Susan M. Knoblach

Early neuronal expression of tumor necrosis factor-α after experimental brain injury contributes to neurological impairment

Tumor necrosis factor-alpha (TNF alpha) is a pleiotropic cytokine involved in inflammatory cascades associated with CNS injury. To examine the role of TNF alpha in the acute pathophysiology of traumatic brain injury (TBI), we studied its expression, localization and modulation in a clinically relevant rat model of non-penetrating head trauma. TNF alpha levels increased significantly in the injured cortex at 1 and 4, but not at 12, 24 or 72 h after severe lateral fluid-percussion trauma (2.6-2.7 atm). TNF alpha was not elevated after mild injury. At 1 and 4 h after severe TBI, marked increases of TNF alpha were localized immunocytochemically to neurons of the injured cerebral cortex. A small population of astrocytes, ventricular cells and microvessels, also showed positive TNF alpha staining, but this expression was not injury-dependent. Macrophages that were present in a hemorrhagic zone along the external capsule, corpus callosum and alveus hippocampus at 4 h after TBI did not express TNF alpha. Intracerebroventricular administration of a selective TNF alpha antagonist--soluble TNF alpha receptor fusion protein (sTNFR:Fc) (37.5 microg)--at 15 min before and 1 h after TBI, improved performance in a series of standardized motor tasks after injury. In contrast, intravenous administration of sTNFR:Fc (0.2, 1 or 5 mg/kg) at 15 min after trauma did not improve motor outcome. Collectively, this evidence suggests that enhanced early neuronal expression of TNF alpha after TBI contributes to subsequent neurological dysfunction.

Interleukin-10 Improves Outcome and Alters Proinflammatory Cytokine Expression after Experimental Traumatic Brain Injury

Traumatic injury to the central nervous system initiates inflammatory processes that are implicated in secondary tissue damage. These processes include the synthesis of proinflammatory cytokines, leukocyte extravasation, vasogenic edema, and blood-brain barrier breakdown. Interleukin-10 (IL-10), a cytokine with antiinflammatory properties, negatively modulates proinflammatory cascades at multiple levels. We examined the hypothesis that IL-10 treatment can improve outcome in a clinically relevant model of traumatic brain injury (TBI). IL-10 was administered via different routes and dosing schedules in a lateral fluid-percussion model of TBI in rats. Intravenous administration of IL-10 (100 micrograms) at 30 min before and 1 h after TBI improved neurological recovery and significantly reduced TNF expression in the traumatized cortex at 4 h after injury. Such treatment was associated with lower IL-1 expression in the injured hippocampus, and to a lesser extent, in the injured cortex. Subcutaneous IL-10 administration (100 micrograms) at 10 min, 1, 3, 6, 9, and 12 h after TBI also enhanced neurological recovery. In contrast, intracerebroventricular administration of IL-10 (1 or 6 micrograms) at 15 min, 2, 4, 6, and 8 h after TBI was not beneficial. These results indicate that IL-10 treatment improves outcome after TBI and suggest that this improvement may relate, in part, to reductions in proinflammatory cytokine synthesis.

Gene profiling in spinal cord injury shows role of cell cycle in neuronal death

Spinal cord injury causes secondary biochemical changes leading to neuronal cell death. To clarify the molecular basis of this delayed injury, we subjected rats to spinal cord injury and identified gene expression patterns by high‐density oligonucleotide arrays (8,800 genes studied) at 30 minutes, 4 hours, 24 hours, or 7 days after injury (total of 26 U34A profiles). Detailed analyses were limited to 4,300 genes consistently expressed above background. Temporal clustering showed rapid expression of immediate early genes (30 minutes), followed by genes associated with inflammation, oxidative stress, DNA damage, and cell cycle (4 and 24 hours). Functional clustering showed a novel pattern of cell cycle mRNAs at 4 and 24 hours after trauma. Quantitative reverse transcription polymerase chain reaction verified mRNA changes in this group, which included gadd45a, c‐myc, cyclin D1 and cdk4, pcna, cyclin G, Rb, and E2F5. Changes in their protein products were quantified by Western blot, and cell‐specific expression was determined by immunocytochemistry. Cell cycle proteins showed an increased expression 24 hours after injury and were, in part, colocalized in neurons showing morphological evidence of apoptosis. These findings suggest that cell cycle–related genes, induced after spinal cord injury, are involved in neuronal damage and subsequent cell death. Ann Neurol 2003

