Arginase overexpression in neurons and its effect on traumatic brain injury.

Arginine is a semi-essential amino acid which serves as a substrate for nitric oxide (NO) production by nitric oxide synthase (NOS) and a precursor for various metabolites including ornithine, creatine, polyamines, and agmatine. Arginase competes with nitric oxide synthase for substrate arginine to produce orthinine and urea. There is contradictory evidence in the literature on the role of nitric oxide in the pathophysiology of traumatic brain injury (TBI). These contradictory perspectives are likely due to different NOS isoforms - endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) which are expressed in the central nervous system. Of these, the role of nNOS in acute injury remains less clear. This study aimed to employ a genetic approach by overexpressing arginase isoforms specifically in neurons using a Thy-1 promoter to manipulate cell autonomous NO production in the context of TBI. The hypothesis was that increased arginase would divert arginine from pathological NO production. We generated 2 mouse lines that overexpress arginase I (a cytoplasmic enzyme) or arginase II (a mitochondrial enzyme) in neurons of FVB mice. We found that two-weeks after induction of controlled cortical injury, overexpressing arginase I but not arginase II in neurons significantly reduced contusion size and contusion index compared to wild-type (WT) mice. This study establishes enhanced neuronal arginase levels as a strategy to affect the course of TBI and provides support for the potential role of neuronal NO production in this condition.

Axon initial segment-associated microglia.

Microglia are the brain's resident immune cells and function as the main defense against pathogens or injury. However, in the absence of disease, microglia have other functions in the normal brain. For example, previous studies showed that microglia contribute to circuit refinement and synaptic plasticity in the developing and adult brain, respectively. Thus, microglia actively participate in regulating neuronal excitability and function. Here, we report that in the cortex, but not other brain regions, a subset of microglia extend a single process that specifically associates and overlaps with the axon initial segment (AIS), the site where action potentials are generated. Similar associations were not observed with dendrites or distal axons. Microglia-AIS interactions appear early in development, persist throughout adulthood, and are conserved across species including mice, rats, and primates. However, these interactions are lost after microglial activation following brain injury, suggesting that such interactions may be part of healthy brain function. Loss of microglial CX3CR1 receptors, or the specialized extracellular matrix surrounding the AIS, did not disrupt the interaction. However, loss of AIS proteins by the neuron-specific deletion of the master AIS scaffold AnkyrinG disrupted microglia-AIS interactions. These results reveal a unique population of microglia that specifically interact with the AIS in the adult cortex.

Improved cerebrovascular function and reduced histological damage with darbepoietin alfa administration after cortical impact injury in rats.

Darbepoetin alfa (darbEpo) is an erythropoietic glycoprotein that activates the erythropoietin receptor. The aim of our study was to determine whether darbEpo is neuroprotective in a cortical impact injury (CII) model and to determine the characteristics of dose response and time window. To better understand the vascular mechanism of darbEpo neuroprotection, the reactivity of cerebral blood flow (CBF) to l-arginine administration was also studied. Rats were given saline or darbEpo from 2.5 to 50 µg/kg at 5 min after CII or a dose of 25 µg/kg darbEpo at times ranging from 5 min to 24 h after CII. Histological assessment was determined 2 weeks after a severe CII. Other rats were given either darbEpo (25 µg/kg) or saline daily for 3 days before injury. Five minutes after severe CII, they were given either l-arginine or d-arginine. Hemodynamic variables were monitored for 2 h after injury. In the dose-response study, darbEpo in doses of 25 and 50 µg/kg significantly reduced contusion volume from 39.1 ± 6.7 to 8.1 ± 3.1 and 11.2 ± 6.0 mm(3), respectively. In the time window study, darbEpo reduced contusion volume when given in a dose of 25 µg/kg at 5 min to 6 h after the impact injury. In animals pretreated with darbEpo, the CBF response to l-arginine was significantly greater than in the animals pretreated with saline. These data demonstrate that darbEpo has neuroprotective effects in traumatic brain injury in a dose- and time-dependent manner and that vascular effects of darbEpo may have a role in neuroprotection.

Functional and Structural Improvement with a Catalytic Carbon Nano-Antioxidant in Experimental Traumatic Brain Injury Complicated by Hypotension and Resuscitation.

