The purpose of this study was to develop a simple, reproducible model for examining the morphologic, physiologic, and biochemical consequences of stretch-induced injury on tissue-cultured cells of brain origin. Rat cortical astrocytes from 1- to 2-day-old rats were cultured to confluency in commercially available 25-mm-diameter tissue culture wells with a 2-mm-thick flexible silastic bottom. A cell injury controller was used to produce a closed system and exert a rapid positive pressure of known amplitude (psi) and duration (msec). The deformation of the membrane, and thus the stretch of the cells growing on the membrane, was proportional to the amplitude and duration of the air pressure pulse. Extent of cell injury was qualitatively assessed by light and electron microscopy and quantitatively assessed by nuclear uptake of the fluorescent dye propidium iodide, which is excluded from cells with intact membranes. Lactate dehydrogenase (LDH) enzyme release was measured spectrophotometrically. Cell injury was found to be proportional to the extent of the silastic membrane deformation. Increasing cell stretch caused mitochondrial swelling and vacuolization as well as disruption of glial filaments. Stretching also caused increased dye uptake, with maximum dye uptake occurring with a 50 msec pressure pulse duration, whereas deformations produced over longer periods of time (seconds) caused little dye uptake. With increasing postinjury survival fewer cells took up dye, implying cell repair. LDH release was also proportional to the amplitude of cell stretch, with maximum release occurring within 2 h of injury. In summary we have developed a simple, reproducible model to produce graded, strain-related injuries in cultured cells. Our continuing experiments suggest that this model can be used to study the biochemistry and physiology of injury as well as serve as a tool to examine the efficacy of therapeutic agents.

Purified PGH synthase when acting on arachidonic acid in the presence of reduced nicotinamideadenine dinucleotide or reduced nicotinamide-adenine dinucleotide 3'-phosphate generated superoxide in burst-like fashion. In eight experiments using different batches of enzyme, the mean ± SE rate of superoxide generation from 100 U of enzyme measured as the superoxide dismutase-inhibitable reduction of cytochrome c was 5.06 ± 0.19 nmol/min in the first minute and 0.35 ± 0.03 nmol/min subsequently. Optimum rates of superoxide were seen at concentrations of reduced nicotinamideadenine dinucleotide in excess of 80 /xM and reduced nicotinamide-adenine dinucleotide 3-phosphate in excess of 100 juM. Using prostaglandin G 2 or linoleic acid as substrate rather than arachidonate also resulted in superoxide generation. When prostaglandin H 2 was used as substrate, no superoxide was generated. The rate of superoxide generation was markedly inhibited by cyclooxygenase inhibitors. Superoxide generation was also observed during the action of lipoxygenase on either linoleic or arachidonic acid in the presence of reduced nicotinamide-adenine dinucleotide or reduced nicotinamide-adenine dinucleotide 3-phosphate but not in their absence. Indomethacin had no effect on superoxide generation from lipoxygenase. We conclude that PGH synthase and lipoxygenase produce superoxide via a side-chain reaction dependent on the presence of suitable reducing cosubstrate. This mechanism is analogous to that described for peroxidases in general. and topical application of arachidonate 89 cause a spectrum of functional, morphological, and biochemical cerebral arteriolar abnormalities. These consist of sustained dilation, reduced responsiveness to vasoconstrictor and vasodilator influences, focal destructive lesions of the endothelium and vascular smooth muscle, and reduced oxygen consumption of the vessel wall. Superoxide anion and other reactive agents derived from it, such as hydrogen peroxide and the hydroxyl radical, are the likely mediators of the cerebral arteriolar abnormalities seen in these conditions, since the latter are inhibited by scavengers of superoxide anion, hydrogen peroxide, and of the hydroxyl radical. 6 Received January 21, 1986; accepted August 25, 1986. ioles to these agents for more than a few minutes causes vascular injury. The enzymatic source of superoxide anion in the experimental conditions noted above is not firmly established. The following evidence suggests strongly that superoxide is produced via the action of the prostaglandin hydroperoxidase. First, the cerebral arteriolar abnormalities noted above can be reproduced by topical application of prostaglandin G 2 (PGG 2 ) 8 or 15-hydroperoxy-eicosatetraenoic acid (15-HPETE)," which are good substrates of the prostaglandin hydroperoxidase. The cerebral arteriolar abnormalities are not produced by prostaglandin H 2 (PGH 2 ), the product of the reaction catalyzed by prostaglandin hydroperoxidase.8 Second, the vasodilation and cerebral arteriolar abnormalities due to hype...

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.

Gliosis is characterized by hypertrophic and hyperplastic responses of astrocytes to brain injury. To determine whether injury of astrocytes produced by an in vitro model of brain trauma activates extracellular signal-regulated protein kinase (ERK), a key regulator of cellular proliferation and differentiation, astrocytes cultured on deformable SILASTIC membranes were subjected to rapid, reversible strain (stretch)-induced injury. Activation of ERK was observed 1 min after injury, was maximal from 10 to 30 min, and remained elevated for 3 hr. Activation of ERK was dependent on the rate and magnitude of injury; maximum ERK activation was observed after a 20-60 msec, 7.5 mm membrane displacement. ERK activation was blocked by inhibiting MEK, the upstream activator of ERK. Activation of ERK was reduced when calcium influx was diminished. When extracellular ATP was hydrolyzed by apyrase or ATP/P2 receptors were blocked, injury-induced ERK activation was significantly reduced. P2 receptor antagonist studies indicated a role for P2X2 and P2Y1, but not P2X1, P2X3, or P2X7, receptors in injury-induced ERK activation. These findings demonstrate for the first time that ATP released by mechanical injury is one of the signals that triggers ERK activation and suggest a role for extracellular ATP, P2 purinergic receptors, and calcium-dependent ERK signaling in the astrocytic response to brain trauma.

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.