Head-focused Microwave Irradiation Research Articles

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Exogenous oxygen is required for prostanoid induction under brain ischemia as evidence for a novel regulatory mechanism

Previously, we and others reported a rapid and dramatic increase in brain prostanoids (PG), including prostaglandins, prostacyclins, and thromboxanes, under ischemia that is traditionally explained through the activation of esterified arachidonic acid (20:4n6) release by phospholipases as a substrate for cyclooxygenases (COX). However, the availability of another required COX substrate, oxygen, has not been considered in this mechanism. To address this mechanism for PG upregulation through oxygen availability, we analyzed mouse brain PG, free 20:4n6, and oxygen levels at different time points after ischemic onset using head-focused microwave irradiation (MW) to inactivate enzymes in situ before craniotomy. The oxygen half-life in the ischemic brain was 5.32 ± 0.45 s and dropped to undetectable levels within 12 s of ischemia onset, while there were no significant free 20:4n6 or PG changes at 30 s of ischemia. Furthermore, there was no significant PG increase at 2 and 10 min after ischemia onset compared to basal levels, while free 20:4n6 was increased ∼50 and ∼100 fold, respectively. However, PG increased ∼30-fold when ischemia was followed by craniotomy of nonMW tissue that provided oxygen for active enzymes. Moreover, craniotomy performed under anoxic conditions without MW did not result in PG induction, while exposure of these brains to atmospheric oxygen significantly induced PG. Our results indicate, for the first time, that oxygen availability is another important regulatory factor for PG production under ischemia. Further studies are required to investigate the physiological role of COX/PG regulation through tissue oxygen concentration.

Effects of hypercapnia / ischemia and dissection on the rat brain metabolome

It is known that brain energy metabolites such as ATP are quickly depleted during postmortem ischemia; however, a comprehensive assessment on the effects of preceding hypercapnia/ischemia and the dissection process on the larger brain metabolome remains lacking. This study sought to address this unknown by measuring aqueous metabolites impacted by hypercapnia/ischemia and brain dissection using Nuclear Magnetic Resonance. Metabolites were measured in rats subjected to 1) high energy head-focused microwave irradiation (control group); 2) CO2–induced hypercapnia/ischemia followed by immediate microwave irradiation; 3) CO2 followed by decapitation and then microwave irradiation ∼6.4 min later, to simulate a postmortem interval equivalent to typical dissection times; and 4) CO2–induced hypercapnia/ischemia followed by dissection within ∼6 min (no microwave fixation) to test the effects of brain dissection on the metabolome. Compared to control rats subjected to head-focused microwave irradiation, concentrations of high-energy phosphate metabolites and glucose were significantly reduced, while β-hydroxybutyrate and lactate were increased in rats subjected to all other treatments. Several amino acids and neurotransmitters (GABA) increased by hypercapnia/ischemia and dissection. Sugar donors involved in glycosylation decreased and nucleotides decreased or increased following hypercapnia/ischemia and dissection. sn-Glycero-3-phosphocholine decreased and its choline byproduct increased in all groups relative to controls, indicating postmortem changes in lipid turnover. Antioxidants increased following hypercapnia/ischemia but decreased to control levels following dissection. This study demonstrates substantial post-mortem changes in brain energy and glycosylation pathways, as well as protein, nucleotide, neurotransmitter, lipid, and antioxidant turnover due to hypercapnia/ischemia and dissection. Changes in phosphate donors, glycosylation and amino acids reflect post-translational modification and protein degradation processes that persist post-mortem. Microwave irradiation is necessary for accurately capturing in vivo brain metabolite concentrations.

Brain 2-Arachidonoylglycerol Levels Are Dramatically and Rapidly Increased Under Acute Ischemia-Injury Which Is Prevented by Microwave Irradiation.

The involvement of brain 2-arachidonoylglycerol (2-AG) in a number of critical physiological and pathophysiological regulatory mechanisms highlights the importance for an accurate brain 2-AG determination. In the present study, we validated head-focused microwave irradiation (MW) as a method to prevent postmortem brain 2-AG alterations before analysis. We compared MW to freezing to prevent 2-AG induction and estimated exogenous and endogenous 2-AG stability upon exposure to MW. Using MW, we measured, for the first time, true 2-AG brain levels under basal conditions, 30 s after brain removal from the cranium, and upon exposure to 5 min of brain global ischemia. Our data indicate that brain 2-AG levels are instantaneously and dramatically increased approximately 60-fold upon brain removal from the cranium. With 5 min of brain global ischemia 2-AG levels are also, but less dramatically, increased 3.5-fold. Our data indicate that brain tissue fixation with MW is a required technique to measure both true basal 2-AG levels and 2-AG alterations under different experimental conditions including global ischemia, and 2-AG is stable upon exposure to MW.

