Paradoxical increase in neuronal DNA fragmentation after neuroprotective free radical scavenger treatment in experimental traumatic brain injury.

The mechanisms and role of nerve cell death after traumatic brain injury (TBI) are not fully understood. The authors investigated the effect of pretreatment with the oxygen free radical spin trap alpha-phenyl-N-tert-butyl-nitrone (PBN) on the number of neurons undergoing apoptosis after TBI in rats. Apoptotic cells were identified by the TUNEL method combined with the nuclear stain, Hoechst 33258, and immunohistochemistry for the active form of caspase-3. Numerous neurons became positive for activated caspase 3 and TUNEL in the cortex at 24 hours after injury, suggesting ongoing biochemical apoptosis. In PBN-treated rats, a significantly greater number of cells were found to be TUNEL positive at 24 hours compared with controls. However, PBN treatment resulted in a reduced cortical lesion volume and improved behavioral outcome two weeks after injury. The authors conclude that a treatment producing an increase in DNA fragmentation in the early phase may be compatible with an overall beneficial effect on outcome after TBI. This should be considered in the screening process for future neuroprotective remedies.     

Caspase-dependent cell death involved in brain damage after acute subdural hematoma in rats

Traumatic brain injury is associated with acute subdural hematoma (ASDH) that worsens outcome. Although early removal of blood can reduce mortality, patients still die or remain disabled after surgery and additional treatments are needed. The blood mass and extravasated blood induce pathomechanisms such as high intracranial pressure (ICP), ischemia, apoptosis and inflammation which lead to acute as well as delayed cell death. Only little is known about the basis of delayed cell death in this type of injury. Thus, the purpose of the study was to investigate to which extent caspase-dependent intracellular processes are involved in the lesion development after ASDH in rats. A volume of 300microL blood was infused into the subdural space under monitoring of ICP and tissue oxygen concentration. To asses delayed cell death mechanisms, DNA fragmentation was measured 1, 2, 4 and 7 days after ASDH by TUNEL staining, and the effect of the pan-caspase inhibitor zVADfmk on lesion volume was assessed 7 days post-ASDH. A peak of TUNEL-positive cells was found in the injured cortex at day 2 after blood infusion (53.4+/-11.6 cells/mm(2)). zVADfmk (160ng), applied by intracerebroventricular injection before ASDH, reduced lesion volume significantly by more than 50% (vehicle: 23.79+/-7.62mm(3); zVADfmk: 9.06+/-4.08). The data show for the first time that apoptotic processes are evident following ASDH and that caspase-dependent mechanisms play a crucial role in the lesion development caused by the blood effect on brain tissue.     

Therapeutic effect of SN50, an inhibitor of nuclear factor-κB, in treatment of TBI in mice

NF-κB upregulation has been demonstrated in neurons and glial cells in response to experimental injury and neuropathological disorders, where it has been related to both neurodegenerative and neuroprotective activities. It has been generally recognized that NF-κB plays important roles in the regulation of apoptosis and inflammation as well as innate and adaptive immunity. However, the regulatory mechanism of NF-κB in apoptosis remained to be determined. The present study sought to first investigate the effect of a NF-κB inhibitor SN50, which inhibits NF-κB nuclear translocation, on cell death and behavioral deficits in our mice traumatic brain injury (TBI) models. Additionally, we tried to elucidate the possible mechanisms of the therapeutic effect of SN50 through NF-κB regulating apoptotic and inflammatory pathway in vivo. Encouragingly, the results showed that pretreatment with SN50 remarkably attenuated TBI-induced cell death (detected by PI labeling), cumulative loss of cells (detected by lesion volume), and motor and cognitive dysfunction (detected by motor test and Morris water maze). To analyze the mechanism of SN50 on cell apoptotic and inflammatory signaling pathway, we thus assessed expression levels of TNF-α, cathepsin B and caspase-3, Bid cleavage and cytochrome c release in SN50-pretreated groups compared with those in saline vehicle groups. The results imply that through NF-κB/TNF-α/cathepsin networks SN50 may contribute to TBI-induced extrinsic and intrinsic apoptosis, and inflammatory pathways, which partly determined the fate of injured cells in our TBI model.     

