EFFECTS OF TRANEXAMIC ACID ON NEUROPATHOLOGY, ELECTROENCEPHALOGRAPHY, AND CEREBRAL FIBRIN DEPOSITION IN A RAT MODEL OF POLYTRAUMA WITH CONCOMITANT PENETRATING TRAUMATIC BRAIN INJURY.

ABSTRACT: Several studies have demonstrated the clinical utility of tranexamic acid (TXA) for use in trauma patients presenting with significant hemorrhage. Tranexamic acid is an antifibrinolytic that inhibits plasminogen activation, and plasmin activity has been shown to mitigate blood loss and reduce all-cause mortality in the absence of adverse vascular occlusive events. Recent clinical developments indicate TXA is safe to use in patients with concomitant traumatic brain injury (TBI); however, the prehospital effects are not well understood. Importantly, TXA has been associated with seizure activity. Therefore, this study sought to evaluate the effects of early administration of TXA on neurological recovery and electroencephalogram (EEG) abnormalities following penetrating TBI with concomitant hypoxemia and hemorrhagic shock. We hypothesized that early administration of TXA will provide hemodynamic stabilization and reduce intracerebral hemorrhage, which will result in improved neurological function. To test this hypothesis, Sprague-Dawley rats received a unilateral, frontal penetrating ballistic-like brain injury by inserting a probe into the frontal cortex of the anesthetized rat. Five minutes following brain injury, animals underwent 30 min of respiratory distress and 30 min of hemorrhage. Upon completion of the hemorrhage phase, animals received the initial dose of drug intravenously over 10 min after which the prehospital phase was initiated. During the prehospital phase, animals received autologous shed whole blood as needed to maintain a MAP of 65 mm Hg. After 90 min, "in-hospital" resuscitation was performed by administering the remaining shed whole blood providing 100% oxygen for 15 min. Upon recovery from surgery, animals were administered their second dose of vehicle or TXA intravenously over 8 h. Tranexamic acid induced an early improvement in neurologic deficit, which was statistically significant compared with vehicle at 24, 48, and 72 h at three doses tested. Analysis of cerebral hemoglobin content and intracerebral lesion progression revealed 100 mg/kg provided the optimal effects for improvement of neuropathology and was continued for determination of adverse treatment effects. We observed no exacerbation of cerebral thrombosis, but TXA treatment caused an increased risk of EEG abnormalities. These results suggest that TXA following polytrauma with concomitant brain injury may provide mild neuroprotective effects by preventing lesion progression, but this may be associated with an increased risk of abnormal EEG patterns. This risk may be associated with TXA inhibition of glycine receptors and may warrant additional considerations during the use of TXA in patients with severe TBI.

Proteomic analysis and brain-specific systems biology in a rodent model of penetrating ballistic-like brain injury.

Proteomics and systems biology have significantly contributed to biomarker discovery in the field of brain injury. This study utilized 2D-DIGE-PMF-MS as a preliminary screen to detect biomarkers in a rat model of penetrating ballistic-like brain injury (PBBI). Brain-specific systems biology analysis of brain tissue identified 386 proteins having a fold change of more than 2, of which 321 proteins were increased and 65 were decreased 24 h after PBBI compared to sham controls. The majority of upregulated proteins were cytoskeletal (10.5%), nucleic acid binding (9.3%), or kinases (8.9%). Most proteins were involved in protein metabolism (22.7%), signal transduction (20.4%), and development (9.6%). Pathway analysis indicated that these proteins were involved in neurite outgrowth and cell differentiation. Semiquantitative Western blotting of 6, 24, 48, and 72 h after PBBI indicated ubiquitin carboxyl-terminal hydrolase isozyme L1 (a proposed traumatic brain injury biomarker in human clinical trials), tyrosine hydroxylase, and syntaxin-6 were found to be consistently elevated in brain tissue and cerebral spinal fluid after PBBI compared to sham controls. Combining proteomics and brain-specific systems biology can define underlying mechanisms of traumatic brain injury and provide valuable information in biomarker discovery that, in turn, may lead to novel therapeutic targets.

Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic and traumatic brain injury in rats.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).

Post-Traumatic Epilepsy and Seizure Susceptibility in Rat Models of Penetrating and Closed-Head Brain Injury.