The low level of EPA in brain phospholipids is maintained by multiple compensatory mechanisms

ObjectiveTo examine if mitochondrial fatty acid beta‐oxidation is necessary for maintaining low EPA levels in brain phospholipids.MethodsAdult male free‐living Sprague Dawley rats were administered vehicle or methyl palmoxirate (MEP), a carnitine palmitoyltransferase I (CPTI) antagonist prior to being infused intravenously with 150 μCi/kg of 14C‐radiolabeled palmitate, DHA or EPA. Blood samples were collected during the five minute infusion via the jugular vein. At 5 minutes with continual radiotracer infusion, rats were subjected to high‐energy, head‐focused microwave irradiation and brains were collected for radioactive and biochemical analyses.ResultsUsing equations from our fatty acid model, we found that that incorporation coefficient (k*) into the aqueous fraction (marker of β‐oxidation) decreased significantly in MEP‐treated brains compared to vehicle controls for palmitate (3.8‐fold) and EPA (2.2‐fold) but not DHA. This was accompanied by a significant increase in the k* into total phospholipids of MEP‐treated brains as compared to controls for 14C‐EPA infused rats (1.5‐fold) but not 14C‐palmitate and 14C‐DHA infused rats. However, after identification and correction for metabolized 14C‐EPA, the increase in k* was attributed to an increase in synthesis of the EPA elongation product, n‐3 docosapentaenoic acid. Furthermore, the incorporation (Jin) and recycling (FFA) of EPA into brain phospholipids were significantly lower than palmitate and DHA by up to 56‐fold and 9‐fold, respectively.ConclusionThe low level of EPA in brain phospholipids is maintained via beta‐oxidation, elongation and rapid metabolisms.Grant Funding Source: NSERC

Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis

Acetate supplementation in rats increases plasma acetate and brain acetyl-CoA levels. Although acetate is used as a marker to study glial energy metabolism, the effect that acetate supplementation has on normal brain energy stores has not been quantified. To determine the effect(s) that an increase in acetyl-CoA levels has on brain energy metabolism, we measured brain nucleotide, phosphagen and glycogen levels, and quantified cardiolipin content and mitochondrial number in rats subjected to acetate supplementation. Acetate supplementation was induced with glyceryl triacetate (GTA) by oral gavage (6g/kg body weight). Rats used for biochemical analysis were euthanized using head-focused microwave irradiation at 2, and 4h following treatment to immediately stop metabolism. We found that acetate did not alter brain ATP, ADP, NAD, GTP levels, or the energy charge ratio [ECR, (ATP+½ ADP)/(ATP+ADP+AMP)] when compared to controls. However, after 4h of treatment brain phosphocreatine levels were significantly elevated with a concomitant reduction in AMP levels with no change in glycogen levels. In parallel studies where rats were treated with GTA for 28days, we found that acetate did not alter brain glycogen and mitochondrial biogenesis as determined by measuring brain cardiolipin content, the fatty acid composition of cardiolipin and using quantitative ultra-structural analysis to determine mitochondrial density/unit area of cytoplasm in hippocampal CA3 neurons. Collectively, these data suggest that an increase in brain acetyl-CoA levels by acetate supplementation does increase brain energy stores however it has no effect on brain glycogen and neuronal mitochondrial biogenesis.

LC/MS/MS method for analysis of E2 series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂) and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.

Insulin/FOXO Signaling Regulates Ovarian Prostaglandins Critical for Reproduction

Abnormalities in insulin/IGF-1 signaling are associated with infertility, but the molecular mechanisms are not well understood. Here we use liquid chromatography with electrospray ionization tandem mass spectrometry to show that the C. elegans insulin/FOXO pathway regulates the metabolism of locally acting lipid hormones called prostaglandins. C. elegans prostaglandins are synthesized without prostaglandin G/H synthase homologs, the targets of nonsteroidal anti-inflammatory drugs. Our results support the model that insulin signaling promotes the conversion of oocyte polyunsaturated fatty acids (PUFAs) into F-series prostaglandins that guide sperm to the fertilization site. Reduction in insulin signaling activates DAF-16/FOXO, which represses the transcription of germline and intestinal genes required to deliver PUFAs to oocytes in lipoprotein complexes. Nutritional and neuroendocrine cues target this mechanism to control prostaglandin metabolism and reproductive output. Prostaglandins may be conserved sperm guidance factors and widespread downstream effectors of insulin actions that influence both reproductive and nonreproductive processes.

Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain

Inflammatory lipid mediators derived from arachidonic acid (AA) and docosahexaenoic acid (DHA) modify the pathophysiology of brain ischemia. The goal of this work was to investigate the formation of eicosanoids and docosanoids generated from AA and DHA, respectively, during no-flow cerebral ischemia. Rats were subjected to head-focused microwave irradiation 5 min following decapitation (complete ischemia) or prior to decapitation (controls). Brain lipids were extracted and analyzed by reverse-phase liquid chromatography-tandem mass spectrometry. After complete ischemia, brain AA, DHA, and docosapentaenoic acid concentrations increased 18-, 5- and 4-fold compared with controls, respectively. Prostaglandin E(2) (PGE(2)) and PGD(2) could not be detected in control microwaved rat brain, suggesting little endogenous PGE(2)/D(2) production in the brain in the absence of experimental manipulation. Concentrations of thromboxane B(2), E(2)/D(2)-isoprostanes, 5-hydroxyeicosatetraenoic acid (5-HETE), 5-oxo-eicosatetraenoic acid, and 12-HETE were significantly elevated in ischemic brains. In addition, DHA products such as mono-, di- and trihydroxy-DHA were detected in control and ischemic brains. Monohydroxy-DHA, identified as 17-hydroxy-DHA and thought to be the immediate precursor of neuroprotectin D(1), was 6.5-fold higher in ischemic than in control brain. The present study demonstrated increased formation of eicosanoids, E(2)/D(2)-IsoPs, and docosanoids following cerebral ischemia. A balance of these lipid mediators may mediate immediate events of ischemic injury and recovery.

An improved LC-MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction

High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC-MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at -80 degrees C was reduced 2- to 4- fold in 4 weeks; however, PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave-irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.

Assessment of post‐mortem‐induced changes to the mouse brain proteome

This study was designed to assess the influence of high-energy head-focused microwave irradiation and the post-mortem interval on measurements of the mouse brain proteome. Difference gel electrophoresis was used to compare mouse brain protein levels in animals killed by decapitation, where the tissue was held at 25 degrees C for selected time intervals post-mortem, and by high-energy head-focused microwave irradiation followed by immediate resection. Microwave-mediated killing was used because it comprehensively snap-inactivates enzymes while largely retaining brain cytoarchitecture. Of the 912 protein spots common to at least eight of 10 gels analyzed, 35 (3.8%) showed significant differences in levels (t-test; p < 0.05) depending on whether animals were killed by microwave irradiation or decapitation. When animals were killed by decapitation, 43 protein spots (4.7%) showed changes in levels over the post-mortem interval (anova; p < 0.05). The vast majority of the near 1000 proteins evident on a 2D gel were stable for up to 4 h. These data have important implications for studies of proteins in the brain, whether based on analysis of tissue derived from animal models or from humans.

Rapid High-Energy Microwave Fixation is Required to Determine the Anandamide (N-arachidonoylethanolamine) Concentration of Rat Brain

Anandamide (N-arachidonoylethanolamine, AEA) is the putative endogenous ligand for the CB1 receptor. Despite being regulated enzymatically, brain AEA concentrations are quite variable and have been reported to increase in response to ischemia and post-mortem delay. Because these observations are similar to the effects of decapitation on brain concentrations of unesterified arachidonic acid and several of its metabolites, we propose that brain AEA concentrations also increase with decapitation and that immediate head-focused microwave irradiation is necessary to quantify basal brain AEA levels correctly. To test this hypothesis, we measured brain AEA levels in rats that were subjected to head-focused microwave irradiation 5 min. following decapitation (5.5 kW, 3.4 s) (ischemic) and prior to decapitation (controls). Brain AEA concentrations were quantified by LC/MS/MS. AEA concentrations from ischemic animals (10.01 +/- 4.41 pmol/g, mean +/- SD) were significantly higher and more variable than control concentrations (2.45 +/- 0.39 pmol/g). Thus, the basal concentration of AEA in the brain is lower than previously thought and future studies attempting to quantify brain AEA should consider using head-focused microwave fixation to prevent anomalous results.

