Susan Kraydieh

The Significance of Brain Temperature in Focal Cerebral Ischemia: Histopathological Consequences of Middle Cerebral Artery Occlusion in the Rat

The purpose of this study was to determine the effect of selective modulation of brain temperature in the experimental settings of permanent and reversible middle cerebral artery (MCA) occlusion in Sprague–Dawley rats. Three models of proximal MCA occlusion were used, in which the effect of brain-temperature modulations could be studied. These included (a) permanent MCA occlusion with an initial 30-min period of hypotension (30 or 36°C × 4 h), (b) permanent MCA occlusion alone (30, 36, or 39°C × 2 h), and (c) 2 h of reversible MCA occlusion (30, 36, or 39°C × 2 h). In the transient MCA occlusion series, intra- and postischemic cortical blood flow was assessed using a laser–Doppler flowmeter placed over the dorsolateral cortex. After a 3-day survival, all rats were perfusion fixed for histopathological analysis and the determination of infarct volume. In animals with permanent MCA occlusion plus hypotension, no significant difference in infarct volume was demonstrated between the 30 and 36°C groups. In rats with permanent MCA occlusion without hypotension, significant differences in infarct volume were again not demonstrable, but an interaction between infarct area and temperature class was shown by repeated-measures analysis, indicating that hypothermia altered the topographic pattern of the cortical infarct. With 2 h of reversible MCA occlusion, there was a statistically significant reduction in infarct volume in the 30°C group compared to 39°C rats. Although intra- and postischemic CBF were not significantly different among the three temperature groups, the cortical infarct volume was positively correlated with postischemic CBF. The postischemic CBF, in turn, was positively correlated to the intraischemic brain temperature and was negatively correlated to CBF during the ischemic period. These findings demonstrate that moderate manipulations of brain temperature have a greater influence on the resulting cortical infarction in the setting of transient focal ischemia than in the context of permanent vascular occlusion.

Delayed Postischemic Hyperthermia in Awake Rats Worsens the Histopathological Outcome of Transient Focal Cerebral Ischemia

BACKGROUND AND PURPOSE Over the past several years, it has been demonstrated that mild intraischemic or immediate postischemic hyperthermia worsens ischemic outcome in models of global and focal ischemia. Periods of hyperthermia are commonly seen in patients after stroke and cardiac arrest. The hypothesis tested in this study was that a brief hyperthermic period, even when occurring days after an ischemic insult, has detrimental effects on the pathological outcome of focal ischemia. METHODS Rats were subjected to 60 minutes of transient middle cerebral artery occlusion by insertion of an intraluminal filament. Twenty-four hours after reperfusion, awake rats were subjected to temperature modulation for 3 hours in a heating chamber. The brain temperature was equilibrated to either 37 degrees C to 38 degrees C, or 40 degrees C. Changes in rectal temperature and blood glucose concentration were evaluated during and just after temperature modulation. Behavioral tests were also assessed. Three days after temperature modulation, brains were perfusion-fixed, and infarct volumes were determined. RESULTS In animals with 40 degrees C hyperthermia, cortical and total infarct volumes were markedly greater (92.2 +/- 63.1 and 126.5 +/- 72.3 mm3 [mean +/- SD], respectively) than in normothermic rats (14.4 +/- 12.7 and 42.4 +/- 19.2 mm3) and in animals with 39 degrees C hyperthermia (16.5 +/- 28.7 and 40.9 +/- 34.3 mm3) (P < .05), whereas there was no significant difference between normothermic and 39 degrees C hyperthermic animals. In addition, animals with 40 degrees C hyperthermia displayed worsened neurological scores compared with normothermic and 39 degrees C hyperthermic rats. In the 39 degrees C hyperthermia group, rectal temperatures were significantly lower (by 0.2 degree C to 0.5 degree C) than brain temperatures throughout the modulation period. CONCLUSIONS The present findings provide evidence that, after a transient focal ischemic insult, the postischemic brain becomes abnormally sensitive to the effects of delayed temperature elevation, even of moderate degree. The threshold for aggravation of ischemic injury by delayed hyperthermia appears to be approximately 40 degrees C. Body-temperature measurements, in both awake and anesthetized animals, may not accurately reflect brain temperature under these conditions. The present study stresses that fever of even moderate degree in the days following brain ischemia may markedly exacerbate brain injury.

