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Role of nitric oxide in traumatic brain injury in the rat

Kojiro Wada, Katina Chatzipanteli, Raul Busto, and W. Dalton Dietrich

Object. Although nitric oxide (NO) has been shown to play an important role in the pathophysiological process of cerebral ischemia, its contribution to the pathogenesis of traumatic brain injury (TBI) remains to be clarified. The authors investigated alterations in constitutive nitric oxide synthase (NOS) activity after TBI and the histopathological response to pharmacological manipulations of NO.

Methods. Male Sprague—Dawley rats underwent moderate (1.7–2.2 atm) parasagittal fluid-percussion brain injury. Constitutive NOS activity significantly increased within the ipsilateral parietal cerebral cortex, which is the site of histopathological vulnerability, 5 minutes after TBI occurred (234.5 ± 60.2% of contralateral value [mean ± standard error of the mean {SEM}], p < 0.05), returned to control values by 30 minutes (114.1 ± 17.4%), and was reduced at 1 day after TBI (50.5 ± 13.1%, p < 0.01). The reduction in constitutive NOS activity remained for up to 7 days after TBI (31.8 ± 6.0% at 3 days, p < 0.05; 20.1 ± 12.7% at 7 days, p < 0.01). Pretreatment with 3-bromo-7-nitroindazole (7-NI ) (25 mg/kg), a relatively specific inhibitor of neuronal NOS, significantly decreased contusion volume (1.27 ± 0.17 mm3 [mean ± SEM], p < 0.05) compared with that of control (2.52 ± 0.35 mm3). However, posttreatment with 7-NI or pre- or posttreatment with nitro-l-arginine-methyl ester (l-NAME) (15 mg/kg), a nonspecific inhibitor of NOS, did not affect the contusion volume compared with that of control animals (1.87 ± 0.46 mm3, 2.13 ± 0.43 mm3, and 2.18 ± 0.53 mm3, respectively). Posttreatment with l-arginine (1.1 ± 0.3 mm3, p < 0.05), but not 3-morpholino-sydnonimine (SIN-1) (2.48 ± 0.37 mm3), significantly reduced the contusion volume compared with that of control animals.

Conclusions. These data indicate that constitutive NOS activity is affected after moderate parasagittal fluid percussion brain injury in a time-dependent manner. Inhibition of activated neuronal NOS and/or enhanced endothelial NOS activation may represent a potential therapeutic strategy for the treatment of TBI.

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Stilbazulenyl nitrone, a novel azulenyl nitrone antioxidant, improved neurological deficit and reduced contusion size after traumatic brain injury in rats

Ludmila Belayev, David A. Becker, Ofelia F. Alonso, Yitao Liu, Raul Busto, James J. Ley, and Myron D. Ginsberg

Object. Stilbazulenyl nitrone (STAZN) is a second-generation azulenyl nitrone that has markedly enhanced antioxidant properties compared with those of conventional alpha-phenyl nitrones. In this study, the authors assessed the potential efficacy of STAZN in a rodent model of fluid-percussion brain injury, which results in a consistent cortical contusion.

Methods. After anesthesia had been induced in normothermic Sprague—Dawley rats (brain temperature 36–36.5°C) by halothane—nitrous oxide, the animals were subjected to a right parietooccipital parasagittal fluid-percussion injury (1.5–2 atm). The agent (STAZN, 30 mg/kg; eight animals) or vehicle (dimethyl sulfoxide; eight animals) was administered intraperitoneally at 5 minutes and 4 hours after trauma. The neurological status of each rat was evaluated on Days 1, 2, and 7 postinjury (normal score 0, maximum injury 12). Seven days after trauma, the rat brains were perfusion fixed, coronal sections at various levels were digitized, and areas of contusion were measured. Treatment with STAZN significantly improved neurological scores on Days 2 and 7 postinjury compared with vehicle-treated rats. Administration of STAZN also significantly reduced the total contusion area by 63% (1.8 ± 0.5 mm2 in STAZN-treated animals compared with 4.8 ± 2.1 mm2 in vehicle-treated animals; p = 0.04) and the deep cortical contusion area by 60% (1.2 ± 0.2 mm2 in STAZN-treated animals compared with 2.9 ± 1.2 mm2 in vehicle-treated animals; p = 0.03). By contrast, hippocampal cell loss in the CA3 sector was unaffected by STAZN treatment.