Decreases in mouse brain NAD + and ATP induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): prevention by the poly(ADP-ribose) polymerase inhibitor, benzamide

Inhibitors of poly(ADP-ribose) polymerase (PARP), including benzamide, protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine neurotoxicity in vivo [Cosi et al., Brain Res. 729 (1996) 264–269]. In vitro, the activation of PARP by free radical damaged DNA has been shown to be correlated with rapid decreases in the cellular levels of its substrate nicotinamide adenine dinucleotide (NAD +), and ATP. Here, we investigated in vivo whether MPTP acutely caused region- and time-dependent changes in brain levels of NAD +, ATP, ADP and AMP in C57BL/6N mice killed by head-focused microwave irradiation, and whether such effects were modified by treatments with neuroprotective doses of benzamide. At 1 h after MPTP injections (4×20 mg/kg i.p.), NAD + was reduced by 11–13% in the striatum and ventral midbrain, but not in the frontal cortex. The ATP/ADP ratio was reduced by 10% and 32% in the striatum and cortex, respectively, but was unchanged in the midbrain. All of these regional changes were prevented by co-treatment with benzamide (2×160 mg/kg i.p.), which by itself did not alter regional levels of NAD +, ATP, ADP or AMP in control mice. In a time-course study, a single dose of MPTP (30 mg/kg i.p.) resulted in maximal and transient increases in striatal levels of MPP + and 3-methoxytyramine (+540%) at 0.5–2 h, followed by maximal and coincidental decreases in NAD + (−10%), ATP (−11%) and dopamine content (−39%) at 3 h. Benzamide (1×640 mg/kg i.p., 30 min before MPTP) partially reduced MPP + levels by 30% with little or no effect on MPTP or MPDP + levels, did not affect or even slightly potentiated the increase in 3-methoxytyramine, and completely prevented the losses in striatal NAD +, ATP and dopamine content, without by itself causing any changes in these latter parameters in control mice. These results (1) confirm that MPTP reduces striatal ATP levels [Chan et al., J. Neurochem. 57 (1991) 348–351.]; (2) show that MPTP causes a regionally-dependent (striatal and midbrain) loss of NAD +; (3) indicate that the PARP inhibitor benzamide can prevent these losses without interfering with MPTP-induced striatal dopamine release; and (4) provide further evidence to suggest an involvement of PARP in MPTP-induced neurotoxicity in vivo.

Regional and temporal variations in the accumulation of unesterified fatty acids and diacylglycerols in the rat brain during kainic acid induced limbic seizures

These experiments tested the hypothesis that limbic seizures induced by kainic acid (KA) activate mechanisms (e.g. phospholipases) that degrade the cell membrane, causing a release and accumulation of free fatty acids (FFAs) and diacylglycerols (DGs) in brain areas susceptible to seizure-related damage. The possible link between these effects on lipids and the subsequent development of seizure-related brain damage was investigated by studying the temporal and regional relationship between alterations in lipids in the hippocampus, frontal cerebral cortex, amygdala, striatum and cerebellum, and the development and severity of seizures. Rats were treated with 10 mg/kg KA (s.c.) and sacrificed by head focused microwave irradiation at 1 h, 2 h, 24 h or 7 days. Levels of FFAs and DGs were determined by gas liquid chromatography (GLC). Brain regions from control rats differed markedly in the content and composition of both FFA and DG pools. Changes in FFAs and DGs during KA-induced limbic seizures also varied from region to region and over time after drug treatment. The largest increases in FFAs in amygdala, striatum, cortex and hippocampus occured during the peak of seizure activity. Although DG levels were altered in some areas at some time points, there was no apparent correlation between changes in DGs and seizure severity. However, increases in DGs occurred at later time points, coincident with the occurence of neuronal cell loss in amygdala, cortex, hippocampus and striatum. These data indicate that limbic seizures activate the accumulation of FFAs through increased neuronal activity, while accumulation of DGs may be related to the development of seizure-related brain damage.

Effects of streptozotocin-induced diabetes on acetylcholine metabolism in rat brain

