Leela Cherian

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.

Regional cerebral blood flow after controlled cortical impact injury in rats

Regional cerebral blood flow (rCBF) and laser-Doppler perfusion were measured in rats after controlled cortical impact injury (CCII), a model of traumatic brain injury. Male Long-Evans rats were anesthetized with isoflurane and surgically prepared with arterial and venous cannulae. CCII was induced after a craniectomy by deforming the right parietal cortex to a depth of 2 mm with a metal cylinder traveling at a velocity of 5.2 m/s. Laser-Doppler perfusion was monitored from the surface of the left frontal cortex. rCBF, using14 C-isopropyliodoamphetamine, and laser-Doppler perfusion were measured in three groups of rats consisting of a sham group (n = 6), a group 30 min after CCII (n = 6), and a group 4 h after CCII (n = 5). CCII was characterized by cortical ischemia (rCBF < 20 mL centered dot 100 g-1 centered dot min-1) surrounding the site of impact. The occipital cortex, a cortical region distant from the impact site, was also ischemic in some, but not all, injured rats. In the 30-min group, the ischemic zone showed very sharp boundaries with cortical areas of hyperperfusion surrounding the ischemic zone. In the 4-h group, the ischemic boundaries were not as sharp and the hyperperfusion surrounding the ischemic zone was no longer present. The caudate-putamen, hippocampus, and thalamus showed significant reductions in rCBF ranging from 50% to 30% of control 30 min and 4 h postinjury, respectively. We conclude that complex changes in rCBF occur shortly after CCII and persist for at least 4 h. During this time several brain regions, especially the cortical areas, may suffer damage due to the ischemia. (Anesth Analg 1995;80:687-95)

Cerebral hemodynamic effects of phenylephrine and L-arginine after cortical impact injury.

Objective: To determine the effects of a pressor agent (phenylephrine and L-arginine) on the abnormal cerebral hemodynamics and on neurologic outcome after a severe cortical impact injury in rats. Design: Prospective, randomized study. Setting: University Iaboratory. Subjects: Male Long-Evans rats, weighing 300 to 400 g, fasted overnight. Interventions : The animals were anesthetized with isoflurane, and a severe cortical impact injury (velocity, 5 m/sec; deformation, 3 mm) was produced In the right parietal cortex. Five minutes after impact injury, one of the following three treatments were infused: 1 mL saline Intravenously for 10 mlns, 300 mg/kg L-arginine In 1 mL saline intravenously for 10 mins, or 0.3 μg/ kg/min phenylephrine intravenously for 3 hrs. Mean arterial pressure, intracranial pressure (ICP), cerebral perfusion pressure (CPP), and laser Doppler flow (LDF) at the impact site and in the contralateral parietal cortex were monitored for 3 hrs after the impact injury. Histologic examination of the brain was performed at 2 wks after injury In a separate group of L-arginine- and saline-treated animals. Measurements and Main Results: The immediate response to the Impact injury was an increase in ICP, and a decrease in mean arterial pressure, CPP, and LDF. In the saline-treated animals, LDF decreased to <25% of the baseline values at the impact site and stayed at that level for the entire 3-hr monitoring period. On the contralateral side, LDF decreased Initially and recovered gradually to approximately 50% of the preimpact baseline value. Infusion of both phenylephrine and L-arginine increased LDF back to near-baseline levels. However, phenylephrine increased ICP significantly, whereas ICP with L-arginine did not change. L-arginine treatment reduced the contusion volume from a median value of 5.28 mm 3 to 0.63 mm 3 . Conclusions: Phenylephrine increased cerebral blood flow (CBF) by increasing CPP. L-arginine, however, Increased CBF without changing CPP. The improvement in CBF was accompanied by a decrease in neurologic injury. Although the pressor agents are used currently to increase CBF after traumatic brain injury, other strategies may also increase CBF without the potential adverse effects of Induced hypertension.

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 with in 6 h of injury.

Hyperglycemia increases brain injury caused by secondary ischemia after cortical impact injury in rats.

OBJECTIVE To examine the effects of glucose infusion on the histologic brain damage caused by controlled cortical impact injury alone and by cortical impact injury complicated by secondary ischemia. DESIGN Prospective, randomized study. SETTING University laboratory. SUBJECTS Male Long-Evans rats. INTERVENTIONS Three experimental conditions were studied: a) 2.5-mm deformation impact (velocity 4 m/sec) injury followed by 40 mins of bilateral carotid occlusion; b) sham impact injury followed by 40 mins of bilateral carotid occlusion; and c) 2.5-mm deformation impact (velocity 4 m/sec) injury followed by sham carotid occlusion. For each experimental condition, animals were randomized to receive either glucose solution or saline solution before the induced injury and the sham impact injury. Contusion volume and neuron density in the CA1 and CA3 regions of the hippocampus were measured 2 wks after the injury. MEASUREMENTS AND MAIN RESULTS Parenteral administration of 2.2 g/kg glucose solution increased the blood glucose concentration from 6.7 +/- 3.3 to 17.9 +/- 10.6 mmol/L before the impact injury, and to 12.3 +/- 5.6 mmol/L before carotid occlusion. Hyperglycemia had the greatest effect on the consequences of the impact injury complicated by secondary ischemia, increasing contusion volume from 1 to 30.6 mm3 in the animals that received saline or glucose solution, respectively (p = .005), and reducing the density of normal appearing neurons in the CA1 area of the hippocampus from 201 to 144 cells/mm2 in the animals that received saline solution and glucose solution, respectively (p = .038). The impact injury alone and bilateral carotid occlusion alone caused minimal neuronal loss in the hippocampus and minimal contusion or infarction at the impact site. Individually, these mild injuries were not adversely affected by infusion of glucose solution. CONCLUSION Hyperglycemia increases brain damage when traumatic brain injury is complicated by secondary ischemia.

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.

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.

Axon Initial Segment–Associated Microglia

Microglia are the brains 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.

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.

Gangliosides, or sialic acid, antagonize ethanol intoxication

Because ethanol elicits a dose-dependent hydrolysis of brain sialogangliosides, we tested the possibility that injected gangliosides might antagonize intoxicating doses of ethanol. Clear anti-intoxication effects were seen at 24 hr post-injection of mixed mouse-brain gangliosides at 125-130 mg/kg, but not at lower or higher doses. Sleep time was reduced on the order of 50%, and roto-rod agility was significantly enhanced. Sialic acid (SA) similarly antagonized ethanol; however, the precursor of SA, N-acetyl-D-mannosamine, as well as ceramide and asialoganglioside did not.