Enoch P. Wei

Appearance of superoxide anion radical in cerebral extracellular space during increased prostaglandin synthesis in cats.

When increased prostaglandin synthesis was induced in anesthetized cats equipped with cranial windows by topical application of arachidonate (200 μg/ml) or bradykinin (20 μg/ml), there was reduction of nitroblue terrazolium, resulting in deposition of the reduced insoluble form of this dye on the brain surface. The amount of reduced nitroblue terrazolium deposited on the brain surface was measured spectrophotometrically after fixation of the brain by perfusion with aldehydes to eliminate interference from hemoglobin. Topical application of 56 U/ml superoxide dismutase or 20 μg/ml indomethatin inhibited nitroblue terrazolium reduction by 76.5%–82.5% and by 78%–85.5%, respectively. These results show that most of the nitroblue terrazolium reduction was accounted for by superoxide anion radical generated in the course of arachidonate metabolism via the cyclooxygenase pathway. No superoxide production could be detected in the absence of arachidonate or bradykinin. Histological examination showed no evidence of parenchymal cellular damage or vascular damage and no accumulation of leukocytes. Pronounced leukocyte accumulation occurred 24 hours after topical arachidonate in rabbits with chronically implanted cranial windows. Superoxide appearance was reduced severely by 4, 4′-diisothiocyano-2, 2′-stilbene disulfonate and phenylglyoxal, two specific inhibitors of the anion channel. The most likely explanation for these findings is that increased metabolism of exogenous or endogenous arachidonate via cyclooxygenase results in the appearance of superoxide anion radical in cerebral extracellular space. Superoxide crosses the membrane of undamaged cells via the anion channel.

Calcitonin gene-related peptide mediates nitroglycerin and sodium nitroprusside-induced vasodilation in feline cerebral arterioles.

The cerebral vasodilator response induced by topical nitroglycerin and nitroprusside was examined in cats equipped with cranial windows for the observation of the cerebral microcirculation. In cats subjected to chronic unilateral trigeminal ganglionectomy, the vasodilator responses to nitroprusside and nitroglycerin were markedly depressed on the denervated side. Application of a selective calcitonin gene-related peptide (CGRP) antagonist [CGRP(8-37)] on the innervated side reduced the response to nitrodilators to the same extent as seen on the denervated side. The vasodilator response to acetylcholine was unaffected by trigeminal ganglionectomy. CGRP(8-37) almost abolished the vasodilator response to nitroglycerin and sodium nitroprusside and to CGRP, but did not affect the response to adenosine or to adenosine diphosphate. Pretreatment with LY83583, a drug that lowers cyclic GMP levels, diminished the vasodilation to CGRP and to nitroprusside but not to adenosine. We conclude that the nitrovasodilators activate sensory fibers to release CGRP, which in turn relaxes cerebral vascular smooth muscle by activating guanylate cyclase. Hence, nitrovasodilators possess a novel mechanism of action within the cephalic circulation which may explain both the occurrence of vasodilation and headache.

Oxygen radicals mediate the cerebral arteriolar dilation from arachidonate and bradykinin in cats.

Topical application of sodium arachidonate (50–200 μg/ml) or bradykinin (0.1–10 μg/ml) on the brain surface of anesthetized cats caused dose-dependent cerebral arteriolar dilation. This dilation was blocked by 67–100percent; in the presence of superoxide dismutase and catalase. These enzymes did not affect the changes in arteriolar diameter caused by alterations in arterial blood Pco2/ or the arteriolar dilation from topical acetylcholine. Enzymes inactivated by heat had no effect on the vasodilation from arachidonate or bradykinin. Superoxide dismutase alone or catalase alone reduced the dilation during application of 200 Mg/ml of arachidonate for 15 minutes; they also completely prevented the residual dilation seen 1 hour after washout, as well as the reduction in the vasoconstrictive effects of arterial hypocapnia observed at this time. The results show that superoxide anion radical and hydrogen peroxide, or radicals derived from them, such as the hydroxyl radical, are mediators of the cerebral arteriolar dilation from sodium arachidonate or bradykinin. These radicals are not the endothelium-derived relaxant factor released by acetylcholine. The presence of both superoxide anionradical and hydrogen peroxide is required for the production of the vascular damage seen during prolonged application of high concentrations of sodium arachidonate.

Detailed Description of a Cranial Window Technique for Acute and Chronic Experiments

Methods for implantation of cranial windows for the direct observation of the pial microcirculation in experimental animals are described in detail. These techniques are suitable for both acute experiments in anesthetized animals and chronic implantation permitting several months of observation in awake animals. Experience over several years shows that these techniques have an acceptably low rate of failure, are low in cost and can easily be mastered in most laboratories. They make possible observation of the microcirculation and accurate measurement of the diameter of pial vessels, and permit study of the effects on the microcirculation of a variety of maneuvers and vasoactive agents which can be studied by direct application as well as by intravascular administration. Because they preserve the integrity of the skull, the techniques permit study of the cerebral microcirculation under conditions closely approximating the normal environment of these vessels.