Conclusions. Therapy with STAZN, a novel potent antioxidant, administered following traumatic brain injury, markedly improves neurological and histological outcomes. Azulenyl nitrones appear to represent a promising class of neuroprotective agents for combating this devastating condition.

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Diminution of metabolism/blood flow uncoupling following traumatic brain injury in rats in response to high-dose human albumin treatment

Myron D. Ginsberg, Weizhao Zhao, Ludmila Belayev, Ofelia F. Alonso, Yitao Liu, Judith Y. Loor, and Raul Busto

Object. The authors have recently demonstrated that high-dose human albumin is markedly neuroprotective in experimental traumatic brain injury (TBI) and cerebral ischemia. The pathophysiology of TBI involves acute uncoupling of cerebral glucose utilization and blood flow. The intent of this study was to establish whether the use of human albumin therapy in a model of acute TBI would influence this phenomenon.

Methods. Anesthetized, physiologically regulated rats received moderate (1.5–2 atm) fluid-percussion injury to the parietal lobe. Fifteen minutes after trauma or sham injury, rats in one group received human albumin (2.5 g/kg) administered intravenously and those in another group received 0.9% saline vehicle. At 60 minutes and 24 hours posttrauma, autoradiographic studies of local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCMRglu) were conducted, and the LCMRglu/LCBF ratio was determined. Sham-injured rats had normal levels of LCBF and LCMRglu, and no differences between vehicle- and albumin-treated rats were evident. Sixty minutes after TBI, LCBF was moderately reduced bilaterally in vehicle-treated rats, whereas in albumin-treated animals, the LCBF contralateral to the side of injury was generally normal. Despite acutely depressed LCBF, LCMRglu in vehicle-treated rats at 60 minutes was paradoxically normal bilaterally, and foci of elevated LCMRglu were noted in the ipsilateral hippocampus and thalamus. By contrast, in albumin-treated rats studied 60 minutes post-TBI, reduced LCMRglu values were measured in the ipsilateral caudoputamen and parietal cortex, whereas LCMRglu in other ipsilateral and contralateral sites did not differ from that measured in sham-injured animals. The metabolism/blood flow ratio was normal in sham-injured rats, but became markedly elevated in vehicle-treated rats 60 minutes post-TBI (on average, by threefold ipsilaterally and 2.1-fold contralaterally). By contrast, the mean metabolism/blood flow ratio in albumin-treated animals was elevated by only 1.6-fold ipsilaterally and was normal contralaterally. Twenty-four hours after TBI, LCBF contralateral to the side of injury had generally returned to normal levels in the albumin-treated group.

Conclusions. These results demonstrate that human albumin therapy benefits the posttraumatic brain by diminishing the pronounced metabolism > blood flow dissociation that would otherwise occur within the 1st hour after injury. Viewed together with our previous evidence of histological neuroprotection, these findings indicate that human albumin therapy may represent a desirable treatment modality for acute TBI.

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Effect of delayed albumin hemodilution on infarction volume and brain edema after transient middle cerebral artery occlusion in rats

Ludmila Belayev, Raul Busto, Weizhao Zhao, James A. Clemens, and Myron D. Ginsberg

✓ The authors examined the effect of delayed high-concentration albumin therapy on ischemic injury in a highly reproducible model of middle cerebral artery (MCA) occlusion in rats. Male Sprague—Dawley rats weighing 270 to 320 g were anesthetized with halothane and subjected to 120 minutes of temporary MCA occlusion induced by means of a poly-l-lysine—coated intraluminal nylon suture inserted retrograde via the external carotid artery into the internal carotid artery and MCA. The agent (20% human serum albumin [HSA]) or control solution (sodium chloride 0.9%) was administered intravenously at a dosage of 1% of body weight immediately after suture removal following a 2-hour period of MCA occlusion. The animals' neurological status was evaluated during MCA occlusion (at 60 minutes) and daily for 3 days thereafter. The brains were perfusion-fixed, and infarct volumes and brain edema were determined. The HSA significantly improved the neurological score compared with saline at 24 hours after MCA occlusion. The rats treated with HSA also had significantly reduced total infarct volume (by 34%) and brain edema (by 81%) compared with saline-treated rats. There was a strong correlation between hematocrit level and brain edema (p < 0.01), and between total infarct volume or brain edema and neurological score at 24, 48, and 72 hours postinjury (p < 0.0002).

These results strongly support the beneficial effect of delayed albumin therapy in transient focal ischemia and indicate its possible usefulness in treating patients with acute ischemic stroke.