The genomic profile of the cerebral cortex after closed head injury in mice: effects of minocycline

Microarray analysis was used to delineate gene expression patterns and profile changes following traumatic brain injury (TBI) in mice. A parallel microarray analysis was carried out in mice with TBI that were subsequently treated with minocycline, a drug proposed as a neuroprotectant in other neurological disorders. The aim of this comparison was to identify pathways that may be involved in secondary injury processes following TBI and potential specific pathways that could be targeted with second generation therapeutics for the treatment of neurotrauma patients. Gene expression profiles were measured with the compugen long oligo chip and real-time PCR was used to validate microarray findings. A pilot study of effect of minocycline on gene expression following TBI was also carried out. Gene ontology comparison analysis of sham TBI and minocycline treated brains revealed biological pathways with more genes differentially expressed than predicted by chance. Among 495 gene ontology categories, the significantly different gene ontology groups included chemokines, genes involved in cell surface receptor-linked signal transduction and pro-inflammatory cytokines. Expression levels of some key genes were validated by real-time quantitative PCR. This study confirms that multiple regulatory pathways are affected following brain injury and demonstrates for the first time that specific genes and molecular networks are affected by minocycline following brain injury. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of CCL2 (MCP-1) in traumatic brain injury (TBI): evidence from severe TBI patients and CCL2?/? mice

Cerebral inflammation involves molecular cascades contributing to progressive damage after traumatic brain injury (TBI). The chemokine CC ligand-2 (CCL2) (formerly monocyte chemoattractant protein-1, MCP-1) is implicated in macrophage recruitment into damaged parenchyma after TBI. This study analyzed the presence of CCL2 in human TBI, and further investigated the role of CCL2 in physiological and cellular mechanisms of secondary brain damage after TBI. Sustained elevation of CCL2 was detected in the cerebrospinal fluid (CSF) of severe TBI patients for 10 days after trauma, and in cortical homogenates of C57Bl/6 mice, peaking at 4 to 12 h after closed head injury (CHI). Neurological outcome, lesion volume, macrophage/microglia infiltration, astrogliosis, and the cerebral cytokine network were thus examined in CCL2-deficient (−/−) mice subjected to CHI. We found that CCL2−/− mice showed altered production of multiple cytokines acutely (2 to 24 h); however, this did not affect lesion size or cell death within the first week after CHI. In contrast, by 2 and 4 weeks, a delayed reduction in lesion volume, macrophage accumulation, and astrogliosis were observed in the injured cortex and ipsilateral thalamus of CCL2−/− mice, corresponding to improved functional recovery as compared with wild-type mice after CHI. Our findings confirm the significant role of CCL2 in mediating post-traumatic secondary brain damage.     

Continuous infusion of cyclosporin A postinjury significantly ameliorates cortical damage following traumatic brain injury

Traumatic brain injury (TBI) results in the rapid necrosis of cortical tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the original damage resulting in significant neurological dysfunction. Recent reports from our lab demonstrate that a bolus injection of the immunosuppressant cyclosporin A (CsA) is neuroprotective following TBI. CsA transiently inhibits the opening of the mitochondrial permeability transition pore and maintains calcium homeostasis in isolated mitochondria. The present study utilized a unilateral controlled cortical impact model of TBI to assess whether the neuroprotective effects of CsA could be extended by chronic infusion. Adult rats were subjected to a moderate (2 mm) cortical deformation and the extent of cortical damage was assessed using modern stereological techniques. Animals were administrated a 20 mg/kg intraperitoneal bolus of CsA or vehicle 15 min postinjury and osmotic minipumps were implanted subcutaneously to deliver CsA (4.5 or 10 mg/kg/day) or vehicle. All animals receiving CsA demonstrated a significant reduction in lesion volume, with the highest dose offering the most neuroprotection (74% reduction in lesion volume). These results extend our previous findings and demonstrate that chronic infusion of CsA is neuroprotective following TBI. These findings also suggest that the mechanisms responsible for tissue necrosis following TBI are amenable to manipulation.     

Caspase inhibition therapy abolishes brain trauma-induced increases in Abeta peptide: implications for clinical outcome

The detrimental effects of traumatic brain injury (TBI) on brain tissue integrity involve progressive axonal damage, necrotic cell loss, and both acute and delayed apoptotic neuronal death due to activation of caspases. Post-injury accumulation of amyloid precursor protein (APP) and its toxic metabolite amyloid-beta peptide (Abeta) has been implicated in apoptosis as well as in increasing the risk for developing Alzheimer's disease (AD) after TBI. Activated caspases proteolyze APP and are associated with increased Abeta production after neuronal injury. Conversely, Abeta and related APP/Abeta fragments stimulate caspase activation, creating a potential vicious cycle of secondary injury after TBI. Blockade of caspase activation after brain injury suppresses apoptosis and improves neurological outcome, but it is not known whether such intervention also prevents increases in Abeta levels in vivo. The present study examined the effect of caspase inhibition on post-injury levels of soluble Abeta, APP, activated caspase-3, and caspase-cleaved APP in the hippocampus of nontransgenic mice expressing human Abeta, subjected to controlled cortical injury (CCI). CCI produced brain tissue damage with cell loss and elevated levels of activated caspase-3, Abeta(1-42) and Abeta(1-40), APP, and caspase-cleaved APP fragments in hippocampal neurons and axons. Post-CCI intervention with intracerebroventricular injection of 100 nM Boc-Asp(OMe)-CH(2)F (BAF, a pan-caspase inhibitor) significantly reduced caspase-3 activation and improved histological outcome, suppressed increases in Abeta and caspase-cleaved APP, but showed no significant effect on overall APP levels in the hippocampus after CCI. These data demonstrate that after TBI, caspase inhibition can suppress elevations in Abeta. The extent to which Abeta suppression contributes to improved outcome following inhibition of caspases after TBI is unclear, but such intervention may be a valuable therapeutic strategy for preventing the long-term evolution of Abeta-mediated pathology in TBI patients who are at risk for developing AD later in life.     

