Earl F. Ellis

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana,. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.

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.

Inhibition by free radical scavengers and by cyclooxygenase inhibitors of pial arteriolar abnormalities from concussive brain injury in cats.

We studied the role of prostaglandins and free radicals in the induction of the functional and morphological pial arteriolar abnormalities produced by concussive brain injury. Anesthetized cats equipped with a cranial window for the observation of the pial microcirculation were subjected to concussive brain injury using a fluid-percussion device following administration of cyclooxygenase inhibitors (indomethacin or AHR-5850) or the vehicle for the solution of these agents (NaCl or Na2CC>3solution). Pial arterioles from vehicle-treated animals displayed sustained dilation, reduced responsiveness to the vasoconstrictor effect of arterial hypocapnia, and a high density of endothelial lesions. Animals pretreated with cyclooxygenase inhibitors showed less pronounced vasodilation, normal responsiveness to hypocapnia, and a significantly reduced number of lesions. The vasodilation and reduced responsiveness to the vasoconstrictor effects of hypocapnia after brain injury also were inhibited by topical application of free radical scavengers (nitroblue tetrazolium, superoxide dismutase, or mannitol). The vessels from cats pretreated with free radical scavengers also had a lower density of endothelial lesions than controls. The results support the view that the immediate cause of cerebral arteriolar damage in concussive brain injury is the generation of free oxygen radicals associated with increased prostaglandin synthesis. Circ Res 48: 95-103, 1981

Reduction of Voltage-Dependent Mg2+ Blockade of NMDA Current in Mechanically Injured Neurons

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.

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.

Cerebral arterial smooth muscle contraction by thromboxane A2.

The contractile effects of thromboxane A2 (TxAj), a labile arachidonic acid metabolite, were studied in arterial smooth muscle strips. TxA2 was generated upon the addition of 255 nM prostaglandin cyclic endoperoxide H3 to human platelet particles in the muscle bath. Using the isometric contraction produced by 40 mM K+ in isotonic saline as the reference contraction, bovine middle cerebral artery strips contracted to 153 ± 14% of the reference response while bovine coronary and porcine coronary, renal and common carotid strips contracted to 47 ± 3, 26 ± 5, 43 ± 2 and 2 ± 1% of reference, respectively. The cerebral artery response to the TxA2 generating system was as great as the maximum response to prostaglandin F2a and two times the maximum response to 5- hydroxytryptamine. Because TxA2 is formed by brain tissue and released from aggregating platelets, it may be important in the pathogenesis of spasm associated with injured brain tissue or pathologic changes leading to platelet aggregation.

Calcium Influx Factor, Further Evidence It Is 5,6-Epoxyeicosatrienoic Acid

We present evidence in astrocytes that 5,6-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid, may be a component of calcium influx factor, the elusive link between release of Ca2+ from intracellular stores and capacitative Ca2+ influx. Capacitative influx of extracellular Ca2+ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A2 and cytochrome P450 metabolism of arachidonic acid. AAOCF3, which inhibits cytosolic phospholipase A2, blocked thapsigargin-stimulated release of arachidonic acid as well as thapsigargin-stimulated elevation of intracellular free calcium. Inhibition of P450 arachidonic acid metabolism with SKF525A, econazole, orN-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca2+ influx. Nano- to picomolar 5,6-epoxyeicosatrienoic acid induced [Ca2+] i elevation consistent with capacitative Ca2+ influx. We have previously shown that 5,6-epoxyeicosatrienoic acid is synthesized and released by astrocytes. When 5,6-epoxyeicosatrienoic acid was applied to the rat brain surface, it induced vasodilation, suggesting that calcium influx factor may also serve a paracrine function. In summary, our results suggest that 5,6-epoxyeicosatrienoic acid may be a component of calcium influx factor and may participate in regulation of cerebral vascular tone.

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons.

Abstract: Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch‐induced injury alters mitochondrial membrane potential (Δ?m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, Δ?m was measured using rhodamine‐123, and ATP was quantified with a luciferin‐luciferase assay. In astrocytes, Δ?m dropped significantly, and ATP content declined 43‐52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, Δ?m declined at 15 min only in the severely stretched group. At 48 h postinjury, Δ?m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, Δ?m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24‐48 h. ATP content in mixed cultures declined 22‐28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.

Isoprostanes: Free Radical–Generated Prostaglandins With Constrictor Effects on Cerebral Arterioles

BACKGROUND AND PURPOSE Isoprostanes are generated by cyclooxygenase-independent free radical attack of arachidonic acid and are potent constrictors of the peripheral vasculature. Traumatic brain injury stimulates oxygen radical production and is associated with cerebral blood flow reduction. However, no specific vasoconstrictor has been identified as the cause of posttraumatic blood flow reduction. The purpose of this study was to determine whether isoprostanes constrict cerebral arterioles. METHODS The effects of 10(-9) to 10(-5) mol/L 8-iso-prostaglandin F2 alpha (8-iso-PGF2 alpha), 8-iso-prostaglandin E2 (8-iso-PGE2), and prostaglandin F2 alpha (PGF2 alpha) on pial arteriolar diameter were measured in anesthetized rats using a closed cranial window and in vivo microscopy. RESULTS All prostanoids produced vasoconstriction. Of these, 8-iso-PGF2 alpha produced the greatest vasoconstriction (34% +/- 2), followed by 8-iso-PGE2 (25% +/- 4) and PGF2 alpha (20% +/- 2). After six cerebrospinal fluid washouts of the cranial window, both 8-iso-PGF2 alpha- and 8-iso-PGE2-treated vessels remained slightly constricted, whereas the PGF2 alpha-treated vessels returned to control diameter. Coapplication of the semiselective thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 completely blocked the vasoconstriction induced by 8-iso-PGF2 alpha and 8-iso-PGE2. CONCLUSIONS Isoprostanes are potent constrictors of cerebral arterioles and appear to act at a receptor that is similar to the thromboxane A2/prostaglandin H2 receptor. Isoprostanes may play a role in the reduction of cerebral blood flow that occurs after brain injury and subsequent oxygen radical production.

Intracellular free calcium dynamics in stretch-injured astrocytes.

Abstract: We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca2+]i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura‐2 microphotometry. Our results indicate that astrocyte [Ca2+]i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury‐induced [Ca2+]i elevation is not sustained and returns to near‐basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca2+]i returns to normal after injury, we have identified persistent injury‐induced alterations in calcium‐mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate‐sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor‐mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate‐linked metabotropic glutamate receptor agonist trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid with an elevation in [Ca2+]i. We hypothesize that changes in calcium‐mediated signaling pathways, rather than an absolute elevation in [Ca2+]i, is responsible for some of the pathological consequences of traumatic brain injury.