Ricardo Prado

Hyperglycemia Increases Infarct Size in Collaterally Perfused but Not End-Arterial Vascular Territories

Hyperglycemia exacerbates neuronal injury in the setting of reversible brain ischemia, but its effect on focal thrombotic infarction has been less extensively characterized. We investigated this problem in two rat models of focal vascular occlusion. In Model I, the right middle cerebral artery (MCA) was exposed via a subtemporal craniotomy in halothane- and nitrous oxide-anesthetized Wistar rats and was occluded photochemically by irradiation with an argon ion laser the intravenous administration of the photosensitizing dye rose bengal. Permanent MCA occlusion was combined with temporary bilateral common carotid artery ligation. In Model II, similarly anesthetized Sprague-Dawley rats were subjected to permanent photochemical occlusion of the right MCA without common carotid occlusion. In both models, rats were food deprived for 24 h and were administered varying amounts of 50% dextrose (or saline) 15 min prior to vascular occlusion to produce a spectrum of plasma glucose values, ranging from 5 to 44 μmol/ml. Brains were examined histologically 7 days following vascular occlusion, and computer-assisted planimetry was used to compute infarct volumes. In Model I, the volume of neocortical infarction ranged from 30.3 to 108.4 mm3 and exhibited a strong linear correlation with increasing preischemic plasma glucose values (r = 0.70). In contrast, the size of the smaller striatal infarct in this model was not correlated with plasma glucose level. In Model II, there was a prominent striatal infarct, ranging in volume from 14.4 to 96.4 mm3, while neocortical infarction occurred inconstantly. As in Model I, striatal infarct volume in Model II showed no correlation with plasma glucose level. These results are consistent with the view that infarcted regions having collateral circulation [neocortex in MCA occlusion (Model I)] are vulnerable to the deleterious effects of hyperglycemia, whereas regions of nonanastomosing (end-arterial) vascular supply are not [striatum in Models I and II, and neocortex in previously reported model of photochemically induced primary microvascular thrombosis (Ginsberg et al., 1987)]. Thus, the harmful effects of elevated plasma glucose in stroke appear to be complex and may depend critically upon the degree to which collateral perfusion is available to the specific brain regions affected, as well as the extent to which local blood flow is reduced and the timing of glucose administration.

Hyperglycemia reduces the extent of cerebral infarction in rats.

Although hyperglycemia is known to exacerbate neuronal injury in the setting of reversible brain ischemia, its effect on irreversible thrombotic infarction is less well understood. In this study, unilateral thrombotic infarction was induced photochemically in the parietal cortex of Wistar rats. Seven days later, brains were perfusion-fixed for light microscopy. Infarct areas were measured by computer-assisted planimetry on multiple coronal sections at 250-micron intervals; these data were integrated to yield infarct volumes. Fasted, normoglycemic rats were compared with hyperglycemic rats that had received 1.2-1.5 ml of 50% dextrose i.p. 15 minutes prior to the induction of infarction. Infarct volume averaged 12.5 +/- 4.0 mm3 (mean +/- SD) in rats (n = 14) with plasma glucose levels of 72-184 mg/dl; this differed statistically from the average volume of 9.3 +/- 3.3 mm3 observed in rats (n = 13) with elevated plasma glucose (range 264-607 mg/dl). Spearman rank correlation analysis confirmed a significant correlation of larger infarct volumes with lower plasma glucose levels. In contrast, rats receiving mannitol i.p. to produce an osmotic load comparable with that of the dextrose-pretreated animals showed larger infarct volumes than saline-treated controls. The small but definite beneficial effect of hyperglycemia in this end-arteriolar thrombotic infarction model is possibly attributable to improved local energy metabolism at the periphery of the lesion during the early period of lesion expansion.

Endothelium-derived nitric oxide synthase inhibition. Effects on cerebral blood flow, pial artery diameter, and vascular morphology in rats.