Traumatic brain injury (TBI) carries a risk of developing post-traumatic epilepsy (PTE). Currently, animal models that replicate clinical PTE (delayed spontaneous and recurrent seizures) are limited, which hinders pre-clinical research. In this study, we used two rat models of penetrating ballistic-like brain injury (PBBI) and closed-head injury (CHI) to induce spontaneous seizures and also measure changes in seizure susceptibility. In the PBBI model, two trajectories (frontal and lateral) and two injury severities for each trajectory, were evaluated. In the CHI model, a single projectile impact to the dorsal/lateral region of the head was tested. Continuous video-electroencephalographic (EEG) recordings were collected for 10 days at 1 or 6 month(s) post-injury. After EEG recording, all rats were given a sub-convulsant dose of pentylenetetrazole (PTZ) to challenge the seizure susceptibility. The video-EEG recording did not detect PTE following the PBBI. Only one CHI rat demonstrated persistent and recurrent non-convulsive seizures detected at 6 months post-injury. However, after PTZ challenge, 50-100% of the animals across different TBI groups experienced seizures. Seizure susceptibility increased over time from 1 to 6 months post-injury across the majority of TBI groups. Injury severity effects were not apparent within the PBBI model, but were evident between PBBI and CHI models. These results demonstrated the difficulties in detecting delayed spontaneous post-traumatic seizures even in a high-risk model of penetrating brain injury. The PTZ-induced increase in seizure susceptibility indicated the existence of vulnerable risk of epileptogenesis following TBI, which may be considered as an alternative research tool for pre-clinical studies of PTE.

NNZ-2566 treatment inhibits neuroinflammation and pro-inflammatory cytokine expression induced by experimental penetrating ballistic-like brain injury in rats.

BACKGROUND: Inflammatory cytokines play a crucial role in the pathophysiology of traumatic brain injury (TBI), exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair. NNZ-2566, a synthetic analogue of the neuroprotective tripeptide Glypromate, has been shown to be neuroprotective in animal models of brain injury. The goal of this study was to determine the effects of NNZ-2566 on inflammatory cytokine expression and neuroinflammation induced by penetrating ballistic-like brain injury (PBBI) in rats. METHODS: NNZ-2566 or vehicle (saline) was administered intravenously as a bolus injection (10 mg/kg) at 30 min post-injury, immediately followed by a continuous infusion of NNZ-2566 (3 mg/kg/h), or equal volume of vehicle, for various durations. Inflammatory cytokine gene expression from the brain tissue of rats exposed to PBBI was evaluated using microarray, quantitative real time PCR (QRT-PCR), and enzyme-linked immunosorbent assay (ELISA) array. Histopathology of the injured brains was examined using hematoxylin and eosin (H&E) and immunocytochemistry of inflammatory cytokine IL-1beta. RESULTS: NNZ-2566 treatment significantly reduced injury-mediated up-regulation of IL-1beta, TNF-alpha, E-selectin and IL-6 mRNA during the acute injury phase. ELISA cytokine array showed that NZ-2566 treatment significantly reduced levels of the pro-inflammatory cytokines IL-1beta, TNF-alpha and IFN-gamma in the injured brain, but did not affect anti-inflammatory cytokine IL-6 levels. CONCLUSION: Collectively, these results suggest that the neuroprotective effects of NNZ-2566 may, in part, be functionally attributed to the compound's ability to modulate expression of multiple neuroinflammatory mediators in the injured brain.

Extraluminal cooling of bilateral common carotid arteries as a method to achieve selective brain cooling for neuroprotection.

Systemic cooling to achieve brain hypothermia has been investigated as a neuroprotective therapy but can present serious adverse effects. Here we describe a novel method to selectively cool the rat brain and investigate its neuroprotective effects following transient middle cerebral artery occlusion (MCAo). The novelty of our method of selective brain cooling (SBC) was that the extraluminal cooling of the carotid arterial blood was achieved by using a cooling cuff wrapped around each common carotid artery (CCA). Within 20 min of CCA cooling, brain temperature could be lowered by 2-5 degrees C below the baseline and maintained stable for approximately 2 h while maintaining body temperature at 37 degrees C. No adverse effects of SBC were observed on systemic physiology, regional cerebral blood flow (rCBF), bleeding time, or tissue histology in normal animals. In rats having sustained 2-h MCAo, intra-ischemic SBC for 90 min, initiated 30 min following the onset of ischemia, significantly reduced infarction measured at 24 h post-injury (normothermic rats=312+/-51 mm3, SBC rats=139+/-83 mm3). In subgroup experiments, the incidence of peri-infarct depolarization (PID) was assessed during the MCAo and cooling period. Compared to normothermic but ischemic rats, SBC significantly reduced the number of PID events from 6.2+/-2.5 to 2.0+/-2.5, and reduced infarct volumes from 323+/-79 to 139+/-102 mm3. In conclusion, this extralumimal cooling method of SBC provides a safe and efficient approach to rapidly and safely achieve hypothermic neuroprotection.

