Brant D. Watson

Lipid Peroxidation In Vivo Induced by Reversible Global Ischemia in Rat Brain

Abstract: It has been hypothesized that ischemia, followed by reperfusion, facilitates peroxidative free‐radical chain processes in brain. To resolve this question, rats were subjected to reversible global ischemia. From coronal sections of brains frozen in situ, small (ca. 2 mg) amounts of tissue were sampled from neocortex, hippocampus, and thalamus of both cerebral hemispheres of four groups of rats exposed to 30 min cerebral ischemia followed by 0, 30, 60, and 240 min of reperfusion, and from a control group subjected to the same operative procedures, except for the induction of ischemia. Heptane‐solubilized total lipid extracts from these samples were analyzed spectroscopically in the 190–330 nm range for content of isolated (nonconjugated) double bonds and of conjugated diene structures; the latter are formed from isolated double bonds during peroxidation of unsaturated fatty acids. Spectra derived from tissue regions of rats subjected to ischemia, or ischemia followed by reperfusion, were compared to averaged, region‐specific control spectra and were normalized to the original content of isolated double bonds in the peroxidized samples. The resultant difference spectra were analyzed in terms of ratios of conjugated diene concentration to the concentration of isolated double bonds originally at risk in the specific tissue zones considered. The peak representing conjugated diene formation was centered at 238 ± 1 nm and was usually well resolved when the molar ratio [conjugated diene]/[isolated double bonds], expressed as a percentage ([CD]/[IDB]), was greater than 0.25%. The incidence of resolvable conjugated diene peaks was much greater in the reperfused groups (18 of 124 samples total; 0.25% [CD]/[IDB] 1.34%), compared to the nonreperfused group (1 of 38 samples; [CD]/[IDB] = 0.62%). No specific regional susceptibility to conjugated diene formation was observed. It also was shown by computer averaging over all the tissue zones sampled that, if one had used large samples consisting of pooled brain tissue for analysis of lipid peroxides and/or their reaction products, the conjugated diene peak would have been attenuated below distinctly resolvable levels. These results constitute the first demonstration of lipid peroxidation induced by reversible global ischemia. The observation that the effect is highly focal and not generalized throughout the brain suggests that special conditions of reperfusibility and reoxygenation must be satisfied for lipid peroxidation to be detectable in an ischemic milieu.

Induction of spreading depression in the ischemic hemisphere following experimental middle cerebral artery occlusion: effect on infarct morphology.

This study was undertaken to test whether transient depolarizations occurring in periinfarct regions are important in contributing to infarct spread and maturation. Following middle cerebral artery (MCA) occlusion we stimulated the ischemic penumbra with recurrent waves of spreading depression (SD) and correlated the histopathological changes with the electrophysiological recordings. Halothane-anesthetized, artificially ventilated Sprague–Dawley rats underwent repetitive stimulation of SD in intact brain (Group 1; n = 8) or photothrombotic MCA occlusion coupled with ipsilateral common carotid artery occlusion (Groups 2 and 3, n = 9 each). The electroencephalogram and direct current (DC) potential were recorded for 3 h in the parietal cortex, which represented the periinfarct border zone in ischemic rats. In Group 2, only spontaneously occurring negative DC shifts occurred; in Group 3, the (nonischemic) frontal pole of the ischemic hemisphere was electrically stimulated to increase the frequency of periinfarct DC shifts. Animals underwent perfusion-fixation 24 h later, and volumes of complete infarction and scattered neuronal injury (“incomplete infarction”) were assessed on stained coronal sections by quantitative planimetry. Electrical induction of SD in Group 1 did not cause morphological injury. During the initial 3 h following MCA occlusion, the number of spontaneous periinfarct depolarizations in Group 2 (7.0 ±1.5 DC shifts) was doubled in Group 3 by frontal current application (13.4 ± 2.7 DC shifts; p < 0.001). The duration as well as the integrated negative amplitude of DC shifts over time were significantly greater in Group 3 than in Group 2 rats (duration, 5.7 ± 3.8 vs. 4.1 ± 2.5 min; p < 0.05). Histopathological examination disclosed well-defined areas of pannecrosis surrounded by a cortical rim exhibiting selectively damaged acidophilic neurons and astrocytic swelling in otherwise normal-appearing brain. Induction of SD in the ischemic hemisphere led to a significant increase in the volume of incomplete infarction (19.0 ± 6.1 mm3 in Group 3 vs. 10.3 ± 5.1 mm3 in Group 2; p < 0.01) and of total ischemic injury (100.7 ± 41.0 mm3 in Group 3 vs. 66.5 ± 24.7 mm3 in Group 2; p < 0.05). The integrated magnitude of DC negativity per experiment correlated significantly with the volume of total ischemic injury (r = 0.780, p < 0.0001). Thus, induction of SD in the ischemic hemisphere accentuated the development of scattered neuronal injury and increased the volume of total ischemic injury. This observation may be explained by the fact that, with limited perfusion reserve, periinfarct depolarizations are associated with episodic energy failure in the acute ischemic penumbra.