Background and Purpose We determined the effects of inhibiting the production of cerebral endothelium- derived nitric oxide on pial artery diameter, cortical blood flow, and vascular morphology. Methods An inhibitor of endothelium-derived nitric oxide synthesis, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME), or an equivalent volume of 0.9% saline was infused into rats intra-arterially in a retrograde fashion via the right external carotid artery at a rate of 3 mg/kg/min to a total dose of 190 mg/kg or intravenously at 1 mg/kg/min to a total dose of 15 mg/kg. Large pial arteries were continuously visualized through an operating microscope, and cortical cerebral blood flow was monitored by laser-Doppler flowmetry. To localize areas of morphological interest, the protein tracer horseradish peroxidase was injected 15 minutes before termination of the L-NAME infusion and the rats were perfusion-fixed 15 minutes later for light and electron microscopic analysis. Results Infusion of L-NAME significantly raised arterial blood pressure at both doses (for 190 mg/kg, from 103.2±3.4 to 135±3.4 mm Hg; for 15 mg/kg, from 125±2.8 to 144.4±4.0 mm Hg). Pial arteries constricted within 10 minutes after the start of the intracarotid infusion to 40% of the preinfusion diameter, while cortical cerebral blood flow decreased to an average of 72.5% of that at baseline. Morphological abnormalities in the experimental rats included microvascular stasis and focal areas of blood–brain barrier disruption to protein. Ultrastructural examination of cortical leaky sites revealed constricted arterioles with many endothelial pinocytotic vesicles and microvilli. Conclusions These observations suggest that inhibition of endothelium-derived nitric oxide synthesis affects the relation between cerebral arterial diameter and cerebral blood flow and can lead to subtle cerebral vascular pathological changes consistent with focal brain ischemia.

Posttraumatic Cerebral Ischemia after Fluid Percussion Brain Injury: An Autoradiographic and Histopathological Study in Rats

OBJECTIVES Mild-to-moderate reductions in local cerebral blood flow (ICBF) have been reported to occur in rats after moderate (1.7-2.2 atm) fluid percussion brain injury. The purpose of this study was to determine whether evidence for severe ischemia (i.e., mean ICBF < 0.25 ml/g/min) could be demonstrated after severe brain injury. In addition, patterns of indium-labeled platelet accumulation and histopathological outcome were correlated with the hemodynamic alterations. METHODS Sprague-Dawley rats (n = 23), anesthetized with halothane and maintained on a 70:30 mixture of nitrous oxide:oxygen and 0.5% halothane, underwent normothermic (37 degrees C) parasagittal fluid percussion brain injury (2.4-2.6 atm). Indium-111-tropolone-labeled platelets were injected 30 minutes before traumatic brain injury (TBI), while 14C-iodoantipyrine was infused 30 minutes after trauma for ICBF determination. Sham-operated animals (n = 8) underwent similar surgical procedures but were not injured. For histopathological analysis, traumatized rats (n = 5) were perfusion-fixed 3 days after TBI. RESULTS In autoradiographic images of indium-labeled platelets, abnormal platelet accumulation that was most pronounced overlying the pial surface was commonly associated with severe reductions in ICBF within underlying cortical regions 30 minutes after TBI. For example, within the lateral parietal cortex, ICBF was significantly reduced from 1.67 +/- 0.11 ml/g per minute (mean +/- standard error of the mean) in sham-operated animals to 0.23 +/- 0.03 ml/g per minute within the traumatized group. In addition to focal severe ischemia, moderate reductions in ICBF were detected throughout the traumatized hemisphere, including the frontal and occipital cortices, hippocampus, thalamus, and striatum. Mild decreases in ICBF were also observed throughout the contralateral cerebral cortex. At 3 days after severe TBI, histopathology demonstrated intracerebral and subarachnoid hemorrhage associated with cerebral contusion and selective neuronal necrosis. CONCLUSION These data indicate that multiple cerebrovascular abnormalities, including subarachnoid hemorrhage, focal platelet accumulation, and severe ischemia, are important early events in the pathogenesis of cortical contusion formation after TBI. Injury severity is expected to be a critical factor in determining what therapeutic strategies are attempted in the clinical setting.