Hypotension worsens outcome after all severities of traumatic brain injury (TBI), with loss of cerebral autoregulation being a potential contributor. Previously, we demonstrated that intravenous injection of a high capacity catalytic antioxidant, poly(ethylene)glycol conjugated hydrophilic carbon clusters (PEG-HCCs) rapidly restored cerebral perfusion and acutely restored brain oxidative balance in a TBI model complicated by hemorrhagic hypotension without evidence of toxicity. Here, we tested whether these acute effects translated into behavioral and structural benefit. TBI was generated by a cortical contusion impactor in 38 Long Evans rats, followed by blood withdrawal to a target mean arterial pressure of 40 mm Hg. PEG-HCC (2 mg/kg) or diluent was injected intravenously 80 min later at the onset of blood resuscitation followed by another injection 2 h later (doses determined in prior studies). Performance on beam walking (performed on days 1-5) and Morris water maze (MWM) (performed on days 11-15) was tested, and lesion size was determined at the termination. PEG-HCC treatment nearly completely prevented motor dysfunction (p < 0.001 vs. diluent), improved MWM performance (p < 0.001; treatment vs. time interaction) and reduced lesion size by 61% (p = 0.054). Here we show that treatment with PEG-HCCs at a clinically realistic time point (onset of resuscitation) prevented a major portion of the neurological dysfunction induced in this TBI model, and that PEG-HCCs are candidates for additional study as a potential therapeutic agent.

Cerebral microdialysis of interleukin (IL)-1beta and IL-6: extraction efficiency and production in the acute phase after severe traumatic brain injury in rats.

BACKGROUND: As a research tool, cerebral microdialysis might be a useful technique in monitoring the release of cytokines into the extracellular fluid (ECF) following traumatic brain injury (TBI). We established extraction efficiency of Interleukin(IL)-1ss and Interleukin(IL)-6 by an in vitro microdialysis-perfusion system, followed by in vivo determination of the temporal profile of extracellular fluid cytokines after severe TBI in rats. MATERIALS AND METHODS: In vitro experiments using a polyether sulfon (PES) microdialysis probe especially developed for recovery of macromolecules such as cytokines, were carried out to establish the extraction efficiency of IL-1ss and IL-6 from artificial cerebrospinal fluid (CSF) with defined IL-1ss and IL-6 concentrations. In vivo experiments in which rats were subjected to TBI or sham and microdialysis samples were collected from the parietal lobe for measurement of cytokines. FINDINGS: The extraction efficiency was maximal 6.05% (range, 5.97-6.13%) at 0.5 microl/min(-1) and decreased at higher flow rates. Both cytokines were detectable in the dialysates. Highest IL-1ss levels were found within 200 min, highest IL-6 concentrations were detected at later intervals (200-400 min). No differences were found between the TBI and control groups. CONCLUSIONS: Cerebral microdialysis allows measurement of cytokine secretion in the ECF of brain tissue in rats.

Nitric oxide in traumatic brain injury.

Nitric oxide (NO) is a gaseous chemical messenger which has functions in the brain in a variety of broad physiological processes, including control of cerebral blood flow, interneuronal communications, synaptic plasticity, memory formation, receptor functions, intracellular signal transmission, and release of neurotransmitters. As might be expected from the numerous and complex roles that NO normally has, it can have both beneficial and detrimental effects in disease states, including traumatic brain injury. There are two periods of time after injury when NO accumulates in the brain, immediately after injury and then again several hours-days later. The initial immediate peak in NO after injury is probably due to the activity of endothelial NOS and neuronal NOS. Pre-injury treatment with 7-nitroindazole, which probably inhibits this immediate increase in NO by neuronal NOS, is effective in improving neurological outcome in some models of traumatic brain injury (TBI). After the initial peak in NO, there can be a period of relative deficiency in NO. This period of low NO levels is associated with a low cerebral blood flow (CBF). Administration of L-arginine at this early time improves CBF, and outcome in many models. The late peak in NO after traumatic injury is probably due primarily to the activity of inducible NOS. Inhibition of inducible NOS has neuroprotective effects in most models.

L-arginine and free radical scavengers increase cerebral blood flow and brain tissue nitric oxide concentrations after controlled cortical impact injury in rats.