Influence of transient ischemia on lipid-soluble antioxidants, free fatty acids and energy metabolites in rat brain

Abstract Cerebral ischemia was induced in anesthetized rats by occluding both common carotid arteries; the vertebral arteries had been permanently occluded one day earlier. The levels of lipid-soluble antioxidants (alpha-tocopherol, reduced ubiquinones), free fatty acids and energy metabolites were measured in forebrain tissue after 30 min of ischemia, and after 15 min or 30 min of postischemic recirculation. Alpha-tocopherol decreased by 7% at the end of ischemia and decreased further during postischemic recirculation, indicating that free radical reactions were initiated during ischemia. All measured saturated, mono- and polyunsaturated free fatty acids accumulated markedly during ischemia. However, particularly rapid decrements were observed in polyunsaturated free fatty acids (arachidonic and docosahexaenoic acids) during an initial 15-min period of recirculation, while other free fatty acids gradually returned toward their preischemic levels after 30 min of recirculation. Both reduced and oxidized ubiquinone-9 tended to increase at the end of ischemia. During recirculation, reduced ubiquinone-9 declined below control by 20–24%,accompanied by a rise in the oxidized form. Profiles of free fatty acids and the changes in ubiquinones were compatible with the postischemic occurrence of lipid peroxidation. High energy phosphates were nearly depleted by ischemia, and recovery of adenosine triphosphate was limited to 73% of control after 30 min of recirculation. The results are compatible with the view that free radical reactions are initiated during ischemia, and that overt peroxidative processes become manifest during reflow when cerebral tissue is reoxygenated. Lipid peroxidation by free radical reactions may be a factor restricting postischemic recovery of energy metabolism, and lipid-soluble antioxidants may act to mitigate the extent of eventual brain damage.

Photochemically induced cerebral infarction - I. Early microvascular alterations

SummaryCerebral ischemia leading to infarction was produced in rats by intravascular thrombosis induced by a photochemical reaction between systemically injected rose bengal and green light (560 nm) transmitted through the intact skull for a 2-min period. At 2 or 15 min following photochemical sensitization, animals were perfusion-fixed for scanning (SEM) and transmission (TEM) electron microscopic analyses of the cerebral vasculature. At 2 minutes, ultrastructural examination of cortical regions destined to undergo infarction revealed numerous platelet aggregates within both pial and intraparenchymal vessels. Platelets close to the endothelial walls were routinely degranulated with pseudopodia. Endothelial cells were frequently swollen and contained dilated mitochondria and granular endoplasmic reticulum. The endothelial luminal membrane structure was shown by high-power TEM to be focally damaged. If brain temperature was reduced by 4°C during the photochemical sensitization period, the platelet response was inhibited without interfering with other ultrastructural changes. These results are consistent with the hypothesis that photochemically induced endothelial alterations stimulate platelet activation and implicate abnormal endothelial function as a primary event in the pathogenesis of photochemically induced cerebral infarction.

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.

