Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI.

Traumatic brain injury (TBI) produces a rapid and robust inflammatory response in the brain characterized in part by activation of microglia. A novel histone deacetylase (HDAC) inhibitor, 4-dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]benzamide (DMA-PB), was administered (0, 0.25, 2.5, 25 mg/kg) systemically immediately after lateral fluid percussion TBI in rats. Hippocampal CA2/3 tissue was processed for acetyl-histone H3 immunolocalization, OX-42 immunolocalization (for microglia), and Fluoro-Jade B histofluorescence (for degenerating neurons) at 24 h after injury. Vehicle-treated TBI rats exhibited a significant reduction in acetyl-histone H3 immunostaining in the ipsilateral CA2/3 hippocampus compared to the sham TBI group (p<0.05). The reduction in acetyl-histone H3 immunostaining was attenuated by each of the DMA-PB dosage treatment groups. Vehicle-treated TBI rats exhibited a high density of phagocytic microglia in the ipsilateral CA2/3 hippocampus compared to sham TBI in which none were observed. All doses of DMA-PB significantly reduced the density of phagocytic microglia (p<0.05). There was a trend for DMA-PB to reduce the number of degenerating neurons in the ipsilateral CA2/3 hippocampus (p=0.076). We conclude that the HDAC inhibitor DMA-PB is a potential novel therapeutic for inhibiting neuroinflammation associated with TBI.

A number of potential traumatic brain injury (TBI) biomarkers have been proposed and evaluated in the laboratory and clinic. This study investigated the temporal profile of circulating biomarkers of astrocytic and neuronal injury over the first 24 h and relevant histopathological changes after experimental moderate TBI. Twenty male rats were randomly assigned to either moderate parasagittal fluid percussion or sham injury. Blood serum samples were collected 2 d prior to TBI (baseline) and at 3, 6, and 24 h after TBI. A single cerebrospinal fluid (CSF) sample was collected from the cisterna magna 24 h after TBI, followed by euthanasia and brain harvesting for histology. Serum and CSF samples were analyzed for neuronal (ubiquitin carboxy-terminal hydrolase L1 [UCH-L1]) and astroglial (glial fibrillary acidic protein [GFAP]) protein levels using enzyme-linked immunosorbent assay. Brain histology included GFAP immunostaining and Fluoro-Jade histofluorescence. Serum and CSF levels of GFAP were near zero in sham animals. Serum GFAP levels were significantly elevated at 3 and 6 h post-TBI, compared with baseline and time-matched sham values, while UCH-L1 was significantly elevated only at 3 h post-TBI. Both CSF GFAP and UCH-L1 at 24 h post-TBI were significantly elevated, compared with sham. GFAP immunohistochemistry and FJ histofluorescence of degenerating neurons were performed in the same animals after 24 h survival. Histology revealed characteristic acute neuronal degeneration in the ipsilateral hippocampus and parietal cortex and reduction in GFAP immunostaining in areas of neuronal cell loss. The data provide evidence of a causal relationship between TBI-induced acute brain pathology and circulating neuronal and glial markers, further demonstrating their role as candidate markers for TBI. Studies of relative changes in biomarker levels in CSF and serum suggest that different mechanisms may underlie the transport and/or clearance of UCH-L1 and GFAP in these two compartments.

This study evaluated the effects of clinically relevant concentrations of amantadine (AMT) on cognitive outcome and hippocampal cell survival in adult rats after lateral fluid percussion traumatic brain injury (TBI). AMT is an antagonist of the N-methyl-D-aspartate-type glutamate receptor, increases dopamine release, blocks dopamine reuptake, and has an inhibitory effect on microglial activation and neuroinflammation. Currently, AMT is clinically used as an antiparkinsonian drug. Amantadine or saline control was administered intraperitoneally, starting at 1 h after TBI followed by dosing three times daily for 16 consecutive days at 15, 45, and 135 mg/kg/day. Terminal blood draws were obtained from TBI rats at the time of euthanasia at varying time points after the last amantadine dose. Pharmacokinetics analysis confirmed that the doses of AMT achieved serum concentrations similar to those observed in humans receiving therapeutic doses (100-400 mg/day). Acquisition of spatial learning and memory retention was assessed using the Morris water maze (MWM) on days 12-16 after TBI. Brain tissues were collected and stained with Cresyl-violet for long-term cell survival analysis. Treatment with 135mg/kg/day of AMT improved acquisition of learning and terminal cognitive performance on MWM. The 135-mg/kg/day dosing of AMT increased the numbers of surviving CA2-CA3 pyramidal neurons at day 16 post-TBI. Overall, the data showed that clinically relevant dosing schedules of AMT affords neuroprotection and significantly improves cognitive outcome after experimental TBI, suggesting that it has the potential to be developed as a novel treatment of human TBI.