Cyclooxygenase Products of Arachidonic Acid Metabolism in Cat Cerebral Cortex After Experimental Concussive Brain Injury

Abstract Previous studies have suggested that following experimental fluid percussion brain injury, increased prostaglandin (PG) synthesis, with its concomitant production of oxygen free radicals, causes functional and morphological abnormalities of the cerebral arterioles. The purpose of this study was to chemically determine if PGs are altered following this injury. To facilitate interpretation of neurochemical measurements the cats were ventilated, blood pressure was measured, and a cranial window, for microscopic observation of pial arteriolar diameter was inserted. PG levels were determined in quick‐frozen cortical tissue removed from control and 3 groups of injured cats at 1.5, 8.0, and 60 min after injury. Analysis of PGE2, PGF2α, and 6‐keto‐PGFlα was performed by HPLC and GC/MS. The control levels of PGE2, PGF2α, and 6‐keto‐PGF1α, were 216 ± 44, 210 ± 48, and 48 ± 12 ng/g wet weight, respectively. Following injury, produced by a 22 ms increase in intracranial pressure, the pial arterioles dilated irreversibly and a transient hypertensive response occurred, thereby producing hyperemia. During the maximum hyperemic response, the total PGs were 75% of control. At 8 min after injury, when blood pressure returned to control level, the PGs were 158% of control and PGs fell to 111% of control at 60 min. These experiments supported our previous studies implicating increased PG synthesis in the genesis of the physiologic and morphologic sequelae of experimental concussive brain injury.

Hydroxyl radical-dependent inactivation of guanylate cyclase in cerebral arterioles by methylene blue and by LY83583.

Background and Purpose Methylene blue and 6-anilino,5,8-quinolinedione (LY83583) are used extensively to block activation of guanylate cyclase. Both agents generate oxygen radicals. Therefore, it appeared profitable to investigate whether the generation of oxygen radicals by these agents is responsible for the blockade of responses to nitrodilators that act via activation of guanylate cyclase to relax vascular smooth muscle and cause vasodilation. Methods We tested in anesthetized cats equipped with cranial windows responses to topical application of nitroglycerin, nitroprusside, and adenosine before and during topical application of methylene blue (5 μM).Responses to the vasoactive agents were tested during application of methylene blue after permeabilization of the cell membrane with a detergent to allow methylene blue to enter vascular smooth muscle. Responses were also tested in the presence of superoxide dismutase, catalase, deferoxamine, or dimethyl sulfoxide to scavenge reactive products of oxygen metabolism or to eliminate catalytic iron. In additional experiments we tested the effects of topical application of nitroprusside or adenosine before and after application of LY83583. The responses to the vasoactive agents were also tested in the presence of superoxide dismutase, catalase, or dimethyl sulfoxide in addition to LY83583. We also tested responses to calcitonin gene-related peptide before and in the presence of LY83583 with or without superoxide dismutase. Results Methylene blue eliminated the arteriolar dilation in response to nitroprusside and nitroglycerin after permeabilization of the cell membrane with a detergent but not before. The responses to adenosine were unaffected. The blockade induced by methylene blue was reversed by superoxide dismutase, catalase, or dimethyl sulfoxide but not by deferoxamine. LY83583 blocked responses to nitroprusside but not to adenosine. The blockade was eliminated by superoxide dismutase, catalase, or dimethyl sulfoxide. LY83583 blocked the vasodilation induced by calcitonin gene-related peptide. This blockade was reversed by superoxide dismutase. Conclusions Methylene blue and LY83583 prevent the activation of soluble guanylate cyclase by nitrodilators or by calcitonin gene-related peptide by generating oxygen radicals. The mediator of this response is the hydroxyl radical. Methylene blue does not enter the vascular smooth muscle of cerebral arterioles unless the cell membrane is permeabilized.

Local mechanism of CO2 action of cat pial arterioles.

The effects of local hypercapnic acidosis or local hypocapnic alkalosis on pial arterioles were studied in anesthetized cats equipped with a cranial window for the direct observation of the pial microcirculation of the parietal cortex. Changes in Pco, and pH of the extracellular fluid were induced by perfusing the space under the cranial window with artificial cerebrospinal fluid equilibrated with different concentrations of CO, while Paco, was maintained constant. Hypercapnic acidosis dilated and hypocapnic alkalosis constricted pial arterioles markedly. The results indicate that a basis exists for considering CO, as a mediator for local regulation of brain blood flow. The vasodilation associated with arterial hypercapnia was abolished by a reduction in CSF Pco, equal in magnitude to the rise in arterial blood Pco, suggesting that the action of CO, is entirely local.