Combination therapy of levetiracetam and gabapentin against nonconvulsive seizures induced by penetrating traumatic brain injury.

BACKGROUND: Posttraumatic seizures are a medical problem affecting patients with traumatic brain injury. Yet effective treatment is lacking owing to the limitations of antiepileptic drugs (AEDs) applicable to these patients. METHODS: In this study, we evaluated the dose-response efficacy of levetiracetam (12.5-100.0 mg/kg) and gabapentin (1.25-25.0 mg/kg) administered either individually or in pairs at fixed-dose ratios as a combination in mitigating posttraumatic nonconvulsive seizures induced by severe penetrating ballistic-like brain injury (PBBI) in rats. Seizures were detected by continuous electroencephalogram (EEG) monitoring for 72 hours postinjury. Animals were treated twice per day for 3 days by intravenous injections. RESULTS: Both levetiracetam (25-100 mg/kg) and gabapentin (6.25-25 mg/kg) significantly reduced PBBI-induced seizure frequency by 44% to 73% and 61% to 69%, and seizure duration by 45% to 64% and 70% to 78%, respectively. However, the two drugs manifested different dose-response profiles. Levetiracetam attenuated seizure activity in a dose-dependent fashion, whereas the beneficial effects of gabapentin plateaued across the three highest doses tested. Combined administration of levetiracetam and gabapentin mirrored the more classic dose-response profile of levetiracetam monotherapy. However, no additional benefit was derived from the addition of gabapentin. Furthermore, isobolographic analysis of the combination dose-response profile of levetiracetam and gabapentin failed to reach the expected level of additivity, suggesting an unlikelihood of favorable interactions between these two drugs against spontaneously occurring posttraumatic seizure activities at the particular set of dose ratios tested. CONCLUSION: This study was the first attempt to apply isobolographic approach to studying AED combination therapy in the context of spontaneously occurring posttraumatic seizures. Despite the failure to achieve additivity from levetiracetam and gabapentin combination, it is important to recognize the objectivity of the isobolographic approach in the evaluation of AED combination therapy against seizures directly associated with brain injuries.

Air-puff induced vocalizations: A novel approach to detecting negative affective state following concussion in rats.

BACKGROUND: Negative emotional states resulting from concussion are of increasing concern. In the current study, we developed a model to investigate negative affect following concussion in the projectile concussive impact (PCI) model. High frequency ultrasonic vocalizations (22kHz USVs) are associated with negative affective stimuli in rats. Changes in negative affective state were examined following PCI using a mild air-puff stimulus to elicit 22kHz USVs. NEW METHOD: Forty-eight hours post-injury, animals were placed into a clean acrylic box lined with bedding. A 5min baseline recording was followed by 15 air puffs (55psi) spaced 15s apart aimed at the upper back and neck. RESULTS: Injured animals produced on average 153.5±55.13 more vocalizations than shams, vocalizing on average 4min longer than shams. Additionally, concussed animals vocalized to fewer air-puffs, exhibiting a 1.5 fold lower threshold for the expression of negative affect. COMPARISON WITH EXISTING METHODS: Studies currently used to test negative affective states following concussion in animals, such as the elevated plus maze and forced swim task have, as of yet, been unsuccessful in demonstrating injury effects in the PCI model. While the air-puff test has been applied in other fields, to our knowledge it has not been utilized to study traumatic brain injury. CONCLUSION: The current study demonstrates that the air-puff vocalization test may be a valuable tool in assessing negative mood states following concussion in rat models and may be used to evaluate novel therapies following brain injury for the treatment of mood dysfunction.

Penetrating ballistic-like brain injury in the rat: differential time courses of hemorrhage, cell death, inflammation, and remote degeneration.

