Eric J. Murphy

Cerebral Edema After Temporary and Permanent Middle Cerebral Artery Occlusion in the Rat

BACKGROUND AND PURPOSE The potential of thrombolytic agents to improve outcome after ischemic stroke could be negated if recanalization of an occluded artery exacerbates cerebral edema. We examined whether infarctions associated with reperfusion have more edema than those without reperfusion and whether the time course for the development of cerebral edema varied with and without reperfusion. METHODS Infarct volumes were measured 24 hours after permanent and 1, 2, and 3 hours of temporary right middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats. Hemispheric volume, water, sodium, and potassium were measured 3, 6, 12, 24, 36, and 48 hours after permanent and 3 hours of temporary MCA occlusion and also determined 24 hours after permanent and 2 and 3 hours of temporary MCA occlusion. RESULTS Minimal tissue damage occurred after 1 hour of temporary ischemia. Infarct sizes were similar after permanent and 3 hours of temporary MCA occlusion and significantly greater than after 2 hours of temporary ischemia. Hemispheric volume, water, and sodium from the infarcted right hemisphere were significantly greater than those from the left hemisphere beginning 6 hours after MCA occlusion and continuing for 48 hours, with a peak at 24 hours. Right hemispheric water measured 24 hours after 2 hours of temporary ischemia was significantly less than after permanent or 3 hours of temporary ischemia. CONCLUSIONS This study demonstrates that cerebral edema after focal stroke is related to infarct size and is independent of reperfusion status. The results suggest that exacerbation of cerebral edema will not occur after thrombolytic treatment or spontaneous recanalization of occluded cerebral vessels.

Acidic hydrolysis of plasmalogens followed by high-performance liquid chromatography

A simple, quantitative method for determining the plasmalogen content of small samples is reported here. The method uses the different susceptibility to acid-catalyzed hydrolysis of the alkyl, alkenyl and acyl linkages to separate the plasmalogen subclass from the other two non-labile subclasses. Hydrolysis of plasmenylethanolamine and plasmenylcholine was complete after 4 and 1 min of acid treatment, respectively. The acid-catalyzed hydrolysis did not alter the phospholipid fatty acid composition, making this method useful for fatty acid compositional analysis of the plasmalogen subclass. High-performance liquid chromatography was used for separations, and phospholipids were quantitated by assay of lipid phosphorus or by direct quantitation of peak area. Using this method, small amounts (10 nmol) of ethanolamine glycerophospholipid and choline glycerophospholipid are subjected to acid-catalyzed hydrolysis and subsequent separation of the resulting lysocompounds obtained from plasmalogens from the more acid-stable alkylacyl and diacyl glycerophospholipid fractions. Our values for plasmalogens from commercial preparations of choline and ethanolamine glycerophospholipids agree with literature values. The usefulness of the method is demonstrated for small glycerophospholipid samples that are equivalent to samples from cultured neural cells.

Extracellular calcium is a mediator of astroglial injury during combined glucose-oxygen deprivation

We tested the hypothesis that extracellular calcium is a mediator of astroglial injury during combined glucose-oxygen deprivation. Both differentiated and undifferentiated astroglial cultures were exposed to combined glucose-oxygen deprivation in the presence and absence of extracellular calcium. Lactate dehydrogenase efflux was used as an index of cellular injury. Both types of cultures exhibited significantly less cellular injury when exposed to combined glucose-oxygen deprivation in the absence of extracellular calcium (e.g. lactate dehydrogenase efflux in undifferentiated cultures after 12 h of exposure: presence of calcium, 65.2 +/- 2.5% vs. absence of calcium, 21.4 +/- 1.3%). To further elucidate the mechanism by which extracellular calcium produces injury, we studied the effect of nimodipine, an L-type calcium channel blocker, on astroglial injury resulting from combined glucose-oxygen deprivation. Nimodipine decreased cellular injury in both types of cultures (e.g. lactate dehydrogenase efflux in undifferentiated cultures after 12 h of exposure: untreated, 65.4 +/- 2.2% vs. 10 nM nimodipine, 44.6 +/- 4.2%). Extracellular calcium appears to be a mediator of astroglial injury during combined glucose-oxygen deprivation. These results suggest that influx of extracellular calcium via L-type voltage-gated calcium channels may contribute to astroglial injury during cerebral ischemia.

