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.
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.
Children with prenatal alcohol exposure (PAE) show deficits in verbal learning and spatial memory, as well as abnormal hippocampal development. The relationship between their memory and neuroanatomic impairments, however, has not been directly explored. Given that the hippocampus is integral for the synthesis and retrieval of learned information and is particularly vulnerable to the teratogenic effects of alcohol, we assessed whether reduced learning and recall abilities in children with fetal alcohol spectrum disorders (FASDs) are associated with abnormal hippocampal volumes. Nineteen children with FASDs and 18 typically developing controls aged 9 to 15 years were assessed for verbal learning and verbal and spatial recall and underwent structural magnetic resonance imaging. Images were analyzed for total intracranial volume and for right and left hippocampal volumes. Results revealed smaller left hippocampi and poorer verbal learning and verbal and spatial recall performance in children with FASDs than controls, as well as positive correlations between selective memory indices and hippocampal volumes only in the FASD group. Additionally, hippocampal volumes increased significantly with age in controls only, suggesting that PAE may be associated with long-term abnormalities in hippocampal development that may contribute to impaired verbal learning and verbal and spatial recall. (JINS, 2008(JINS, , 14, 1022(JINS, -1033
Anandamide (AN, arachidonyl ethanolamide) has been isolated from the brain and shown to be an endogenous ligand for the delta 9-tetrahydrocannabinol (delta 9-THC) receptor. The purpose of these studies was to determine whether AN or delta 9-THC can affect the cerebral circulation. With the use of the closed cranial window AN and delta 9-THC (10(-13)-10(-3) M) were topically applied to rabbit cerebral arterioles and effects on diameter were measured with a microscope. AN and delta 9-THC similarly induced a dose-dependent dilation starting at concentrations as low as 10(-12) M. Maximum dilation for AN was 25% and that for delta 9-THC 22%. Topical coapplication of indomethacin, a cyclooxygenase inhibitor, completely blocked dilation, whereas the free radical scavengers superoxide dismutase and catalase or the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) had no effect on AN-induced dilation. The cerebrospinal fluid level of prostaglandin E2 increased only in response to 10(-7) M and greater AN and was not affected by delta 9-THC. [3H]AN superfused through the cranial window was 20% converted to arachidonic acid. These results show that AN and delta 9-THC can modulate cerebral arterioles, likely by stimulating release and metabolism of endogenous arachidonic acid. Whether dilation is due to vasodilator eicosanoids, or other vasoactive agents whose synthesis or release is cyclooxygenase dependent, is uncertain.
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 Ca 2؉ from intracellular stores and capacitative Ca 2؉ influx. Capacitative influx of extracellular Ca 2؉ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A 2 and cytochrome P450 metabolism of arachidonic acid. AAOCF 3 , which inhibits cytosolic phospholipase A 2 , 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, or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca 2؉ influx. Nanoto picomolar 5,6-epoxyeicosatrienoic acid induced [Ca 2؉ ] i elevation consistent with capacitative Ca 2؉ 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.
Few studies have examined both episodic and semantic autobiographical memory (AM) performance during late childhood and early adolescence. Using the newly developed Children’s Autobiographical Interview (CAI), the present study examined the effects of age and sex on episodic and semantic AM and everyday memory in 182 children and adolescents. Results indicated that episodic and semantic AM both improved between 8 and 16 years of age; however, age-related changes were larger for episodic AM than for semantic AM. In addition, females were found to recall more episodic AM details, but not more semantic AM details, than males. Importantly, this sex difference in episodic AM recall was attenuated under conditions of high retrieval support (i.e., the use of probing questions). The ability to clearly visualize past events at the time of recollection was related to children’s episodic AM recall performance, particularly the retrieval of perceptual details. Finally, similar age and sex effects were found between episodic AM and everyday memory ability (e.g., memory for everyday activities). More specifically, older participants and females exhibited better episodic AM and everyday memory performance than younger participants and males. Overall, the present study provides important new insight into both episodic and semantic AM performance, as well as the relation between episodic AM and everyday memory, during late childhood and adolescence.
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