Traumatic brain injury (TBI) leads to a rapid and excessive increase in glutamate concentration in the extracellular milieu, which is strongly associated with excitotoxicity and neuronal degeneration. N-acetylaspartylglutamate (NAAG), a prevalent peptide neurotransmitter in the vertebrate nervous system, is released along with glutamate and suppresses glutamate release by actions at pre-synaptic metabotropic glutamate autoreceptors. Extracellular NAAG is hydrolyzed to N-acetylaspartate and glutamate by peptidase activity. In the present study PGI-02776, a newly designed di-ester prodrug of the urea-based NAAG peptidase inhibitor ZJ-43, was tested for neuroprotective potential when administered intraperitoneal 30 min after lateral fluid percussion TBI in the rat. Stereological quantification of hippocampal CA2-3 degenerating neurons at 24 hrs post injury revealed that 10 mg/kg PGI-02776 significantly decreased the number of degenerating neurons (p<0.05). Both average latency analysis of Morris water maze performance as well as assessment of 24-hour memory retention revealed significant differences between sham-TBI and TBI-saline. In contrast, no significant difference was found between sham-TBI and PGI-02776 treated groups in either analysis indicating an improvement in cognitive performance with PGI-02776 treatment. Histological analysis on day 16 post-injury revealed significant cell death in injured animals regardless of treatment. In vitro NAAG peptidase inhibition studies demonstrated that the parent compound (ZJ-43) exhibited potent inhibitory activity while the mono-ester (PGI-02749) and di-ester (PGI-02776) prodrug compounds exhibited moderate and weak levels of inhibitory activity, respectively. Pharmacokinetic assays in uninjured animals found that the di-ester (PGI-02776) crossed the blood-brain barrier. PGI-02776 was also readily hydrolyzed to both the mono-ester (PGI-02749) and the parent compound (ZJ-43) in both blood and brain. Overall, these findings suggest that post-injury treatment with the ZJ-43 prodrug PGI-02776 reduces both acute neuronal pathology and longer term cognitive deficits associated with TBI.

Hypoxia frequently occurs in patients with traumatic brain injury (TBI) and is associated with increased morbidity and mortality. This study examined the effects of immediate or delayed post-traumatic hypoxia (fraction of inspired oxygen [FiO 2 ] 11%) on acute neuronal degeneration and long-term neuronal survival in hippocampal fields after moderate fluid percussion injury in rats. In Experiment 1, hypoxia was induced for 15 or 30 min alone or immediately following TBI. In Experiments 2 and 3, 30 min of hypoxia was induced immediately after TBI or delayed until 60 min after TBI. In Experiment 1, acute neurodegeneration was evaluated in the hippocampal fields 24 h after insults using Fluoro-Jade staining and stereological quantification. During hypoxia alone, or in combination with TBI, mean arterial blood pressure was significantly reduced by approximately 30%, followed by a rapid return to normal values upon return to pre-injury FiO 2 . Hypoxia alone failed to cause hippocampal neuronal degeneration when measured at 24 h after insult. TBI alone resulted in neuronal degeneration in each ipsilateral hippocampal field, predominantly in CA2-CA3 and the dentate gyrus. Compared to TBI alone, TBI plus immediate hypoxia for either 15 or 30 min significantly increased neuronal loss in most ipsilateral hippocampal fields and in the contralateral hilus and dentate gyrus. In Experiment 2, TBI plus hypoxia delayed 30 min significantly increased degeneration only in ipsilateral CA2-CA3. In Experiment 3, 30 min of immediate hypoxia significantly reduced the numbers of surviving neurons in the CA3 at 14 days after TBI. The greatly increased vulnerability in all hippocampal fields by immediate 30 min post-traumatic hypoxia provides a relevant model of TBI complicated with hypoxia/hypotension. These data underscore the significance of the secondary insult, the necessity to better characterize the range of injuries experienced by the TBI patient, and the importance of strictly avoiding hypoxia in the early management of TBI patients.

Traumatic brain injury (TBI) leads to a rapid and excessive glutamate elevation in the extracellular milieu, resulting in neuronal degeneration and astrocyte damage. Posttraumatic hypoxia is a clinically relevant secondary insult that increases the magnitude and duration of glutamate release following TBI. N-acetyl-aspartyl glutamate (NAAG), a prevalent neuropeptide in the CNS, suppresses presynaptic glutamate release by its action at the mGluR3 (a group II metabotropic glutamate receptor). However, extracellular NAAG is rapidly converted into NAA and glutamate by the catalytic enzyme glutamate carboxypeptidase II (GCPII) reducing presynaptic inhibition. We previously reported that the GCPII inhibitor ZJ-43 and its prodrug di-ester PGI-02776 reduce the deleterious effects of excessive extracellular glutamate when injected systemically within the first 30 minutes following injury. We now report that PGI-02776 (10 mg/kg) is neuroprotective when administered 30-minutes post-injury in a model of TBI plus 30 minutes of hypoxia (FiO2 = 11%). 24-hrs following TBI with hypoxia, significant increases in neuronal cell death in the CA1, CA2/3, CA3c, hilus and dentate gyrus were observed in the ipsilateral hippocampus. Additionally, there was a significant reduction in the number of astrocytes in the ipsilateral CA1, CA2/3 and in the CA3c/hilus/dentate gyrus. Administration of PGI-02776 immediately following the cessation of hypoxia significantly reduced neuronal and astrocytic cell death across all regions of the hippocampus. These findings indicate that NAAG peptidase inhibitors administered post-injury can significantly reduce the deleterious effects of TBI combined with a secondary hypoxic insult.

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