Lipid alterations following impact spinal cord injury in the rat

A computer-controlled impactor was used to produce a severe spinal cord injury in the rat thoracic spinal cord. Cords were rapidly frozen in situ at 5, 15, 30, and 60 min and 6, 12, and 24 h postinjury. Control cords were noninjured cords from animals having undergone a laminectomy and allowed to recover for 90 min postlaminectomy. The cords were assayed for alterations in lipid metabolism. Specifically, there were rapid increases in prostaglandin F2 alpha and thromboxane, with a peak increase in thromboxane levels at 30 min. Prostaglandin F2 alpha levels peaked at 15 min with levels remaining nearly constant for 12 h. There were no detectable changes in phospholipid levels, although diacylglycerol levels and free fatty acid levels were increased. Total free fatty acids were increased at 12 and 24 h postinjury by 2.3- and 3.2-fold over control levels, respectively. Arachidonic acid levels were not significantly elevated at early time points, however, these early time points correspond to elevated eicosanoid synthesis and this may account for the lack of early detectable increases in arachidonic acid. After 6 h postinjury, arachidonic acid levels were 20-fold greater than control levels and remained elevated at 24 h. There were minimal decreases in cholesterol and no decrease in either choline or ethanolamine plasmalogen levels. These results suggest a rapid turnover of arachidonic acid following spinal cord injury with a concomitant increase in vasoconstrictive eicosanoid synthesis. The lack of changes in major membrane constituents suggests the mechanisms may not involve general membrane degradation, but an over-stimulation of phospholipase A2-linked membrane receptors.

Phospholipid composition of cultured human endothelial cells

Detailed analyses of the phospholipid compositions of cultured human endothelial cells are reported here. No significant differences were found between the phospholipid compositions of cells from human artery, saphenous and umbilical vein. However, due to the small sample sizes, relatively large standard deviations for some of the phospholipid classes were observed. A representative composition of endothelial cells is: phosphatidylcholine 36.6%, choline plasmalogen 3.7%, phosphatidylethanolamine 10.2%, ethanolamine plasmalogen 7.6%, sphingomyelin 10.8%, phosphatidylserine 7.1%, lysophosphatidylcholine 7.5%, phosphatidylinositol 3.1%, lysophosphatidylethanolamine 3.6%, phosphatidylinositol 4,5-bisphosphate 1.8%, phosphatidic acid 1.9%, phosphatidylinositol 4-phosphate 1.5%, and cardiolipin 1.9%. The cells possess high choline plasmalogen and lysophosphatidylethanolamine contents. The other phospholipids are within the normal biological ranges expected. Phospholipids were separated by high-performance liquid chromatography and quantified by lipid phosphorus assay.

High-dose methylprednisolone treatment in experimental focal cerebral ischemia.

Previous studies using steroids for experimental focal stroke have demonstrated conflicting results, possibly related to dose used or ischemic models employed. In this study we examined high-dose methylprednisolone treatment following permanent and temporary focal cerebral ischemia in the rat. Focal stroke was induced in spontaneously hypertensive rats by permanent right common carotid and either permanent or 3 h of temporary middle cerebral artery (MCA) occlusion. Methylprednisolone (105 mg/kg) was administered intra-arterially. Infarct volume was measured at 24 h after permanent and temporary MCA occlusion. Cerebral edema was determined by measuring right and left hemispheric volumes and water content 24 h after permanent MCA occlusion in one experiment. Methylprednisolone, whether administered in divided doses over 12 h (n = 15 in each group) or a single bolus (n = 9 per group), had no effect on infarct volume after permanent MCA occlusion. Methylprednisolone treatment also had no influence on cerebral edema (n = 9 per group). In two different experiments, methylprednisolone given in divided doses over 12 h (n = 11, n = 25) after temporary MCA occlusion decreased infarct volume (P < 0.05) by 20% compared with saline controls (n = 10, n = 25). High dose methylprednisolone decreased infarct volume following temporary, but not permanent, focal ischemia. The benefit suggests that high dose methylprednisolone may prove useful clinically if reperfusion can be established with thrombolytic agents. Furthermore, the differential treatment effect in the setting of comparable ischemic insults implies that different modifiable biochemical processes may be present during temporary but not permanent focal ischemia, thus providing indirect evidence for reperfusion injury.