The effect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats.

Background and Purpose Previous in vitro and in vivo studies of the effects of nitric oxide synthase inhibition in the central nervous system have yielded conflicting results concerning the role of nitric oxide in the events that lead to ischemic injury. In this study, we tested the hypothesis that preischemic inhibition of nitric oxide synthase increases infarct volume after reversible focal cerebral ischemia in rats. Methods NG-nitro-L-arginine methyl ester hydrochloride 15 mg/kg IV or an equivalent volume of saline was administered to adult Wistar rats 15 minutes before middle cerebral artery occlusion by the intraluminal suture method. After 2 hours of ischemia, the suture was withdrawn, and rats were allowed to survive for 3 days. Areas of infarction in 10 hematoxylin-eosin-stained sections were measured and used to determine infarct volume. Results Administration of NG-nitro-L-arginine methyl ester hydrochloride increased hemispheric infarct volume by 137% over control (60.9±30.5 to 144.3±19.6 mm3, P<.05; mean±SEM). Cortical and subcortical infarct volumes were increased by 176% (33.8±21.9 to 93.3±15.2 mm3, P<.05) and 103% (25.1±9.4 to 51.0±5.5 mm3, P<.03), respectively. Conclusions Nitric oxide synthase inhibition increases infarct volume and decreases the variability of the response to middle cerebral artery occlusion in Wistar rats, a strain that is normally resistant to focal cerebral ischemic injury owing to extensive collateralization. The mechanism of the deleterious effect of nitric oxide synthase inhibition likely involves a more severe degree of blood flow reduction during and after middle cerebral artery occlusion, primarily by preventing the vasodilatory response of collateral vessels to proximal middle cerebral artery occlusion. Maintenance of nitric oxide synthase activity during and after focal cerebral ischemia appears to minimize ischemic injury.

Photochemically induced spinal cord injury in the rat

We have developed in the rat a minimally invasive model of reproducible spinal cord injury initiated photochemically. With the exposed spinal column intact, 560 nm irradiation of the translucent dorsal surface induces excitation of the systemically injected dye, rose Bengal, in the spinal cord microvasculature. The resultant photochemical reaction leads to vascular stasis. Histopathological changes at 7 days include hemorrhagic necrosis of the central gray matter, edematous pale-staining white matter tracts and vascular congestion. At the level of cord irradiation (T8) the entire cord thickness is necrosed except for the periphery of the anterior funiculus. Voluntary motor function is consistently lost in the subacute phase of injury.

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.

Remote organ ischemic preconditioning protect brain from ischemic damage following asphyxial cardiac arrest.

Ischemic preconditioning (IPC) is a phenomenon whereby an organs adaptive transient resistance to a lethal ischemic insult occurs by preconditioning this organ with a sub-lethal/mild ischemic insult of short duration. Besides IPC, recent studies reported that a short sub-lethal ischemia and reperfusion in various organs can induce ischemic tolerance in another organ as well. This phenomenon is known as remote ischemic preconditioning (RPC). In the present study we tested the hypothesis that tolerance for ischemia can be induced in brain by RPC and IPC in a rat model of asphyxial cardiac arrest (ACA). RPC was induced by tightening the upper two-thirds of both hind limbs using a tourniquet for 15 or 30 min and IPC was induced by tightening bilateral carotid artery ligatures for 2 min. Eight minutes of ACA was induced 48 h after RPC or IPC. After 7 day of resuscitation, brains were extracted and examined for histopathological changes. In CA1 hippocampus, the number of normal neurons was 63% lower in cardiac-arrested rats as compared to the control group. The number of normal neurons in the 15 min RPC, 30 min RPC, and IPC groups was higher than the ACA group by 54, 70, and 67%, respectively. This study demonstrates that RPC and IPC are able to provide neuroprotection in a rat model of ACA. Besides direct application of RPC or IPC paradigms, the exploration of the mechanisms of observed neuroprotection by RPC and IPC may also lead to a possible therapy for CA patients.