Acute and delayed cerebral injury was assessed in a recently developed rat model of a penetrating ballistic-like brain injury (PBBI). A unilateral right frontal PBBI trajectory was used to induce survivable injuries to the frontal cortex and striatum. Three distinct phases of injury progression were observed. Phase I (primary injury, 0-6 h) began with immediate (<5 min) intracerebral hemorrhage (ICH) that reached maximal volumetric size at 6 h (27.0 +/- 2.9 mm(3)). During Phase II (secondary injury, 6-72 h), a core lesion of degenerate neurons surrounding the injury track expanded into peri-lesional areas to reach a maximal volume of 69.9 +/- 6.1 mm(3) at 24 h. The core lesion consisted of predominately necrotic cell death and included marked infiltration of both neutrophils (24 h) and macrophages (72 h). Phase III (delayed degeneration, 3-7 days) involved the degeneration of neurons and fiber tracts remote from the core lesion including the thalamus, internal capsule, external capsule, and cerebral peduncle. Overall, different time courses of hemorrhage, lesion evolution, and inflammation were consistent with complementary roles in injury development and repair, providing key information about these mediators of primary, secondary, and delayed brain injury development. The similarities/differences of PBBI to other focal brain injury models are discussed.

Neuroprotection and anti-seizure effects of levetiracetam in a rat model of penetrating ballistic-like brain injury.

PURPOSE: We assessed the therapeutic efficacy of FDA-approved anti-epileptic drug Levetiracetam (LEV) to reduce post-traumatic nonconvulsive seizure (NCS) activity and promote neurobehavioral recovery following 10% frontal penetrating ballistic-like brain injury (PBBI) in male Sprague-Dawley rats. METHODS: Experiment 1 anti-seizure study: 50 mg/kg LEV (25 mg/kg maintenance doses) was given twice daily for 3 days (LEV3D) following PBBI; outcome measures included seizures incidence, frequency, duration, and onset. Experiment 2 neuroprotection studies: 50 mg/kg LEV was given twice daily for either 3 (LEV3D) or 10 days (LEV10D) post-injury; outcome measures include motor (rotarod) and cognitive (water maze) functions. RESULTS: LEV3D treatment attenuated seizure activity with significant reductions in NCS incidence (54%), frequency, duration, and delayed latency to seizure onset compared to vehicle treatment. LEV3D treatment failed to improve cognitive or motor performance; however extending the dosing regimen through 10 days post-injury afforded significant neuroprotective benefit. Animals treated with the extended LEV10D dosing regimen showed a twofold improvement in rotarod task latency to fall as well as significantly improved spatial learning performance (24%) in the MWM task. CONCLUSIONS: These findings support the dual anti- seizure and neuroprotective role of LEV, but more importantly identify the importance of an extended dosing protocol which was specific to the therapeutic targets studied.

Intravenous Administration of Simvastatin Improves Cognitive Outcome following Severe Traumatic Brain Injury in Rats.

Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor commonly used to reduce serum cholesterol. The beneficial effects of oral simvastatin have been reported in pre-clinical models of traumatic brain injury (TBI). The current study was designed to evaluate the potential beneficial effects of simvastatin in a model of severe penetrating TBI using an intravenous (IV) route of administration. Rats were subjected to unilateral frontal penetrating ballistic-like brain injury (PBBI), and simvastatin was delivered intravenously at 30 min and 6 h post-injury and continued once daily for either 4 or 10 days post-PBBI. Motor function was assessed on the rotarod and cognitive performance was evaluated using the Morris water maze (MWM) task. Serum levels of inflammatory cytokines and the astrocytic biomarker, glial fibrillary acidic protein (GFAP), were quantified at 1 h, 4 h, and 24 h post-injury. Histopathological damage was assessed at the terminal end-point. Rotarod testing revealed significant motor deficits in all injury groups but no significant simvastatin-induced therapeutic benefits. All PBBI-injured animals showed cognitive impairment on the MWM test; however, 10-day simvastatin treatment mitigated these effects. Animals showed significantly improved latency to platform and retention scores, whereas the 4-day treatment regimen failed to produce any significant improvements. Biomarker and cytokine analysis showed that IV simvastatin significantly reduced GFAP, interleukin (IL)-1α, and IL-17 serum levels by 4.0-, 2.6-, and 7.0-fold, respectively, at 4 h post-injury. Collectively, our results demonstrate that IV simvastatin provides significant protection against injury-induced cognitive dysfunction and reduces TBI-specific biomarker levels. Further research is warranted to identify the optimal dose and therapeutic window for IV delivery of simvastatin in models of severe TBI.