H2O2 and endothelium-dependent cerebral arteriolar dilation. Implications for the identity of endothelium-derived relaxing factor generated by acetylcholine.

We studied the mechanism of the vasodilator effect of H2O2 on cerebral arterioles and its effect on endothelium-dependent responses to acetylcholine. Topical application of H2O2 (0.1-1 microM) on the brain surface of anesthetized cats equipped with cranial windows induced dose-dependent arteriolar dilation, which was markedly inhibited by topical deferoxamine, showing that it was probably mediated by generation of hydroxyl radical. Higher concentrations of H2O2 (3 microM) also induced dilation, which was unaffected by deferoxamine, indicating the participation of other mechanisms. After topical application of H2O2, endothelium-dependent responses to acetylcholine were eliminated or converted to vasoconstriction, and in bioassay experiments, acetylcholine-mediated endothelium-derived relaxing factor (EDRF) was absent. Superoxide dismutase plus catalase restored the appearance of transferable EDRF after 1 microM H2O2 but not after 3 microM H2O2. Application of H2O2 in the assay window eliminated the responses to nitroprusside and nitric oxide but did not affect responses to adenosine, to EDRF from the donor window, or responses to S-nitroso-L-cysteine. The inhibiting effect of H2O2 on the response to nitroprusside was partially eliminated after topical application of N-acetyl-L-cysteine. The results show that H2O2 inhibits the vasodilator action of nitroprusside and nitric oxide probably because it oxidizes thiols in vascular smooth muscle and prevents the formation of a nitrosothiol. EDRF from acetylcholine and S-nitroso-L-cysteine still produce dilation in the presence of the blockade induced by H2O2. The findings suggest strongly that the EDRF from acetylcholine in cerebral vessels is a nitrosothiol like S-nitroso-L-cysteine.

Determinants of Response of Pial Arteries to Norepinephrine and Sympathetic Nerve Stimulation

Feline pial arteries larger than 100 μ in diameter constricted in response to cervical sympathetic nerve stimulation or in response to topical application of norepinephrine. Smaller pial arteries were unresponsive to norepinephrine. This unresponsiveness persisted when norepinephrine was dissolved in CSF with high calcium ion concentration, or in CSF with both high calcium ion and zero magnesium ion concentration, or when it was dissolved in the acid fluid used by Wahl et al. and applied by constant infusion or by intermittent application. Comparison of the responses of the larger pial vessels to norepinephrine and to sympathetic nerve stimulation suggests that maximal activation of sympathetic nerves achieves a concentration of released norepinephrine equal to 5.9 × 10-4 M. The constriction of the larger pial vessels in response to sympathetic nerve stimulation could account for modest reductions in cerebral blood flow.

Posttraumatic hypothermia followed by slow rewarming protects the cerebral microcirculation.

In the clinical and laboratory setting, multiple reports have suggested the efficacy of hypothermia in blunting the damaging consequences of traumatic brain injury (TBI). With the use of posttraumatic hypothermia, it has been recognized that the time of initiation and duration of hypothermia are important variables in determining the degree of neuroprotection provided. Further, it has been recently recognized that the rate of posttraumatic rewarming is an important variable, with rapid rewarming exacerbating neuronal/axonal damage in contrast to slow rewarming which appears to provide enhanced neuroprotection. Although these findings have been confirmed in the brain parenchyma, no information exists for the cerebral microcirculation on the potential benefits of posttraumatic hypothermia followed by either slow or rapid rewarming. In the current communication we assess these issues in the pial circulation using a well-characterized model of TBI. Rats were prepared for the placement of cranial widows for direct assessment of the pial microcirculation prior to and after the induction of impact acceleration injury followed by moderate hypothermia with either subsequent slow or rapid rewarming strategies. The cranial windows allowed for the measurement of pial vessel diameter to assess ACh-dependent and CO2 reactivity in the chosen paradigms. ACh was applied topically to assess ACh-dependent dilation, while CO2 reactivity was assessed by changing the concentration of the inspired gas. Through this approach, it was found that posttraumatic hypothermia followed by slow rewarming maintained normal arteriolar vascular responses in terms of ACh-dependent dilation and CO2 reactivity. In contrast, arterioles subjected to TBI followed by normothermia or hypothermia and rapid rewarming showed impaired vasoreactivity in terms of their ACh-dependent and CO2 responses. This study provides additional evidence of the benefits of posttraumatic hypothermia followed by slow rewarming, demonstrating for the first time that the previously described neuroprotective effects extend to the cerebral microcirculation.