Cytotoxicity of aluminum silicates in primary neuronal cultures

To study their cytotoxicity, clays containing aluminum silicates were added to cultures of primary murine spinal cord neurons and differentiated N1E-115 neuroblastoma cells. Bentonite (0.1 mg/ml) and montmorillonite (0.1 mg/ml) rapidly associated with the outer membrane of both N1E-115 and neuronal cells. Erionite (0.1 mg/ml) was randomly distributed throughout the culture. Both bentonite and montmorillonite caused complete cell lysis in the neuronal cultures within 60 min following addition. Erionite had no effect. None of the clays appeared to be cytotoxic to the differentiated N1E-115 cells even though bentonite and montmorillonite were closely associated with the cell membrane. N1E-115 cell lysis did not occur up to 18 h after addition of the clay. Aluminum silicate-containing clays caused a rapid lysis of primary neuronal cells. Differentiated N1E-115 neuroblastoma cells were not susceptible to clay-induced lysis, suggesting that the lytic mechanism is not a general phenomenon that affects all cell types equally.

Effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells.

The effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells were determined. The cellular lipids were extracted on days 0, 3 and 7, following the addition of 1.2% dimethylsulfoxide to induce cellular differentiation. Proportions of ethanolamine glycerophospholipids (EtnGpl), phosphatidylinositol (PtdIns) and sphingomyelin (CerPCho) were significantly elevated following differentiation. The mole percentage of choline glycerophospholipids (ChoGpl) decreased with differentiation. The plasmalogens, both choline and ethanolamine, increased by 1.3- and 2.3-fold, respectively, during differentiation. The fatty acid composition of the phospholipid classes was also altered. PtdIns and ChoGpl had decreased proportions of polyenoic fatty acids, while these proportions were increased in EtnGpl. Both ChoGpl and EtnGpl had increased n-3/n-6 series fatty acid ratios, but this ratio was decreased in PtdIns. The mole percentage of arachidonic acid was significantly decreased in both PtdIns and ChoGpl, but elevated in EtnGpl and may be a result of the increase in ethanolamine plasmalogen. Thus, differentiation did not increase the overall mole percentage of polyenoic FA in the cells nor increase the n-6 series fatty acid proportions. We speculate plasmalogens may have a role in the differentiation process or in maintaining the cell in the differentiated state.

Purification of plasmalogens usingRhizopus delemar lipase andNaja naja naja phospholipase A2

Bovine heart ChoGpl (choline glycerophospholipid) and bovine brain EtnGpl (ethanolamine glycerophospholipid) contain diacyl, alkenylacyl and alkylacyl analogs. Purification of plasmalogens was achieved usingR. delemar lipase andN. naja naja phospholipase A2 digestion. TheR. delemar lipase hydrolyzes the acyl bond at the 1-position of 1,2-diacyl glycerophospholipids. TheN. naja naja phospholipase A2 has greater activity with diacyl and alkylacyl than with alkenylacyl glycerophospholipids. These enzymes were mainly used to remove diacyl and alkylacyl analogs respectively. When the diacyl types were removed by double incubation withR. delemar lipase, the plasmalogen content was 94.2%±0.21% (mean±S.E.M., n=4) for PlsCho (plasmenylcholine) and 94.9%±0.19% (mean±S.E.M., n=3) for PlsEtn (plasmenylethanolamine). Recoveries were 74% and 88% respectively. These partially purified plasmalogens were treated withN. naja naja phospholipase A2. Finally, 97.7%±0.24% (mean±S.E.M., n=4) and 98.8%±0.27% (mean±S.E.M., n=3) pure plasmalogens were obtained for PlsCho and PlsEtn respectively. Plasmalogens were recovered in an overall yield of 7.7%±0.7% (mean±S.E.M., n=4) and 10.2%±1.2% (mean±S.E.M., n=3) for PlsCho and PlsEtn.

Mechanisms of hypoxic and ischemic injury : use of cell culture models

Cell cultures are useful tools to study the mechanisms involved in cell death following hypoxia or ischemia. By manipulating the extracellular environment, conditions that closely mimic the conditions that are thought to occur in vivo can be produced. These conditions permit study of cells reaction to the trauma under specific conditions. Monitoring of the extracellular pH and ionic environment in cell cultures is much easier than in vivo. Further, metabolites produced by injured cells can be quantitated easier from cultures than from tissues in vivo. Cell cultures have recently been used to examine in detail the neurotoxicity of glutamate. Intracellular Ca2+ increases appear to be involved in the mechanisms of neurotoxic cell death. This Ca2+ entry appears to be through the NMDA receptors Ca2+ channel. Ischemic and hypoxic injury produced by mechanisms other than glutamate neurotoxicity appear to involve increases in intracellular Ca2+ by releasing internal Ca2+ stores or by the influx of extracellular Ca2+. This Ca2+ entry may be through voltage-gated channels of the NMDA channel, or may be attributable to membrane perturbations. Through the use of cell cultures, each of the mechanisms involvement in the injury can be delineated.