Comprehensive Evaluation of Neuroprotection Achieved by Extended Selective Brain Cooling Therapy in a Rat Model of Penetrating Ballistic-Like Brain Injury.

Brain hypothermia has been considered as a promising alternative to whole-body hypothermia in treating acute neurological disease, for example, traumatic brain injury. Previously, we demonstrated that 2-hours selective brain cooling (SBC) effectively mitigated acute (≤24 hours postinjury) neurophysiological dysfunction induced by a penetrating ballistic-like brain injury (PBBI) in rats. This study evaluated neuroprotective effects of extended SBC (4 or 8 hours in duration) on sub-acute secondary injuries between 3 and 21 days postinjury (DPI). SBC (34°C) was achieved via extraluminal cooling of rats' bilateral common carotid arteries (CCA). Depending on the experimental design, SBC was introduced either immediately or with a 2- or 4-hour delay after PBBI and maintained for 4 or 8 hours. Neuroprotective effects of SBC were evaluated by measuring brain lesion volume, axonal injury, neuroinflammation, motor and cognitive functions, and post-traumatic seizures. Compared to untreated PBBI animals, 4 or 8 hours SBC treatment initiated immediately following PBBI produced comparable neuroprotective benefits against PBBI-induced early histopathology at 3 DPI as evidenced by significant reductions in brain lesion volume, axonal pathology (beta-amyloid precursor protein staining), neuroinflammation (glial fibrillary acetic protein stained-activated astrocytes and rat major histocompatibility complex class I stained activated microglial cell), and post-traumatic nonconvulsive seizures. In the later phase of the injury (7-21 DPI), significant improvement on motor function (rotarod test) was observed under most SBC protocols, including the 2-hour delay in SBC initiation. However, SBC treatment failed to improve cognitive performance (Morris water maze test) measured 13-17 DPI. The protective effects of SBC on delayed axonal injury (silver staining) were evident out to 14 DPI. In conclusion, the CCA cooling method of SBC produced neuroprotection measured across multiple domains that were evident days/weeks beyond the cooling duration and in the absence of overt adverse effects. These "proof-of-concept" results suggest that SBC may provide an attractive neuroprotective approach for clinical considerations.

Enantiospecificity of glutamate carboxypeptidase II inhibition.

Two representative glutamate carboxypeptidase II (GCP II) inhibitors, 2-(hydroxypentafluorophenylmethyl-phosphinoylmethyl)pentanedioic acid 2 and 2-(3-mercaptopropyl)pentanedioic acid 3, were synthesized in high optical purities (>97%ee). The two enantiomers of 2 were prepared from previously reported chiral intermediates, (R)- and (S)-2-(hydroxyphosphinoylmethyl)pentanedioic acid benzyl esters 8. The synthesis of (R)- and (S)-3 involves the hydrolysis of (R)- and (S)-3-(2-oxo-tetrahydro-thiopyran-3-yl)propionic acids, (R)- and (S)-11, the corresponding optically pure thiolactones delivered by chiral chromatographic separation of the racemic 11. GCP II inhibitory assay revealed that (S)-2 is 40-fold more potent than (R)-2. In contrast, both enantiomers of 3 inhibited GCP II with nearly equal potency. The efficacy observed in subsequent animal studies with these enantiomers correlated well with the inhibitory potency in a GCP II assay.

Recovery from ischemic brain injury in the rat following a 10 h delayed injection with MLN519.

In the present study, we evaluated delayed treatment effects of the proteasome inhibitor and anti-inflammatory agent MLN519 (initiated 10 h post-injury) to improve recovery following ischemic brain injury in rodents. Male rats were exposed to 2 h of middle cerebral artery occlusion (MCAo) and treated with MLN519 (1.0 mg/kg, i.v. @ 10, 24, and 48 h post-occlusion) or vehicle. By 2 weeks post-injury, 60% (6/10) of vehicle animals survived, which was improved (although non-significantly) to 78% (7/9) following MLN519 treatment. The percent loss of tissue in the ipsilateral brain hemisphere (at 2 weeks) was significantly reduced from 27+/-4% (vehicle) to 15+/-4% (MLN519). MLN519 treated animals also lost significantly less body weight (39%) and showed significant improvement in overall neurological function across the 2-week recovery period. However, no significant treatment effects were observed to reduce foot-fault deficits (balance beam) or improve recovery of operant performance (active avoidance test). Overall, delayed treatment with MLN519 provided significant improvement in 3 of 6 test metrics (histopathology, body weight, and neurological dysfunction) supporting improved outcome for brain-injured subjects.