Temporal and regional patterns of axonal damage following traumatic brain injury: a beta-amyloid precursor protein immunocytochemical study in rats.

Diffuse axonal injury (DAI) is an important consequence of human head trauma. This experimental investigation utilized the immunocytochemical visualization of beta-amyloid precursor protein (beta-APP) to document regional patterns of axonal injury after traumatic brain injury (TBI) and to determine the importance of injury severity on the magnitude of axonal damage. Rats underwent moderate (1.84-2.11 atm) or severe (2.38-2.52 atm) parasagittal fluid-percussion (F-P) brain injury or sham procedures. At 1, 3, 7 or 30 days after TBI, rats were perfusion-fixed and sections immunostained for the visualization of beta-APP. A regionally specific axonal response to TBI was documented after moderate F-P injury. Within the dorsolateral striatum, an early increase in beta-APP-positive axonal profiles at 24 hours (h) was followed by a significant decline at subsequent survival periods. In contrast, the frequency of reactive profiles was initially low within the thalamus, but increased significantly by day 7. Within the external capsule at the injury epicenter, numbers of immunoreactive axons increased significantly at 24 h and remained elevated throughout the subsequent survival periods. At multiple periods after TBI, selective cortical and thalamic neurons displayed increased staining of the perikarya. A significant increase in the overall frequency of beta-APP profiles was documented in the severe vs moderately injured rats at 72 h after TBI. These data indicate that parasagittal F-P brain injury (a) results in widespread axonal damage, (b) that axonal damage includes both reversible and delayed patterns, and (c) that injury severity is an important factor in determining the severity of the axonal response to TBI.

Changes in Amino Acid Neurotransmitters and Cerebral Blood Flow in the Ischemic Penumbral Region following Middle Cerebral Artery Occlusion in the Rat: Correlation with Histopathology

We simultaneously measured neurotransmitter amino acids by the microdialysis technique and cortical CBF by laser-Doppler flowmetry in the ischemic penumbral cortex of rats subjected to 2-h normothermic (36.5–37.5°C) transient middle cerebral artery (MCA) clipocclusion. Brains were perfusion-fixed 3 days later and infarct volume measured. CBF (% of preischemic values) fell to 32 ± 2% (mean ± SD) during ischemia and rose to 157 ± 68% during recirculation. Extracellular glutamate levels increased from a baseline value of 7 ± 3 μM to a peak value of 180 ± 247 μM 20–30 min following onset of ischemia but subsequently returned to near baseline levels after 70 min of ischemia despite ongoing MCA occlusion. The threshold CBF for moderate glutamate release was 48%. Massive glutamate release was seen during the first 60 min of MCA occlusion in the two animals showing the largest infarcts and occurred at CBF values ≤20% of control levels. Mean CBF during ischemia exhibited an inverse relationship with infarct volume, and the magnitude of glutamate release during ischemia was positively correlated with infarct volume. Extracellular γ-aminobutyrate and glycine changes were similar to those of glutamate but showed no significant correlation with infarct volume. These results suggest that (a) accumulation of extracellular glutamate is an important determinant of injury in the setting of reversible MCA occlusion and (b) reuptake systems for neurotransmitter amino acids may be functional in the penumbra during transient focal ischemia. We have shown that during temporary MCA occlusion, penumbral levels of amino acid neurotransmitter initially rise but subsequently decline to baseline despite ongoing ischemia and that moderate extracellular glutamate release and massive release of GABA and glycine occur at a CBF threshold of 48%. It was suggested that massive glutamate release might require a lower ischemic threshold of 20%. The magnitude of glutamate release is correlated to the size of the resultant cortical infarct. These results are consistent with the impressions of previous investigations that have implicated glutamatergic mechanisms in focal ischemic injury. It is well known that MK-801 and other glutamate-related receptor antagonists are effective in reducing infarct size in focal ischemia models (Ozyurt et al., 1988; Park et al., 1988b; Simon and Shiraishi, 1990; Buchan et al., 1991; Smith and Meldrum, 1992). While glutamate is a contributory factor, it is nonetheless unlikely to be the sole cause of infarction during transient focal ischemia, and other neurotransmitter systems as well as nonneuronal events may also participate in infarct formation (Plum, 1983; Dietrich et al., 1991; Globus et al., 1991).