Acute intranasal osteopontin treatment in male rats following TBI increases the number of activated microglia but does not alter lesion characteristics

Intranasal recombinant osteopontin (OPN) has been shown to be neuroprotective in different models of acquired brain injury but has never been tested after traumatic brain injury (TBI). We used a model of moderate-to-severe controlled cortical impact in male adult Sprague Dawley rats and tested our hypothesis that OPN treatment would improve neurological outcomes, lesion and brain tissue characteristics, neuroinflammation, and vascular characteristics at 1 day post-injury. Intranasal OPN administered 1 hr after the TBI did not improve neurological score, lesion volumes, blood–brain barrier, or vascular characteristics. When assessing neuroinflammation, we did not observe any effect of OPN on the astrocyte reactivity but discovered an increased number of activated microglia within the ipsilateral hemisphere. Moreover, we found a correlation between edema and heme oxygenase-1 (HO-1) expression which was decreased in OPN-treated animals, suggesting an effect of OPN on the HO-1 response to injury. Thus, OPN may increase or accelerate the microglial response after TBI, and early response of HO-1 in modulating edema formation may limit the secondary consequences of TBI at later time points. Additional experiments and at longer time points are needed to determine if intranasal OPN could potentially be used as a treatment after TBI where it might be beneficial by activating protective signaling pathways.     

Depth of lesion model in children and adolescents with moderate to severe traumatic brain injury: use of SPGR MRI to predict severity and outcome

OBJECTIVES: The utility of a depth of lesion classification using an SPGR MRI sequence in children with moderate to severe traumatic brain injury (TBI) was examined. Clinical and depth of lesion classification measures of TBI severity were used to predict neurological and functional outcome after TBI. METHODS: One hundred and six children, aged 4 to 19, with moderate to severe TBI admitted to a rehabilitation unit had an SPGR MRI sequence obtained 3 months afterTBI. Acquired images were analyzed for location, number, and size of lesions. The Glasgow coma scale (GCS) was the clinical indicator of severity. The deepest lesion present was used for depth of lesion classification. Speed of injury was inferred from the type of injury. The disability rating scale at the time of discharge from the rehabilitation unit (DRS1) and at 1 year follow up (DRS2) were functional outcome measures. RESULTS: The depth of lesion classification was significantly correlated with GCS severity, number of lesions, and both functional measures, DRS1 and DRS2. This result was more robust for time 1, probably due to the greater number of psychosocial factors impacting on functioning at time 2. Lesion volume was not correlated with the depth of lesion model. In multivariate models, depth of lesion was most predictive of DRS1, whereas GCS was most predictive of DRS2. CONCLUSIONS: A depth of lesion classification of TBI severity may have clinical utility in predicting functional outcome in children and adolescents with moderate to severe TBI.     

Cytochrome c release and caspase activation after traumatic brain injury

Experimental traumatic brain injury (TBI) results in a rapid and significant necrosis of cortical tissue at the site of injury. In the ensuing hours and days, secondary injury exacerbates the primary damage resulting in significant neurological dysfunction. The identification of cell death pathways that mediate this secondary traumatic injury have not been elucidated, however recent studies have implicated a role for apoptosis in the neuropathology of traumatic brain injury. The present study utilized a controlled cortical impact model of brain injury to assess the involvement of apoptotic pathways: release of cytochrome c from mitochondria and the activation of caspase-1- and caspase-3-like proteases in the injured cortex at 6, 12 and 24 h post-injury. Collectively, these results demonstrate cytochrome c release from mitochondria and its redistribution into the cytosol occurs in a time-dependent manner following TBI. The release of cytochrome c is accompanied by a time-dependent increase in caspase-3-like protease activity with no apparent increase in caspase-1-like activity. However, pretreatment with a general caspase inhibitor had no significant effect on the amount of cortical damage observed at 7 days post-injury. Our data suggest that several pro-apoptotic events occur following TBI, however the translocation of cytochrome c itself and/or other events upstream of caspase activation/inhibition may be sufficient to induce neuronal cell death.