Elena Martinez

Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain.

We have demonstrated previously that mild intraischemic hypothermia confers a marked protective effect on the final histopathological outcome. The present study was carried out to evaluate whether this protective effect involves changes in the degree of local cerebral blood flow reductions, tissue accumulation of free fatty acids, or alterations in the extracellular release of glutamate and dopamine. Rats whose intraischemic brain temperature was maintained at 36 degrees C, 33 degrees C, or 30 degrees C were subjected to 20 minutes of ischemia by four-vessel occlusion combined with systemic hypotension. Levels of local cerebral blood flow, as measured autoradiographically, were reduced uniformly in all experimental animals at the end of ischemia by gas chromatography after tissue extraction and separation by thin layer chromatography. A massive ischemia-induced accumulation of individual free fatty acids was observed in animal groups whose intraischemic brain temperature was maintained at either 36 degrees C or 30 degrees C. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. In rats whose intraischemic brain temperature was maintained at 36 degrees C, dopamine and glutamate increased significantly during ischemia and the early period of recirculation (by 500-fold and sevenfold, respectively). In animals whose brain temperature was maintained at 33 degrees C and 30 degrees C, the release of glutamate was completely inhibited, and the release of dopamine was significantly attenuated (by 60%). These results suggest that mild intraischemic hypothermia does not affect the ischemia-induced local cerebral blood flow reduction or free fatty acid accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Ischemia on the In Vivo Release of Striatal Dopamine, Glutamate, and γ‐Aminobutyric Acid Studied by Intracerebral Microdialysis

Abstract: We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesioning. The present study was carried out to evaluate whether the protective effect of the lesion involves changes in the degree of local cerebral blood flow (1CBF) reduction, energy metabolite depletion, or alterations in the extracellular release of striatal dopamine (DA), glutamate (Glu), or γ‐aminobutyric acid (GABA). Control and SN‐lesioned rats were subjected to 20 min of forebrain ischemia by four‐vessel occlusion combined with systemic hypotension. Levels of 1CBF, as measured by the autoradiographic method, and energy metabolites were uniformly reduced in both the ipsi‐ and contralateral striata at the end of the ischemic period, a finding implying that the lesion did not affect the severity of the ischemic insult itself. Extracellular neurotransmitter levels were measured by microdialysis; the perfusate was collected before, during, and after ischemia. An ∼ 500‐fold increase in DA content, a 7‐fold increase in Giu content, and a 5‐fold increase in GABA content were observed during ischemia in nonlesioned animals. These levels gradually returned to baseline by 30 min of reperfusion. In SN‐lesioned rats, the release of DA was completely prevented, the release of GABA was not affected, and the release of Glu was partially attenuated. However, excessive extracellular Glu concentrations were still attained, which are potentially toxic. This, taken together with the previous neuropathological findings, suggests that excessive release of DA is important for the development of ischemic cell damage in the striatum.

Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ-Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat

We evaluated whether regional differences in the magnitude of glutamate, γ‐aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia‐induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10‐ and 20‐min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathoiogical neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathoiogical outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10‐min insult but severely damaged after 20 min of ischemia. Ischemia‐induced increases in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10‐ and 20‐min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20‐min insult Although ischemic neuronal damage was not related to glutamate release, it correlated with the „excitotoxic index,” whose value was derived from the following equation: [glutamate] X [glycine]/[GABA]. No significant changes were observed in the excitotoxic index during ischemia. However, a significant increase in the index was observed in vulnerable brain regions during the early and late recirculation periods. These results suggest that the imbalance between excitation and inhibition, reflected by changes in the excitotoxic index, may account for regional vulnerability to ischemia.

Changes in Amino Acid Neurotransmitters and Cerebral Blood Flow in the Ischemic Penumbral Region following Middle Cerebral Artery Occlusion in the Rat: Correlation with Histopathology