Inducible nitric oxide synthase expression after traumatic brain injury and neuroprotection with aminoguanidine treatment in rats

OBJECTIVE We investigated the time course of inducible nitric oxide synthase (iNOS) enzymatic activity and immunocytochemical localization of iNOS expression after traumatic brain injury (TBI), as well as the possible role of iNOS in the pathogenesis of TBI. METHODS Male Sprague-Dawley rats were anesthetized and underwent moderate parasagittal fluid-percussion brain injury. Rats were decapitated 5 minutes, 6 hours, 1 day, 3 days, 7 days, or 14 days later, and iNOS enzymatic activities were measured (n = 6-8). To determine whether nitric oxide produced by iNOS contributed to the histopathological consequences of TBI, inhibition of iNOS activity using aminoguanidine (intraperitoneal injections of 100 mg/kg aminoguanidine [n = 9] or vehicle [n = 8], twice each day) was conducted for 3 days. RESULTS Significantly elevated iNOS activity was detected at 3 days (276.8+/-72.3% of contralateral value, means +/- standard errors; P < 0.05), and the most robust increase occurred 7 days after TBI (608.0+/-127.0%, P < 0.01) in the injured parietal cerebral cortex. Immunostaining for iNOS and glial fibrillary acidic protein, at 3 and 7 days after TBI, revealed that the major cellular sources of iNOS expression were cortical Layer 1 astrocytes and macrophages within the subarachnoid space. Administration of aminoguanidine did not reduce contusion volume significantly; however, treatment reduced total cortical necrotic neuron counts (1367.6+/-210.3; P < 0.01, compared with vehicle, 2808.5+/-325.1). CONCLUSION These data indicate that iNOS is expressed after moderate parasagittal fluid-percussion brain injury, in a time-dependent manner, and that inhibition of iNOS synthesis improves histopathological outcomes. Thus, inhibition of iNOS activation may represent a potential therapeutic strategy for the treatment of TBI.

Importance of posttraumatic hypothermia and hyperthermia on the inflammatory response after fluid percussion brain injury : Biochemical and immunocytochemical studies

The purpose of this study was to investigate: 1) the temporal and regional profile of polymorphonuclear leukocyte (PMNL) infiltration after moderate traumatic brain injury using the parasagittal fluid percussion model and 2) the effects of posttraumatic hypothermia (30°C) and hyperthermia (39°C) on the acute and subacute inflammatory response. We hypothesized that posttraumatic hypothermia would reduce the degree of PMNL accumulation whereas hyperthermia would exacerbate this response to injury. In the first series of experiments we quantitated the temporal profile of altered myeloperoxidase activity under normothermic (37°C) conditions (n = 20). The rats were allowed to survive for 3 hours, 24 hours, 3 days, or 7 days after trauma, and brains were dissected into cortical and subcortical regions ipsilateral and contralateral to injury. Additional animals were perfused and fixed for the immunocytochemical visualization of myeloperoxidase (n = 15). In the second series of experiments, rats (n = 25) were killed 3 hours or 3 days after the 3-hour monitoring period of normothermia (36.5°C), hypothermia (30°C), or hyperthemia (39°C) (n = 4 to 5 per group), and myeloperoxidase activity was again quantitated. In normothermic rats, the enzymatic activity of myeloperoxidase was significantly increased (P < 0.05) at 3 hours within the anterior cortical segment (213.97 ± 56.2 versus control 65.5 ± 52.3 U/g of wet tissue; mean ± SD) and posterior (injured) cortical and subcortical segments compared to shamoperated rats (305.76 ± 27.8 and 258.67 ± 101.4 U/g of wet tissue versus control 62.8 ± 24.8 and 37.28 ± 35.6 U/g of wet tissue; P < 0.0001, P < 0.05, respectively). At 24 hours and 7-days after trauma only the posterior cortical region (P < 0.005, P < 0.05, respectively) exhibited increased myeloperoxidase activity. However, 3 days after trauma, myeloperoxidase activity was also significantly increased within the anterior cortical segment (P < 0.05) and in posterior cortical and subcortical regions compared to sham-operated cortex (P < 0.0001, P < 0.05, respectively). Immunocytochemical analysis of myeloperoxidase reactivity at 3 hours, 24 hours, 3- and 7-days demonstrated large numbers of immunoreactive leukocytes within and associated with blood vessels, damaged tissues, and subarachnoid spaces. Posttraumatic hypothermia and hyperthermia had significant effects on myeloperoxidase activity at both 3 hours and 3 days after traumatic brain injury. Posttraumatic hypothermia reduced myeloperoxidase activity in the injured and noninjured cortical and subcortical segments compared to normothermic values (P < 0.05). In contrast, posttraumatic hyperthermia significantly elevated myeloperoxidase activity in the posterior cortical region compared to normothermic values at both 3 hours and 3 days (473.5 ± 258.4 and 100.11 ± 27.58 U/g of wet tissue, respectively, P < 0.05 versus controls). These results indicate that posttraumatic hypothermia decreases early and more prolonged myeloperoxidase activation whereas hyperthermia increases myeloperoxidase activity. Temperature-dependent alterations in PMNL accumulation appear to be a potential mechanism by which posttraumatic temperature manipulations may influence traumatic outcome.