Widespread hemodynamic depression and focal platelet accumulation after fluid percussion brain injury : A double-label autoradiographic study in rats

Cerebrovascular damage leading to subsequent reductions in local cerebral blood flow (lCBF) may represent an important secondary injury mechanism following traumatic brain injury (TBI). We determined whether patterns of 111indium-labeled platelet accumulation were spatially related to alterations in lCBF determined autoradiographically 30 min after TBI. Sprague–Dawley rats (n = 8), anesthetized with halothane and maintained on a 70:30 (vol/vol) mixture of nitrous oxide/oxygen and 0.5% halothane, underwent parasagittal fluid percussion brain injury (1.7–2.2 atm). 111Indiumtropolone–labeled platelets were injected 30 min prior to TBI while [l4C]-iodoantipyrine was infused 30 min after trauma. Sham-operated animals (n = 7) underwent similar surgical procedures but were not injured. In autoradiographic images of the indium-labeled platelets, focal sites of platelet accumulation within the traumatized hemisphere were restricted to the pial surface (five of eight rats), the external capsule underlying the lateral parietal cortex (five of eight rats), and within cerebrospinal fluid (CSF) compartments (six of eight rats). In contrast, mild-to-moderate reductions in lCBF, not restricted to sites of platelet accumulation, were seen throughout the traumatized hemisphere. Flow reductions were most severe in coronal sections underlying the impact site. For example, within the lateral parietal cortex and hippocampus, lCBF was significantly reduced [p < 0.01; analysis of variance (ANOVA)] from 1.71 ± 0.34 (mean ± SD) and 0.78 ± 0.12 ml/g/min, respectively, versus 0.72 ± 0.17 and 0.41 ± 0.06 ml/g/min within the traumatized hemisphere. Significant flow reductions were also seen in remote cortical and subcortical areas, including the right frontal cortex and striatum. These results indicate that focal platelet accumulation and widespread hemodynamic depression are both early consequences of TBI. Therapeutic strategies directed at these early microvascular consequences of TBI may be neuroprotective by attenuating secondary ischemic processes.

Ischemic preconditioning ameliorates excitotoxicity by shifting glutamate/γ-aminobutyric acid release and biosynthesis

Excitotoxicity is recognized to play a major role in cerebral ischemia‐induced cell death. The main goal of the present study was to define whether our model of ischemic preconditioning (IPC) promotes a shift from excitatory to inhibitory neurotransmission during the test ischemia to diminish metabolic demand during the reperfusion phase. We also determined whether γ‐aminobutyric acid (GABA) played a role in IPC‐induced neuroprotection. Ten minutes of cerebral ischemia was produced by tightening the carotid ligatures bilaterally following hypotension. Samples of microdialysis perfusate, representing extracellular fluid, were analyzed for amino acid content by HPLC. IPC promoted a robust release of GABA after lethal ischemia compared with control rats. We also observed that the activity of glutamate decarboxylase (the predominant pathway of GABA synthesis in the brain) was higher in the IPC group compared with control and ischemic groups. Because GABAA receptor up‐regulation has been shown to occur following IPC, and GABAA receptor activation has been implicated in neuroprotection against ischemic insults, we tested the hypothesis that GABAA or GABAB receptor activation was neuroprotective during ischemia or early reperfusion by using an in vitro model (organotypic hippocampal slice culture). Administration of the GABAB agonist baclofen during test ischemia and for 1 hr of reperfusion provided significant neuroprotection. We concluded that increased GABA release in preconditioned animals after ischemia might be one of the factors responsible for IPC neuroprotection. Specific activation of GABAB receptor contributes significantly to neuroprotection against ischemia in organotypic hippocampal slices.