Multiple caspases are involved in β-amyloid-induced neuronal apoptosis

β‐amyloid peptide (Aβ) has been implicated in the pathogenesis of Alzheimer disease and has been reported to induce apoptotic death in cell culture. Cysteine proteases, a family of enzymes known as caspases, mediate cell death in many models of apoptosis. Multiple caspases have been implicated in Aβ toxicity; these reports are conflicting. We show that treatment of cerebellar granule cells (CGC) with Aβ25–35 causes apoptosis associated with increased activity of caspases‐2, ‐3 and ‐6. Selective inhibition of each of these three caspases provides significant protection against Aβ‐mediated apoptosis. In contrast, no change in caspase‐1 activity was seen after Aβ25–35 application, nor was inhibition of caspase‐1 neuroprotective. Similar to CGC, cortical neuronal cultures treated with Aβ25–35 demonstrate increased caspase‐3 activity but not caspase‐1 activity. Furthermore, significant neuroprotection is elicited by selective inhibition of caspase‐3 in cortical neurons administered Aβ25–35, whereas selective caspase‐1 inhibition has no effect. Taken together, these findings indicate that multiple executioner caspases may be involved in neuronal apoptosis induced by Aβ. J. Neurosci. Res. 65:45–53, 2001.

Diffusion and high resolution MRI of traumatic brain injury in rats: time course and correlation with histology.

Although widely employed in studies of cerebral ischemia, the use of diffusion-weighted imaging (DWI) for traumatic brain injury (TBI) has been both limited and primarily confined to the first few hours after injury. Therefore, the present study examined the temporal evolution of magnetic resonance imaging (MRI) signal changes from hours to weeks after moderate fluid-percussion TBI in rats. We used isotropic diffusion along three directions and high resolution (HR) spin-echo pulse sequences to visualize DWI and HR MRI changes, respectively. Late changes were compared to histopathological and neurological outcome. A significant decrease (P<0.05) in the apparent diffusion coefficients (ADC) below preinjury levels was found in the left cortex and left hippocampus (ipsilateral to injury) at 1-2 h post-TBI. At 2 weeks post-TBI, ADCs were significantly elevated (P<0.05) above preinjury levels in both cortex and hippocampus. Regions of hypo- and hyperintensity detected in HR MRI scans also showed evidence of tissue damage by histological evaluation. Neurological assessment indicated that such changes were observed at a level of injury which produced moderate impairment 2 weeks after the insult. These results indicate that alterations in DWI and HR MRI signals occur both early (hours) and late (weeks) after lateral fluid-percussion injury. Furthermore, the study showed that DWI was sensitive to MR signal change at 1-2 h post TBI (in select ROIs), whereas HR scans showed MR signal change primarily at later time points (3-4 h and later). Moreover, regions which demonstrate late changes are associated with histological damage and neurological impairment. The study demonstrates the utility of MRI to detect early changes, in some cases, that are predictive of long-lasting damage verified histologically.

Multiple Caspases Are Activated after Traumatic Brain Injury: Evidence for Involvement in Functional Outcome

Caspase-3 is a cysteine protease that is strongly implicated in neuronal apoptosis. Activation of caspase-3 may be induced by at least two major initiator pathways: a caspase-8-mediated pathway activated through cell surface death receptors (extrinsic pathway), and a caspase-9-mediated pathway activated by signals from the mitochondria that lead to formation of an apoptosomal complex (intrinsic pathway). In the present studies, we compare the activation of caspases-3, -8, and -9 after lateral fluid-percussion traumatic brain injury (TBI) in rats. Immunoblot analysis identified cleaved forms of caspases-3 and -9, but not caspase-8, at 1, 12, and 48 h after injury. Immunocytochemistry specific for cleaved caspases-3 and -9 revealed their expression primarily in neurons. These caspases were also frequently localized in TUNEL-positive cells, some of which demonstrated morphological features of apoptosis. However, caspases-3 and -9 were also found in neurons that were not TUNEL-positive, and other TUNEL-positive cells did not show activated caspases. In contrast to caspases-3 or -9, caspase-8 expression was only minimally changed by injury. An increase in expression of this caspase was undetectable by immunoblotting methods, and appeared as positive immunostaining restricted to a few cells within the injured cortex. Treatment with the pan-caspase inhibitor z-VAD-fmk at 15 min after TBI improved performance on motor and spatial learning tests. These data suggest that several caspases may be involved in the pathophysiology of TBI and that pan-caspase inhibition strategies may improve neurological outcomes.