Dual Therapeutic Effects of C-10068, a Dextromethorphan Derivative, Against Post-Traumatic Nonconvulsive Seizures and Neuroinflammation in a Rat Model of Penetrating Ballistic-Like Brain Injury.

Post-traumatic seizures can exacerbate injurious outcomes of severe brain trauma, yet effective treatments are limited owing to the complexity of the pathology underlying the concomitant occurrence of both events. In this study, we tested C-10068, a novel deuterium-containing analog of (+)-N-methyl-3-ethoxymorphinan, in a rat model of penetrating ballistic-like brain injury (PBBI) and evaluated the effects of C-10068 on PBBI-induced nonconvulsive seizures (NCS), acute neuroinflammation, and neurofunctional outcomes. NCS were detected by electroencephalographic monitoring. Neuroinflammation was evaluated by immunohistochemical markers, for example, glial fibrillary acidic protein and major histocompatibility complex class I, for activation of astrocytes and microglia, respectively. Neurofunction was tested using rotarod and Morris water maze tasks. Three infusion doses of C-10068 (1.0, 2.5, and 5.0 mg/kg/h × 72 h) were tested in the antiseizure study. Neuroinflammation and neurofunction were evaluated in animals treated with 5.0 mg/kg/h × 72 h C-10068. Compared to vehicle treatment, C-10068 dose dependently reduced PBBI-induced NCS incidence (40-50%), frequency (20-70%), and duration (30-82%). The most effective antiseizure dose of C-10068 (5.0 mg/kg/h × 72 h) also significantly attenuated hippocampal astrocyte activation and perilesional microglial reactivity post-PBBI. Within C-10068-treated animals, a positive correlation was observed in reduction in NCS frequency and reduction in hippocampal astrocyte activation. Further, C-10068 treatment significantly attenuated astrocyte activation in seizure-free animals. However, C-10068 failed to improve PBBI-induced motor and cognitive functions with the dosing regimen used in this study. Overall, the results indicating that C-10068 exerts both potent antiseizure and antiinflammatory effects are promising and warrant further investigation.

Post-treatment with a novel PARG inhibitor reduces infarct in cerebral ischemia in the rat.

Poly(ADP-ribose) is synthesized from nicotinamide adenine dinucleotide (NAD(+)) by poly(ADP-ribose) polymerase (PARP) and degraded by poly(ADP-ribose) glycohydrolase (PARG). Overactivation of the poly(ADP-ribose) pathway increases nicotinamide and decreases cellular NAD(+)/ATP, which leads to cell death. Blocking poly(ADP-ribose) metabolism by inactivating PARP has been shown to reduce ischemia injury. We investigated whether disrupting the poly(ADP-ribose) cycle by PARG inhibition could achieve similar protection. We demonstrate that either pre- or post-ischemia treatment with 40 mg/kg of N-bis-(3-phenyl-propyl)9-oxo-fluorene-2,7-diamide, a novel PARG inhibitor, significantly reduces brain infarct volumes by 40-53% in a rat model of focal cerebral ischemia. Our result provides the first evidence that PARG inhibitors can ameliorate ischemic brain damage in vivo, in support of PARG as a new therapeutic target for treating ischemia injury.

Neuroprotection assessment by topographic electroencephalographic analysis: effects of a sodium channel blocker to reduce polymorphic delta activity following ischaemic brain injury in rats.