To examine the mechanism of the increase in cerebral blood flow induced by L-arginine administration after traumatic brain injury, the cerebral hemodynamic effects of L-arginine, D-arginine, and the free radical scavengers superoxide dismutase (SOD) and catalase were compared in the controlled cortical impact injury model in rats. Animals were anesthetized with isoflurane. Measured parameters included mean blood pressure, intracranial pressure, cerebral blood flow using laser Doppler flowmetry (LDF) and brain tissue nitric oxide (NO) concentrations using an NO electrode. L-arginine, but not D-arginine, administration resulted in a significant increase in tissue NO concentrations and an improvement in LDF at the impact site, compared to control animals given saline. Administration of SOD alone and in combination with catalase resulted in a significant increase in brain tissue NO concentrations. However, LDF was consistently improved only when both SOD and catalase were given. These studies support the theory that L-arginine administration improves post-traumatic cerebral blood flow by increasing NO production. Free radical production after trauma may also contribute to the reduction in CBF by inactivating NO.

Comparison of tetrahydrobiopterin and L-arginine on cerebral blood flow after controlled cortical impact injury in rats.

The purpose of this study was to compare the effects of L-arginine and tetrahydrobiopterin administration on post-traumatic cerebral blood flow (CBF) and tissue levels of NO in injured brain tissue. Rats were anesthetized with isoflurane. Mean blood pressure, intracranial pressure, cerebral blood flow using laser Doppler flowmetry (LDF) and brain tissue nitric oxide (NO) concentrations were measured prior to, and for 2 h after a controlled cortical impact injury. L-arginine, 300 mg/kg, tetrahydrobiopterin, 10 mg/kg, or equal volume of saline was given at 5 min after injury. In the saline-treated animals, LDF decreased to 34 +/- 4% of baseline values after injury. NO concentration also decreased by approximately 20 pmol/ml from baseline values. L-arginine and tetrahydrobiopterin administration both resulted in a significant preservation of tissue NO concentrations and an improvement in LDF, compared to control animals given saline. These studies demonstrate that tetrahydrobiopterin administration has a beneficial effect on cerebral blood flow that is similar to L-arginine administration, and may suggest that depletion of tetrahydrobiopterin plays a role in the post-traumatic hypoperfusion of the brain.

The role of endothelial nitric oxide synthase in the cerebral hemodynamics after controlled cortical impact injury in mice.

Traumatic brain injury causes a reduction in cerebral blood flow, which may cause additional damage to the brain. The purpose of this study was to examine the role of nitric oxide produced by endothelial nitric oxide synthase (eNOS) in these vascular effects of trauma. To accomplish this, cerebral hemodynamics were monitored in mice deficient in eNOS and wild-type control mice that underwent lateral controlled cortical impact injury followed by administration of either L-arginine, 300 mg/kg, or saline at 5 min after the impact injury. The eNOS deficient mice had a greater reduction in laser Doppler flow (LDF) in the contused brain tissue at the impact site after injury, despite maintaining a higher blood pressure. L-Arginine administration increased LDF post-injury only in the wild-type mice. L-Arginine administration also resulted in a reduction in contusion volume, from 2.4 +/- 1.5 to 1.1 +/- 1.2 mm(3) in wild-type mice. Contusion volume in the eNOS deficient mice was not significantly altered by L-arginine administration. These differences in cerebral hemodynamics between the eNOS-deficient and the wild-type mice suggest an important role for nitric oxide produced by eNOS in the preservation of cerebral blood flow in contused brain following traumatic injury, and in the improvement in cerebral blood flow with L-arginine administration.

Histopathological and behavioral effects of immediate and delayed hemorrhagic shock after mild traumatic brain injury in rats.