We simultaneously measured neurotransmitter amino acids by the microdialysis technique and cortical CBF by laser-Doppler flowmetry in the ischemic penumbral cortex of rats subjected to 2-h normothermic (36.5–37.5°C) transient middle cerebral artery (MCA) clipocclusion. Brains were perfusion-fixed 3 days later and infarct volume measured. CBF (% of preischemic values) fell to 32 ± 2% (mean ± SD) during ischemia and rose to 157 ± 68% during recirculation. Extracellular glutamate levels increased from a baseline value of 7 ± 3 μM to a peak value of 180 ± 247 μM 20–30 min following onset of ischemia but subsequently returned to near baseline levels after 70 min of ischemia despite ongoing MCA occlusion. The threshold CBF for moderate glutamate release was 48%. Massive glutamate release was seen during the first 60 min of MCA occlusion in the two animals showing the largest infarcts and occurred at CBF values ≤20% of control levels. Mean CBF during ischemia exhibited an inverse relationship with infarct volume, and the magnitude of glutamate release during ischemia was positively correlated with infarct volume. Extracellular γ-aminobutyrate and glycine changes were similar to those of glutamate but showed no significant correlation with infarct volume. These results suggest that (a) accumulation of extracellular glutamate is an important determinant of injury in the setting of reversible MCA occlusion and (b) reuptake systems for neurotransmitter amino acids may be functional in the penumbra during transient focal ischemia. We have shown that during temporary MCA occlusion, penumbral levels of amino acid neurotransmitter initially rise but subsequently decline to baseline despite ongoing ischemia and that moderate extracellular glutamate release and massive release of GABA and glycine occur at a CBF threshold of 48%. It was suggested that massive glutamate release might require a lower ischemic threshold of 20%. The magnitude of glutamate release is correlated to the size of the resultant cortical infarct. These results are consistent with the impressions of previous investigations that have implicated glutamatergic mechanisms in focal ischemic injury. It is well known that MK-801 and other glutamate-related receptor antagonists are effective in reducing infarct size in focal ischemia models (Ozyurt et al., 1988; Park et al., 1988b; Simon and Shiraishi, 1990; Buchan et al., 1991; Smith and Meldrum, 1992). While glutamate is a contributory factor, it is nonetheless unlikely to be the sole cause of infarction during transient focal ischemia, and other neurotransmitter systems as well as nonneuronal events may also participate in infarct formation (Plum, 1983; Dietrich et al., 1991; Globus et al., 1991).

Intra-ischemic extracellular release of dopamine and glutamate is associated with striatal vulnerability to ischemia

We have previously described a marked attenuation of postischemic striatal neuronal death by prior substantia nigra (SN) lesion, and have shown that lowering the brain temperature by only a few degrees during ischemia also confers a marked protective effect. The present study was carried out to evaluate whether the protective effect of these manipulations involves changes in extracellular release of striatal dopamine (DA) and glutamate (Glu) during ischemia. Four animal subgroups were investigated, including unilateral SN-lesioned rats whose intra-ischemic brain temperature was maintained at 36 degrees C, and non-lesioned animals whose brain temperature was not regulated, or was maintained at 33 or 36 degrees C during ischemia. Striatal extracellular sampling was performed by a microdialysis probe in rats subjected to 20 min of ischemia by 4-vessel occlusion. In rats whose intra-ischemic brain temperature was 36 degrees C, both DA and Glu increased significantly. In SN-lesioned rats no changes were found in extracellular levels of DA. However, significant increases in Glu were measured. In animals whose brain temperature was not regulated (the intra-ischemic brain temperature fell to 30 degrees C) or maintained at 33 degrees C there was a significant increase of DA release, but no changes were found in extracellular levels of Glu. These results, taken together with the neuropathological findings, suggest that release of both DA and Glu during ischemia is necessary for the development of postischemic striatal damage.

Direct Evidence for Acute and Massive Norepinephrine Release in the Hippocampus During Transient Ischemia

Recent studies suggest the norepinephrine (NE) may play a regulatory role in neuronal cell death in the hippocampus after transient ischemia. However, ischemia-induced changes in extracellular NE release have not been demonstrated. In the present study, we utilized the microdialysis technique to measure extracellular NE levels in the hippocampus before, during, and after 20 min of global ischemia induced by two-vessel occlusion combined with systemic hypotension in the rat. Stable basal concentrations of extracellular NE were detected in three consecutive samples collected prior to ischemia (1.86 ± 1.21 pmol/ml of perfusate mean ± SEM). During ischemia, NE levels increased to 30.1 ± 5.5 pmol/ml, representing an 18-fold increase. The levels gradually returned to baseline by 40 min of reperfusion. These results are the first to demonstrate that acute and massive extracellular release of NE occurs in the hippocampus during ischemia and early recirculation. These results support the hypothesis that the activation of the noradrenergic system may play a significant role in modulating the development of ischemic neuronal damage.

Cerebral phosphoinositide, triacylglycerol, and energy metabolism in reversible ischemia: origin and fate of free fatty acids.