Potentiation of lipid peroxides by ischemia in rat brain

Post-ischemic changes in energy metabolites and natural antioxidant compounds have been measured in rat brain in vitro concurrent with two different assays for peroxidized lipids. No exogenous free radical initiators were employed. In vitro oxygenation of minced brain preparations for periods of 10 minutes to 4 hours, following 5 minutes of preparatory ischemia, yielded increased levels of lipid conjugated dienes and TBA-reactive material, in contrast to anaerobically incubated preparations. However, either aerobic or anaerobic incubation of brain minces facilitated increased ratios of lactate: pyruvate and glutathione (oxidized): glutathione (reduced), as well as increased total ubiquinone content and loss of α-tocopherol. Observation of lipid radical formation in vivo was then attempted using rats given embolic stroke in one hemisphere and left in the post-ischemic condition for times up to 24 hours. Conjugated dienes were found in lipids extracted from the ipsilateral hemisphere but not from the contralateral hemisphere. These observations of conjugated dienes in vivo (formed presumably during post-ischemic reperfusion) and in vitro (facilitated by oxygenation of brain minces), indicate that lipid radical intermediates and associated chain peroxidation processes are potentiated by ischemia and occur during tissue reoxygenation.

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.

Postischemic Cerebral Lipid Peroxidation In Vitro: Modification by Dietary Vitamin E

Abstract: Using an in vitro system, we studied the effect of postischemic reoxygenation on cerebral lipid peroxidation in relation to the dietary intake of vitamin E (VE) in rats. Homogenates prepared from VE‐deficient, ‐normal, and ‐supplemented brains, which were previously rendered ischemic for 30 min by decapitation, were incubated under air or nitrogen gas for 60 min. The extent of peroxidation in brain tissue was estimated by a thiobarbituric acid (TBA) test and by diene conjugation in total lipid extracts. The brain levels of α‐tocopherol and of total and free fatty acids (FAs) were also determined. Aerobic incubation increased TBA reactants in all dietary groups; the effect was largest in the VE‐deficient group, intermediate in the VE‐normal group, and smallest in the VE‐supplemented group. In contrast, nitrogen incubation did not alter the basal levels of TBA reactants except for a small rise associated with VE deficiency. Conjugated dienes changed in parallel with TBA reactants. α‐Tocopherol decreased after aerobic incubation and also, to a lesser degree, after nitrogen incubation in each dietary group. Only in the reoxygenated samples of the VE‐deficient group was there a significant fall in total polyunsaturated FAs. The levels of free FAs continuously increased throughout ischemia and subsequent incubation. However, the level of free polyunsaturated FAs was similar after aerobic and nitrogen incubation in each dietary group, and was not affected by VE. Thus, cerebral reoxygenation after ischemia propagates peroxidative reactions within esterified polyunsaturated FAs. The modification by VE of reoxygenation‐induced lipid peroxidation suggests free radical mediation.

Photochemically induced cerebral infarction. II: Edema and blood-brain barrier disruption

SummaryAlterations in the blood-brain barrier to proteins, and regional water and electrolyte content were documented in a rat model of photochemically induced small-vessel thrombosis leading to infarction. Horseradish peroxidase (HRP) or Evans blue was given immediately following a 2-min photochemical sensitization period. At 5 min following irradiation, multifocal sites of peroxidase extravasation were noted within the irradiated area. Ultrastructural examination revealed endothelial cells filled with HRP which in some cases extended into the basal lamina and extracellular spaces. At 15 min, protein leakage was more pronounced within the irradiated zone and reaction product was also apparent within the subarachnoid and perivascular spaces of brain regions remote from the site of irradiation. Widespread staining on the surface of the irradiated hemisphere was apparent in rats perfused 8 h following Evans blue infusion. Water content increased significantly by 15 min within the irradiated zone but not in brain regions remote from this site. Although vasogenic edema is an early event in this stroke model, increases in water content are restricted to the irreversibly damaged site. In contrast, protein tracer escaping from microvessels coursing within the irradiated zone was widely distributed. These findings implicate endothelial barrier dysfunction in the genesis of tissue injury in this model. Morphological evidence for the capability of macromolecules to escape from a site of evolving infarction and to migrate to distances remote from the area of primary microvascular damage is also discussed.