The main objective of this study was to determine whether uncontrolled hyperglycemia, as a consequence of diabetes, altered the metabolism of acetylcholine (ACh) in rat brain. To accomplish this, rats received injections of streptozotocin (STZ, 60 mg/kg, i.v.) or vehicle, and were maintained for up to 7 weeks after the injections. Various indices of ACh metabolism were determined in striatum and hippocampus, two brain regions densely innervated by cholinergic neurons. STZ induced diabetes in 96% of the rats injected, as evidenced by glucose spillage into the urine within 48 hours. Serum glucose levels increased to 326% of control values by 1 week and remained at this level for the duration of the study. The steady-state concentrations of ACh and choline, determined in brain tissue from animals killed by head-focused microwave irradiation, did not differ between the control and STZ-injected groups. However, the synthesis and release of neurotransmitter by striatal slices, measured in vitro, decreased in a time-dependent manner. Although the basal release of ACh was unaltered at 1 week, neurotransmitter release decreased significantly by 21% at 5 weeks and by 26% at 7 weeks. The release of ACh evoked by incubation with 35 mM KCl was inhibited significantly by 20% at all time points studied. ACh synthesis by slices incubated under basal conditions decreased by 13% and 27% at 5- and 7-weeks, respectively, the latter significantly less than controls. Synthesis by striatal slices incubated with 35 mM KCl was inhibited by 17% at 7 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid, stearic acid, and diacylglycerol accumulation correlates with the loss of phosphatidylinositol 4,5-bisphosphate in cerebrum 2 seconds after electroconvulsive shock: complete reversion of changes 5 minutes after stimulation.

The effects of electroconvulsive shock (750 msec, 130 V, 150 pps) on the endogenous content of rat cerebral lipids were studied 2, 5, 10, 20, 30, 60, and 300 sec after stimulation. Rapid enzyme inactivation in situ was attained by high-power head-focused microwave irradiation (6.5 kW, 2450 MHz). At 10 sec, phosphatidylinositol 4,5-bisphosphate (PIP2) mass had decreased by 249 nmol per g wet wt, mainly due to loss of arachidonate and stearate. At the same time, the stearoyl-arachidonoyl glycerol accumulated, although to a lesser extent than the loss exhibited in PIP2. Changes in phosphatidylinositol and in phosphatidylinositol 4-phosphate mass were not statistically significant. Free fatty acids and diacylglycerols accumulated to 395 nmol per g wet wt; arachidonic and stearic acids composed 322 nmol of these lipids. Hence, the reduction in content of PIP2 is sufficient to account for 80% of the increases in free fatty acid and diacylglycerol mass. Thirty-three and 12 nmol of accumulated free palmitic and docosahexaenoic acids, respectively, are not accounted for by the loss of PIP2. Sixty seconds after stimulation, PIP2 content returned to 90% of control levels, while diacylglycerol tended to remain below control levels. Free fatty acids had not returned to control levels by 60 sec, with the exception of docosahexaenoic acid. At 300 sec, PIP2, diacylglycerol, and free fatty acids had all returned to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced inositide turnover in brain during bicuculline-induced status epilepticus

Because brain inositides are enriched in the 1-stearoyl-2-arachidonoyl species, they form a likely source for the tetraenoic free fatty acids (FFA) and diacylglycerols (DG) that are accumulated during seizures. To study inositide turnover during bicuculline-induced seizures, rats were injected intraventricularly and bilaterally with 10–20 μCi 32P, mechanically ventilated and sacrificed by 6.5 KW head-focused microwave irradiation. Seizure activity was recorded by electroencephalography. Bicuculline-induced seizure activity resulted in: a) almost 50% increase in 32P labeling of phosphatidic acid (PA); phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP 2) also increased (24% and 36%, respectively); b) no change in other lipids; and c) water-soluble phosphodiesteratic degradation products, analyzed by high voltage paper electrophoresis, increased 24% in the amount of radiotracer recovered as inositol 1,4-bisphosphate (IP 2) and by 44% in the amount recovered as inositol 1,4,5-trisphosphate (IP 3). These data indicate that during experimental status epilepticus the cerebral inositide cycle is accelerated: PIP 2 → ( (IP 3 → IP 2 → IP → I) + DG → PA → PI → PIP → PIP 2.

The effect of acute and subacute ammonia intoxication on regional cerebral acetylcholine levels in rats

Cerebral regional concentrations of acetylcholine (ACh) were measured in two acute (with or without portacaval shunt) and one subacute (average 3 days) models of ammonia-induced coma in rats. The animals were sacrificed by total-body or head-focused microwave irradiation which, respectively, decreases or essentially eliminates post mortem degradation of ACh. In the two acute models of coma, normal levels of ACh were found in cortex, midbrain, and brainstem. A small decrease in striatal ACh was noted in rats without portacaval shunt given ammonia, but not in the other acutely intoxicated group. By contrast, in the subacute model (portacaval shunted rat given ammoniated resin by gavage) ACh fell by 14, 20, 33 and 39%, respectively, in the cortex, corpus striatum, midbrain, and brainstem. It is suggested that this decrease in brain ACh is a concomitant finding and not the cause of the coma.