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Comparative neuroprotective efficacy of prolonged moderate intraischemic and postischemic hypothermia in focal cerebral ischemia

Pil W. Huh, Ludmila Belayev, Weizhao Zhao, Sebastian Koch, Raul Busto, and Myron D. Ginsberg

Object. The purpose of this study was to compare the effects of prolonged hypothermia on ischemic injury in a highly reproducible model of middle cerebral artery (MCA) occlusion in rats.

Methods. Male Sprague—Dawley rats were anesthetized with halothane and subjected to 120 minutes of temporary MCA occlusion by retrograde insertion of an intraluminal nylon suture coated with poly-l-lysine through the external carotid artery into the internal carotid artery and the MCA. Two levels of prolonged postischemic cranial hypothermia (32°C and 27°C) and one level of intraischemic cranial hypothermia (32°C) were compared with the ischemic normothermia (37°C) condition. Target cranial temperatures were maintained for 3 hours and then gradually restored to 35°C over an additional 2-hour period. The animals were evaluated using a quantitative neurobehavioral battery of tests before inducing MCA occlusion, during occlusion (at 60 minutes postonset in all rats except those in the intraischemic hypothermia group), and at 24, 48, and 72 hours after reperfusion. The rat brains were perfusion fixed at 72 hours after ischemia, and infarct volumes and brain edema were determined. Both intraischemic and postischemic cooling to 32°C led to similar significant reductions in cortical infarct volume (by 89% and 88%, respectively) and total infarct volume (by 54% and 69%, respectively), whereas postischemic cooling to 27°C produced lesser reductions (64% and 49%, respectively), which were not statistically significant. All three hypothermic regimens significantly lessened hemispheric swelling and improved the neurological score at 24 hours. The authors' data confirm that a high degree of histological neuroprotection is conferred by postischemic cooling to 32°C, which is virtually equivalent to that observed with intraischemic cooling to the same level.

Conclusions. These results may be relevant to the design of future clinical trials of therapeutic hypothermia for acute ischemic stroke.

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Influence of early posttraumatic hypothermia therapy on cerebral blood flow and glucose metabolism after fluid-percussion brain injury

Weizhao Zhao, Ofelia F. Alonso, Judith Y. Loor, Raul Busto, and Myron D. Ginsberg

Object. Using autoradiographic image averaging, the authors recently described prominent foci of marked glucose metabolism-greater-than-blood-flow uncoupling in the acutely traumatized rat brain. Because hypothermia is known to ameliorate injury in this and other injury models, the authors designed the present study to assess the effects of posttraumatic therapeutic hypothermia on the local cerebral metabolic rate of glucose (LCMRglu) and local cerebral blood flow (LCBF) following moderate parasagittal fluid-percussion head injury (FPI) in rats.

Methods. Either cranial hypothermia (30°C) or normothermia (37°C) was induced for 3 hours in matched groups of rats immediately after FPI; LCMRglu and LCBF were assessed 3 hours after concluding these temperature manipulations.

In rats subjected to FPI, regardless of whether normothermia or hypothermia ensued, LCBF was reduced relative to the sham-injury groups. In addition, when FPI was followed by hypothermia (FPI—30°C group), the subsequent LCBF was significantly lower (35–38% on average) than in FPI—37°C rats. Statistical mapping of LCBF difference imaging data revealed confluent cortical and subcortical zones of significantly reduced LCBF (largely ipsilateral to the prior injury) in FPI—30°C rats relative to the FPI—37°C group. Local glucose utilization was reduced in both hemispheres of FPI—37°C rats relative to the sham-injury group and was lower in the right (traumatized) hemisphere than in the left. However, LCMRglu values were largely unaffected by temperature manipulation in either the FPI or sham-injury groups. The LCMRglu/LCBF ratio was nearly doubled in FPI—30°C rats relative to the FPI—37°C group, in a diffuse and bihemispheric fashion. Linear regression analysis comparing LCMRglu and LCBF revealed that the FPI—37°C and FPI—30°C data sets were completely nonoverlapping, whereas the two sham-injury data sets were intermixed.

Conclusions. Despite its proven neuroprotective efficacy, early posttraumatic hypothermia (30°C for 3 hours) nonetheless induces a moderate decline in cerebral perfusion without the (anticipated) improvement in cerebral glucose utilization, so that a state of mild metabolism-greater-than-blood-flow dissociation is perpetuated.