Comparative histopathologic consequences of photothrombotic occlusion of the distal middle cerebral artery in Sprague-Dawley and Wistar rats.

Background and Purpose We have developed a minimally invasive model of photothrombotic occlusion of the distal middle cerebral artery in rats and have evaluated the patterns and features of the resulting histopathologic injury in two normotensive strains. Methods Food-deprived male Sprague-Dawley (n = 14) and Wistar (n = 10) rats anesthetized with halothane/nitrous oxide underwent a small craniotomy to expose the right distal middle cerebral artery just above the rhinal fissure. The animals were injected intravenously with the photosensitizing dye rose bengal, and the distal middle cerebral artery was irradiated with light from an argon laser-activated dye laser at three separate points to induce thrombotic occlusion. The ipsilateral common carotid artery was then permanently occluded, and the contralateral common carotid artery was occluded for 60 minutes. Three days later, the brains were perfusion-fixed and prepared for histopathologic examination, and infarct volume was determined by quantitative planimetry. Results In Sprague-Dawley rats, a large consistent temporoparietal cortical infarct was observed; mean ± SD infarct volume was 130.5 ± 40.0 mm3 (coefficient of variation, 30.7%) and a relatively small adjacent zone of selective neuronal necrosis (“incomplete infarction”), amounting to only 9.1% of the total injury volume, was also seen. By contrast, Wistar rats had smaller and more variable cortical infarcts (volume, 48.4±26.9 mm3; coefficient of variation, 55.6%) but displayed a much more substantial zone of incomplete cortical infarction (volume, 20.8±10.1 mm3; 30.1% of the total injury volume). In neither strain was infarct size related to alterations of blood pressure. In both strains, infarcts were limited to the cortex, typically involving the parietal cortex, somatosensory cortex, and forelimb region. Three rats exhibited infarcts in the contralateral hemisphere. Conclusions This model has the advantages of necessitating only minimal surgery, allowing the dura to remain intact, and avoiding mechanical trauma to the brain surface. In Sprague-Dawley rats, the resulting large cortical infarct exhibited relatively small interanimal variation, making the model suitable, for example, for replicate studies of pharmacotherapy. In Wistar rats, the large zone of incomplete infarction, a unique feature heretofore undescribed in rodent models of permanent focal ischemia, lends the model to the study of the pathomechanisms underlying graded cortical ischemic injury.

Temporal and segmental distribution of constitutive and inducible nitric oxide synthases after traumatic spinal cord injury: effect of aminoguanidine treatment.