Caspase Inhibitor z-DEVD-fmk Attenuates Calpain and Necrotic Cell Death in Vitro and after Traumatic Brain Injury:

In studies designed to evaluate the therapeutic window for treatment of traumatic brain injury, the caspase 3 inhibitor z-DEVD-fmk improved neurologic function and reduced lesion volumes when administered at 1 but not at 4, 8, or 24 hours after injury. Moreover, neither caspase 3 nor PARP, a caspase 3 substrate, were cleaved in injured, untreated cortex from 1 to 72 hours after injury. Few cortical neurons expressed active caspase 3 or were TUNEL positive from 6 to 24 hours after injury, and TUNEL staining was primarily Type I (necrotic). Nissl staining revealed extensive neuronal necrosis in the injured cortex from 6 to 24 hours after impact. Considered together, these data suggested that z-DEVD-fmk may reduce neuronal necrosis, so we used an in vitro model of necrotic cell death induced by maitotoxin to test this further and explore the potential mechanism(s) involved. Z-DEVD-fmk (1 nM-100 μM) significantly attenuated maitotoxin induced neuronal cell death and markedly reduced expression of the 145 kD calpain-mediated α-spectrin breakdown product after maitotoxin injury. Neither the 120 kD caspase-mediated α-spectrin cleavage product nor cathepsin B were expressed after maitotoxin injury. In a cell free assay, z-DEVD-fmk reduced hydrolysis of casein by purified calpain I. Finally, z-DEVD-fmk reduced expression of the 145 kD calpain-mediated α-spectrin cleavage fragment after traumatic brain injury in vivo. These data suggest that neuroprotection by z-DEVD-fmk may, in part, reflect inhibition of calpain-related necrotic cell death.

Ceramide induces neuronal apoptosis through mitogen-activated protein kinases and causes release of multiple mitochondrial proteins.

Ceramide accumulates in neurons during various disorders associated with acute or chronic neurodegeneration. In these studies, we investigated the mechanisms of ceramide-induced apoptosis in primary cortical neurons using exogenous C(2) ceramide as well as inducing endogenous ceramide accumulation using inhibitors of glucosylceramide synthetase. Ceramide induced the translocation of certain, but not all, pro-apoptotic mitochondrial proteins: cytochrome c, Omi, SMAC, and AIF were released from the mitochondria, whereas Endonuclease G was not. Ceramide also selectively altered the phosphorylation state of members of the MAPK superfamily, causing dephosphorylation of ERK1/2 and hyperphosphorylation of p38 MAP kinases, but not affecting the phosphorylation of JNK or ERK5. Inhibitors of the p38 MAP kinase pathway (SB-202190 or SB-203580) and an inhibitor of the ERK1/2 pathway (U0126) reduced ceramide-induced neuronal death. These p38 and ERK1/2 inhibitors appear to block ceramide-activated apoptotic signaling upstream of the mitochondria, as they attenuated mitochondrial release of cytochrome c, Omi, AIF, and SMAC, as well as reducing ceramide-induced caspase-3 activation.

Neuronal plasticity after spinal cord injury: identification of a gene cluster driving neurite outgrowth

Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady‐state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re‐induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule‐ associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth.

Activation of group I metabotropic glutamate receptors reduces neuronal apoptosis but increases necrotic cell death in vitro.

Glutamate released during acute CNS insults acts at metabotropic glutamate receptors (mGluR), including group I mGluR. Blockade of group I mGluR during in vitro neuronal trauma provides neuroprotection, whereas activation exacerbates such injury. However, the effects of group I mGluR agonists or antagonists have been primarily studied in in vitro models characterized by necrotic cell death. We examined the role of group I mGluR in the modulation of neuronal injury induced during oxygen-glucose deprivation (OGD), a well-studied model of necrosis, and by application of two well established pro-apoptotic agents: staurosporine and etoposide. Inhibition of group I mGluR attenuated necrosis induced by OGD, whereas selective activation of group I mGluR exacerbated such injury. In contrast, activation of group I mGluR, including selective activation of mGluR5, significantly attenuated apoptotic cell death induced by both staurosporine and etoposide. This effect was completely reversed by co-application of a group I mGluR antagonist. Thus, group I mGluR appear to exhibit opposite effects on necrotic and apoptotic neuronal cell death. Our findings suggest that activation of mGluR1 exacerbates neuronal necrosis whereas both mGluR1 and mGluR5 play a role in attenuation of neuronal apoptosis. Cell Death and Differentiation (2000) 7, 470–476