The purpose of this study was to investigate the increased susceptibility of the brain, after a controlled mild cortical impact injury, to a secondary ischemic insult. The effects of the duration and the timing of the secondary insult after the initial cortical injury were studied. Rats anesthetized with isoflurane underwent a 3 m/sec, 2.5-mm deformation cortical impact injury followed by hypotension to 40 mm Hg induced by withdrawing blood from a femoral vein. The duration of hypotension was varied from 40 to 60 min. The timing of 60 min of hypotension was varied from immediately post-injury to 7 days after the injury. Outcome was assessed by behavioral tasks and histological examination at 2 weeks post-injury. A separate group of animals underwent measurement of the acute physiology including mean blood pressure (MAP), intracranial pressure (ICP), and cerebral blood flow (CBF) using a laser Doppler technique. Increasing durations of hypotension resulted in marked expansion of the contusion, from 6.5±1.8 mm³ with sham hypotension to 27.1±3.9 mm³ with 60 min of hypotension. This worsening of the contusion was found only when then hypotension occurred immediately after injury or at 1 h after injury. CA3 neuron loss followed a similar pattern, but the injury group differences were not significant. Motor tasks, including beam balance and beam walking, were significantly worse following 50 and 60 min of hypotension. Performance on the Morris water maze task was also significantly related to the injury group. Studies of the acute cerebral hemodynamics demonstrated that CBF was significantly more impaired during hypotension in the animals that underwent the mild TBI compared to those that underwent sham TBI. The perfusion deficit was worst at the impact site, but also significant in the pericontusional brain. With 50 and 60 min of hypotension, CBF did not recover following resuscitation at the impact site, and recovered only transiently in the pericontusional brain. These results demonstrate that mild TBI, like more severe levels of TBI, can impair the brain's ability to maintain CBF during a period of hypotension, and result in a worse outcome.

Neuroprotection with an erythropoietin mimetic peptide (pHBSP) in a model of mild traumatic brain injury complicated by hemorrhagic shock.

Pyroglutamate helix B surface peptide (pHBSP) is an 11 amino acid peptide, designed to interact with a novel cell surface receptor, composed of the classical erythropoietin (EPO) receptor disulfide linked to the beta common receptor. pHBSP has the cytoprotective effects of EPO without stimulating erythropoiesis. Effects on early cerebral hemodynamics and neurological outcome at 2 weeks post-injury were compared in a rat model of mild cortical impact injury (3m/sec, 2.5 mm deformation) followed by 50 min of hemorrhagic hypotension (MAP 40 mm Hg for 50 min). Rats were randomly assigned to receive 5000 U/kg of EPO, 30 µg/kg of pHBSP, or an inactive substance every 12 h for 3 days, starting at the end of resuscitation from the hemorrhagic hypotension, which was 110 min post-injury. Both treatments reduced contusion volume at 2 weeks post-injury, from 20.8±2.8 mm(3) in the control groups to 7.7±2.0 mm(3) in the EPO-treated group and 5.9±1.5 mm(3) in the pHBSP-treated group (p=0.001). Both agents improved recovery of cerebral blood flow in the injured brain following resuscitation, and resulted in more rapid recovery of performance on beam balancing and beam walking tests. These studies suggest that pHBSP has neuroprotective effects similar to EPO in this model of combined brain injury and hypotension. pHBSP may be more useful in the clinical situation because there is less risk of thrombotic adverse effects.

Antioxidant carbon particles improve cerebrovascular dysfunction following traumatic brain injury.

Injury to the neurovasculature is a feature of brain injury and must be addressed to maximize opportunity for improvement. Cerebrovascular dysfunction, manifested by reduction in cerebral blood flow (CBF), is a key factor that worsens outcome after traumatic brain injury (TBI), most notably under conditions of hypotension. We report here that a new class of antioxidants, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), which are nontoxic carbon particles, rapidly restore CBF in a mild TBI/hypotension/resuscitation rat model when administered during resuscitation--a clinically relevant time point. Along with restoration of CBF, there is a concomitant normalization of superoxide and nitric oxide levels. Given the role of poor CBF in determining outcome, this finding is of major importance for improving patient health under clinically relevant conditions during resuscitative care, and it has direct implications for the current TBI/hypotension war-fighter victims in the Afghanistan and Middle East theaters. The results also have relevancy in other related acute circumstances such as stroke and organ transplantation.

Neuroprotection with erythropoietin administration following controlled cortical impact injury in rats.

This study was designed to determine the effect of erythropoietin (Epo) on cerebral blood flow (CBF), nitric oxide (NO) concentration, and neurological outcome after traumatic brain injury. In one experiment, the hemodynamic effects of Epo were determined after controlled cortical impact injury (CCII) by measuring mean arterial pressure, intracranial pressure, CBF using laser Doppler flowmetry, and brain tissue NO concentrations using an NO electrode. In total, 41 rats were given either Epo (5000 U/kg) or saline s.c. 3 days before injury. In animals pretreated with saline, L-arginine but not D-arginine administration resulted in a significant increase in tissue NO concentrations and an improvement in CBF at the impact site. Likewise, in animals pretreated with Epo, L-arginine but not D-arginine given postinjury increased brain tissue NO concentrations and increased CBF. In another experiment, 74 rats underwent CCII (3-mm deformation, velocity 5 m/s), and they were given saline or Epo 5000 U/kg s.c. at 5 min, 1 h, 3 h, 6 h, 9 h, or 12 h postinjury. The contusion volume and cell counts of viable neurons in the CA1 and CA3 regions of the hippocampus were assessed at 2 weeks postinjury. The contusion volume was significantly reduced at 5 min, 1 h, 3 h, and 6 h postinjury Epo administration. The neuron density in the CA1 and CA3 region of the hippocampus was increased at 1, 3, and 6 h after injury. These data demonstrate the neuroprotective effects of Epo in traumatic injury, and the effects are optimal when Epo is given within 6 h of injury.