Nitric oxide (NO) has been shown to play an important role in the pathophysiology of traumatic brain injury (TBI) and cerebral ischemia. However, its contribution to the pathogenesis of traumatic spinal cord injury (SCI) remains to be clarified. This study determined the time course of constitutive and inducible nitric oxide synthases (cNOS and iNOS, respectively) after SCI. Rats underwent moderate SCI at T10 using the NYU impactor device and were allowed to survive for 3, 6, or 24 h and 3 days after SCI (n = 5 in each group). For the determination of enzymatic activities, spinal cords were dissected into five segments, including levels rostral and caudal (remote) to the injury site. Other rats were perfusion fixed for the immunohistochemical localization of iNOS protein levels. cNOS activity was significantly decreased at 3 and 6 h within the traumatized T10 segment and at 3, 6, and 24 h at the rostral (T9) level (p < 0.05). Rostral (T8) and caudal (T11, T12) to the injury site cNOS activity was also decreased at 3 h after injury (p < 0.05). However, cNOS activity returned to control levels within 6 h at T8, T11 and T12 and at one day at T10 and T9 segments. iNOS enzymatic activity was elevated at all time points tested (p < 0.05), with the most robust increase observed at 24 h. Immunostaining for iNOS at 24 h revealed that a significant cellular source of iNOS protein appeared to be invading polymorphonuclear leukocytes (PMNLs). To assess the functional consequences of iNOS inhibition, aminoguanidine treatment was initiated 5 min after SCI and rats tested using the BBB open field locomotor score. Treated rats demonstrated significantly improved hindlimb function up to 7 weeks after SCI. Histopathological analysis of contusion volume showed that aminoguanidine treatment decreased lesion volume by 37% (p < 0.05). In conclusion, these results indicate that (1) cNOS and iNOS activities are regionally and temporally affected after moderate SCI, (2) the early accumulation of PMNLs are a potentially significant source of NO-induced cytotoxic products, and (3) acute aminoguanidine treatment significantly improves functional and histopathological outcome after SCI.

Effect of Posttraumatic Hyperglycemia on Contusion Volume and Neutrophil Accumulation after Moderate Fluid-Percussion Brain Injury in Rats

The purpose of this study was to evaluate the effects of posttraumatic hyperglycemia on contusion volume and neutrophil accumulation following moderate traumatic brain injury (TBI) in rats. A parasagittal fluid-percussion (F-P) brain injury (1.8-2.1 atm) was induced in male Sprague-Dawley rats. Rats were then randomized into four trauma groups (n = 7/group) by the timing of dextrose injection (2.0 gm/kg/ip), which included (1) early (E) group: 5 min after TBI; (2) delayed (D) group: 4 h after TBI; (3) 24-h group: 24 h after TBI; or (4) control (C) group: no dextrose injection. A sham operated control group also received dextrose to document physiological parameters (n = 4). Rats were perfusion fixed 3 days following TBI, and the brains were processed for routine histopathological and immunocytochemical analysis. Contusion areas and volumes, as well as the frequency of myeloperoxidase immunoreactive polymorphonuclear leukocytes (PMNLs) were determined. Dextrose injections significantly increased blood glucose levels (p < 0.005) in all treated groups. Although acute hyperglycemia following TBI did not significantly affect total contusion volume, contusion area was significantly elevated in the early treatment group. In addition, early posttraumatic hyperglycemia enhanced neutrophil accumulation in the area of the cortical contusion (p < 0.005). In contrast, delayed induced hyperglycemia (i.e., 4 h, 24 h) did not significantly affect histopathological outcome or neutrophil accumulation. Taken together, these findings indicate that acute but not delayed hyperglycemia aggravates histopathological outcome and increased accumulation of PMNLs. Posttraumatic hyperglycemia in the acute phase may worsen traumatic outcome by enhancing secondary injury processes, including inflammation.

Induction of tolerance against traumatic brain injury by ischemic preconditioning

We tested the hypothesis that a transient non-lethal ischemic insult lasting 2 min would protect against subsequent moderate traumatic brain injury. Sprague-Dawley rats were randomized into three experimental groups, including sham ischemia procedures and ischemic preconditioning (IPC) followed 48 h later by moderate traumatic brain injury (TBI) provoked by parasagittal fluid percussion injury (1.8-2.1 atm) and IPC followed by 48 h sham TBI. Seven days after the secondary insult, animals were perfusion-fixed for quantitative histopathological analysis. The CA3 necrotic cell count was decreased by 63% in TBI animals that had undergone IPC as compared to TBI animals that underwent sham IPC. TBI animals that had undergone IPC demonstrated significantly smaller contusion volumes than the TBI alone group (6.44 +/- 1.51 vs 1.37 +/- 0.63 mm3, mean +/- s.e.m.) These data indicate that IPC applied 2 days before moderate fluid percussion brain injury increases the brain resistance to traumatic brain damage.