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Comparative neuroprotective efficacy of prolonged moderate intraischemic and postischemic hypothermia in focal cerebral ischemia

Pil W. Huh, Ludmila Belayev, Weizhao Zhao, Sebastian Koch, Raul Busto, and Myron D. Ginsberg

Object

The purpose of this study was to compare the effects of prolonged hypothermia on ischemic injury in a highly reproducible model of middle cerebral artery (MCA) occlusion in rats.

Methods

Male Sprague-Dawley rats were anesthetized with halothane and subjected to 120 minutes of temporary MCA occlusion by retrograde insertion of an intraluminal nylon suture coated with poly-L-lysine through the external carotid artery into the internal carotid artery and the MCA. Two levels of prolonged postischemic cranial hypothermia (32°C and 27°C), and one level of intraischemic cranial hypothermia (32°C) were compared with the ischemic normothermia (37°C) condition. Target cranial temperatures were maintained for 3 hours and then gradually restored to 35°C over an additional 2-hour period. The animals were evaluated using a quantitative neurobehavioral battery of tests before inducing MCA occlusion, during occlusion (at 60 minutes postonset in all rats except those in the intraischemic hypothermia group), and at 24, 48, and 72 hours after reperfusion. The rat brains were perfusion fixed at 72 hours after ischemia and infarct volumes and brain edema were determined. Both intraischemic and postischemic cooling to 32°C led to similar significant reductions in cortical infarct volume (by 89 and 88%, respectively) and total infarct volume (by 54 and 69%, respectively), whereas postischemic cooling to 27°C produced lesser reductions (64 and 49%, respectively), which were not statistically significant. All three hypothermic regimens significantly lessened hemispheric swelling and improved the neurological score at 24 hours. Our data confirm that a high degree of histological neuroprotection is conferred by postischemic cooling to 32°C, which is virtually equivalent to that observed with intraischemic cooling to the same level.

Conclusions

These results may be relevant to the design of future clinical trials of therapeutic hypothermia for acute ischemic stroke.

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Influence of early posttraumatic hypothermia therapy on local cerebral blood flow and glucose metabolism after fluid-percussion brain injury

Weizhao Zhao, Ofelia F. Alonso, Judith Y. Loor, Raul Busto, and Myron D. Ginsberg

Object

Using autoradiographic image averaging, the authors recently described prominent foci of marked glucose metabolism-greater-than-blood-flow uncoupling in the acutely traumatized rat brain. Because hypothermia is known to ameliorate injury in this and other injury models, the authors designed the present study to assess the effects of post-traumatic therapeutic hypothermia on the local cerebral metabolic rate of glucose (LCMRglu) and local cerebral blood flow (LCBF) following moderate parasagittal fluid-percussion head injury (FPI) in rats.

Methods

Either cranial hypothermia (30°C) or normothermia (37°C) was induced for 3 hours in matched groups of rats immediately after FPI; LCMRglu and LCBF were assessed 3 hours after concluding these temperature manipulations.

In rats subjected to FPI, regardless of whether normothermia or hypothermia ensued, LCBF was reduced relative to the sham-injury groups. In addition, when FPI was followed by hypothermia (FPI–30°C group), the subsequent LCBF was significantly lower (35–38% on average) than in FPI–37°C rats. Statistical mapping of LCBF difference imaging data revealed confluent cortical and subcortical zones of significantly reduced LCBF (largely ipsilateral to the prior injury) in FPI–30°C rats relative to the FPI–37°C group. Local glucose utilization was reduced in both hemispheres of FPI–37°C rats relative to the sham-injury group and was lower in the right (traumatized) hemisphere than in the left. However, LCMRglu values were largely unaffected by temperature manipulation in either the FPI or sham-injury groups. The LCMRglu/LCBF ratio was nearly doubled in FPI–30°C rats relative to the FPI–37°C group, in a diffuse and bihemispheric fashion. Linear regression analysis comparing LCMRglu and LCBF revealed that the FPI–37°C and FPI–30°C data sets were completely nonoverlapping, whereas the two sham-injury data sets were intermixed.

Conclusions

Despite its proven neuroprotective efficacy, early posttraumatic hypothermia (30°C for 3 hours) nonetheless induces a moderate decline in cerebral perfusion without the (anticipated) improvement in cerebral glucose utilization, so that a state of mild metabolism-greater-than-blood-flow dissociation is perpetuated.