervation by SNB motoneurons that survive axotomy on P14, or a combination of the two. © 1995 John Wiley & Sons, Inc.     

Protein and DNA oxidation in spinal injury: neurofilaments--an oxidation target

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.     

Glucocorticoids Exacerbate Hypoxic and Hypoglycemic Hippocampal Injury In Vitro: Biochemical Correlates and a Role for Astrocytes

The acute secretion of glucocorticoids is critical for responding to physiological stress. Under normal circumstances these hormones do not cause acute neuronal injury, but they have been shown to enhance ischemic and seizure-induced neuronal injury in the rat brain. Using fetal rat hippocampal cultures, we asked whether hypoxic and hypoglycemic cell damage in vitro could be exacerbated by direct exposure to corticosterone (CORT). Each of these insults alone damaged neuronal cells, whereas 4-6 h of hypoxic treatment could damage age-matched astrocytes if glucose was reduced or omitted. Ischemic-like injury to both cell types could be attenuated by pretreatment with high (30 mM) glucose. Exposure to 100 nM CORT did not affect cell viability under control conditions but enhanced both hypoxic and hypoglycemic neuronal injury. In both cases, pretreatment with high glucose abolished this CORT-mediated synergy. In astrocyte cultures, CORT exacerbated both hypoxic and hypoglycemic injury and this effect was also attenuated by high-glucose pretreatment. Identical 24-h CORT treatment caused a 13% reduction in glucose uptake in astrocytes and a 38% reduction in glycogen content, without affecting the level of intracellular glucose. Thus, CORT could endanger both neurons and astrocytes in mixed hippocampal cultures and this effect emerged only under conditions of substrate depletion. The metabolic disruption in astrocytes by CORT further suggests that the ability of CORT to exacerbate neuronal injury may be due in part to impaired glial cell function.