The spatiotemporal electroencephalogram (EEG) pathology associated with brain injury was studied using high-resolution, 10-electrode cortical EEG mapping in a rat model of middle cerebral artery occlusion (MCAo). Using this model we evaluated the ability of the novel sodium channel blocker and neuroprotective agent RS100642 to resolve injury-induced EEG abnormalities as a measure of neurophysiological recovery from brain injury. The middle cerebral artery (MCA) was occluded for 1 h during which a dramatic loss of EEG power was measured over the injured cortex with near complete recovery upon reperfusion of blood to the MCA region in all rats. The resultant progression of the MCAo/reperfusion injury (6-72 h) included the appearance of diffuse polymorphic delta activity (PDA), as visually indicated by the presence of high-amplitude slow-waves recorded from both brain hemispheres. PDA was associated with large increases in EEG power, particularly evident in outer 'peri-infarct' regions of the ipsilateral parietal cortex as visualized using topographic EEG mapping. Post-injury treatment with RS100642 (1.0 mg/kg, i.v.) significantly reduced the PDA activity and attenuated the increase in EEG power throughout the course of the injury. These effects were associated with a reduction in brain infarct volume and improved neurological function. These methods of EEG analysis may be helpful tools to evaluate the physiological recovery of the brain from injury in humans following treatment with an experimental neuroprotective compound.

Correlations between blood-brain barrier disruption and neuroinflammation in an experimental model of penetrating ballistic-like brain injury.

Abstract Blood-brain barrier (BBB) disruption is a pathological hallmark of severe traumatic brain injury (TBI) and is associated with neuroinflammatory events contributing to brain edema and cell death. The goal of this study was to elucidate the profile of BBB disruption after penetrating ballistic-like brain injury (PBBI) in conjunction with changes in neuroinflammatory markers. Brain uptake of biotin-dextran amine (BDA; 3 kDa) and horseradish peroxidase (HRP; 44 kDa) was evaluated in rats at 4 h, 24 h, 48 h, 72 h, and 7 days post-PBBI and compared with the histopathologic and molecular profiles for inflammatory markers. BDA and HRP both displayed a uniphasic profile of extravasation, greatest at 24 h post-injury and which remained evident out to 48 h for HRP and 7 days for BDA. This profile was most closely associated with markers for adhesion (mRNA for intercellular adhesion molecule-1) and infiltration of peripheral granulocytes (mRNA for matrix metalloproteinase-9 [MMP-9] and myeloperoxidase staining). Improvement of BBB dysfunction coincided with increased expression of markers implicated in tissue remodeling and repair. The results of this study reveal a uniphasic and gradient opening of the BBB after PBBI and suggest MMP-9 and resident inflammatory cell activation as candidates for future neurotherapeutic intervention after PBBI.

Human traumatic brain injury induces autoantibody response against glial fibrillary acidic protein and its breakdown products.

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38-50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0-1 days) to late (7-10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.

Similarities and differences of acute nonconvulsive seizures and other epileptic activities following penetrating and ischemic brain injuries in rats.

The similarities and differences between acute nonconvulsive seizures (NCS) and other epileptic events, for example, periodic epileptiform discharges (PED) and intermittent rhythmic delta activities (IRDA), were characterized in rat models of penetrating and ischemic brain injuries. The NCS were spontaneously induced by either unilateral frontal penetrating ballistic-like brain injury (PBBI) or permanent middle cerebral artery occlusion (pMCAO), and were detected by continuous electroencephalogram (EEG) monitoring begun immediately after the injury and continued for 72 h or 24 h, respectively. Analysis of NCS profiles (incidence, frequency, duration, and time distribution) revealed a high NCS incidence in both injury models. The EEG waveform expressions of NCS and PED exhibited intrinsic variations that resembled human electrographic manifestations of post-traumatic and post-ischemic ictal and inter-ictal events, but these waveform variations were not distinguishable between the two types of brain injury. However, the NCS after pMCAO occurred more acutely and intensely (latency=0.6 h, frequency=25 episodes/rat) compared with the PBBI-induced NCS (latency=24 h, frequency=10 episodes/rat), such that the most salient features differentiating post-traumatic and post-ischemic NCS were the intensity and time distribution of the NCS profiles. After pMCAO, nearly 50% of the seizures occurred within the first 2 h of injury, whereas after PBBI, NCS occurred sporadically (0-5%/h) throughout the 72 h recording period. The PED were episodically associated with NCS. By contrast, the IRDA appeared to be independent of other epileptic events. This study provided comprehensive comparisons of post-traumatic and post-ischemic epileptic profiles. The identification of the similarities and differences across a broad spectrum of epileptic events may lead to differential strategies for post-traumatic and post-stroke seizure interventions.