Neuroprotective effects of L-arginine administration after cortical impact injury in rats: dose response and time window.

Administration of L-arginine has been shown to increase cerebral blood flow and reduce neurological damage after experimental traumatic brain injury. The purpose of this study was to examine the optimal dose and time window for these neuroprotective effects. In a dose response experiment, doses of L-arginine ranging from 37.5 to 600 mg/kg were administered 5 min after a 5-m/s, 3-mm, controlled cortical impact in rats. The amount of brain injury found at 2 weeks after injury, both at the contusion site and in the ipsilateral hippocampus, were inversely related to the dose of L-arginine administered. Both 300- and 600-mg/kg doses of L-arginine significantly reduced contusion volume. The 300-mg/kg dose significantly increased the neuron density in the CA1 region of the hippocampus. Physiological effects of L-arginine were also dose-related. The greatest reduction in intracranial pressure occurred with the 300-mg/kg dose of L-arginine. Doses up to 300 mg/kg were well tolerated, but the 600-mg/kg dose resulted in transient hypotension. In another experiment, 300 mg/kg L-arginine was administered at times varying from 5 min to 48 h after injury. Contusion volume was significantly reduced when the L-arginine was given at 5 min and 1 h after injury. The protective effect was less when the same dose was given at the later times, but there was no evidence of an adverse effect even when the L-arginine was administered 48 h after injury.

Laser Doppler flow and brain tissue PO2 after cortical impact injury complicated by secondary ischemia in rats treated with arginine.

BACKGROUND: Traumatic brain injury (TBI) makes the brain susceptible to secondary insults such as ischemia. This study tested the hypothesis that L-arginine would increase regional CBF (rCBF) and brain tissue PO2 (PbtO2) at the injury site. METHODS: A secondary insult model was employed in rodents. rCBF was measured with laser doppler flowmetry (LDF) and PbtO2 with a PO2 catheter at the impact site. Animals were randomized to receive L-arginine, D-arginine or saline intravenously, 5 minutes after impact. RESULTS: In animals who received L-arginine, the percentage rCBF from baseline (%CBF) was higher at the impact site after impact (p < 0.001), during bilateral carotid occulation (BCO) (p = 0.001) and during reperfusion (p = 0.032). In contrast, PbtO2 was not significantly increased throughout the experiment for the L-arginine group. CONCLUSIONS: Administration of L-arginine increased rCBF in the injured brain tissue, and resulted in better preservation of CBF during BCO than D-arginine and saline.

Oxidative DNA lesions in a rodent model of traumatic brain injury.

BACKGROUND: Oxidative DNA lesions have not been well studied in traumatic brain injury (TBI). METHODS: TBI was induced with a controlled cortical impact injury in rats. Brain tissue was examined for 8-hydroxy-2'-deoxyguanosine (oh8dG) using mono-clonal antibodies at different time frames; 15 minutes (n = 4), 30 minutes (n = 7), 60 minutes (n = 6), and 240 minutes (n = 5). The control group consisted of sham-operated animals undergoing the same surgery without the controlled cortical impact injury (n = 5). RESULTS: An elevation of oh8dG was detected in the nuclear and perinuclear (mitochondrial) regions of the ipsilateral cortex, but seldom in those of the contralateral cortex. The amount of oh8dG in those animals with TBI was significant in all time frames when compared with sham-operated controls (p < 0.001). The oh8dG levels were more prominent at 15 minutes (p < 0.0001) when compared with controls. CONCLUSION: Oxidative DNA lesions occurred in this model of TBI maximally early after TBI. This suggests that oh8dGs may affect genetic material of the brain and that oh8dGs may adversely affect gene expression that occurs early after head injury.