Abstract: Levels of phosphatidylinositol 4,5‐bisphosphate (PIP2), phosphatidylinositol 4‐phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid, diacylglycerol (DAG), triacylglycerol (TAG), and free fatty acids (FFAs), as well as their fatty acid composition, were determined in rat forebrain during ischemia and postischemic recirculation. Cerebral energy state and electroencephalograms (EEGs) were also studied. Fifteen minutes of ischemia resulted in a decrease in PIP2 and PIP contents but not in PI content, concurrent with an enlargement of the FFA and DAG pools. The latter were enriched in stearate and arachidonate. Prolongation of ischemia did not produce further changes in content of any of the inositol phosphalipids, but the increase in levels of FFAs and DAG continued. At the end of 45 min of ischemia, levels of both PIP2 and PIP decreased by 45–50%, and the total phosphoinositide content (PIP2+ PIP + PI) decreased by 21%, whereas levels of FFAs and DAG increased to 14– and 3.6‐fold of control levels, respectively. During ischemia, the TAG‐palmitate level decreased, but the TAG‐arachidonate level increased; the tissue energy state deteriorated severely; and the EEG was suppressed. A 30‐min recirculation period after 15 or 45 min of ischemia led to increases in PIP2, PIP, and total phosphoinositide contents, whereas levels of FFAs and DAG promptly decreased toward control values. The TAG‐arachidonate level peaked and the TAG‐palmitate level returned to a low control value during early recirculation. The ischemic changes in tissue lipids were completely reversed within 3 h of recirculation after both periods of ischemia. Adenylates were fully phosphorylated with as little as 30 min of reflow. The EEG activity partially recovered during reflow after 15 min of ischemia, whereas it remained depressed after prolonged ischemia. Thus, phosphodiesteric cleavage of PIP2 and PIP followed by deacylation of DAG is likely to contribute to the production of FFAs in early ischemia. Deacylation of undetermined lipids plays a role for the increment in levels of FFAs in the later period of ischemia. The rapid postischemic increase in levels of PIP2 and PIP indicates active synthesis not only from existing PI, but probably also by means of accumulated FFAs and DAG. These results indicate that the impaired resynthesis of inositol phospholipids cannot be a cause of the poor EEG activity after prolonged ischemia. Degradation and resynthesis of polyphosphoinositides and formation of TAG‐arachidonate may be important for modulation of free arachidonic acid levels in the brain during temporary ischemia.

Simultaneous Measurement of Salicylate Hydroxylation and Glutamate Release in the Penumbral Cortex Following Transient Middle Cerebral Artery Occlusion in Rats

Using the microdialysis technique and laser-Doppler flowmetry, we performed simultaneous measurement of salicylate hydroxylation and glutamate release along with local CBF in the ischemic penumbral cortex of rat brain subjected to normothermic transient middle cerebral artery (MCA) occlusion. Cortical CBF fell to 24 ± 11% (mean ± SD) during ischemia and recovered to 84 ± 16% during reperfusion. Extracellular glutamate levels increased by 6.5-fold above baseline 10 min following MCA occlusion but subsequently returned to near baseline levels in spite of the persistent ischemia. Increase in 2,3- and 2,5-dihydroxybenzoic acid (DHBA) concentrations in the microdialysis perfusate was confirmed during both ischemia and reperfusion phase. Although the temporal profile and amount of salicylate hydroxylation were heterogeneous among individual animals, integrated 2,3-DHBA concentrations during reperfusion were correlated positively with integrated glutamate concentrations during ischemia and negatively with mean postischemic CBF. These relationships suggest a possible association of the enhanced production of 2,3-DHBA during reperfusion with larger amounts of intraischemic glutamate release and lower levels of postischemic CBF.

Children with asthma have improved pulmonary functions after massage therapy

Thirty-two children with asthma (16 4- to 8-year-olds and 16 9- to 14-year-olds) were randomly assigned to receive either massage therapy or relaxation therapy. The childrens parents were taught to provide one therapy or the other for 20 minutes before bedtime each night for 30 days. The younger children who received massage therapy showed an immediate decrease in behavioral anxiety and cortisol levels after massage. Also, their attitude toward asthma and their peak air flow and other pulmonary functions improved over the course of the study. The older children who received massage therapy reported lower anxiety after the massage. Their attitude toward asthma also improved over the study, but only one measure of pulmonary function (forced expiratory flow 25% to 75%) improved. The reason for the smaller therapeutic benefit in the older children is unknown; however, it appears that daily massage improves airway caliber and control of asthma.

Fn14 Is Upregulated in Cytokine-Stimulated Vascular Smooth Muscle Cells and Is Expressed in Human Carotid Atherosclerotic Plaques Modulation by Atorvastatin

Background and Purpose— Interaction between different members of the tumor necrosis factor superfamily and their receptors elicits diverse biologic actions that are implicated in the pathogenesis of atherosclerosis. We have analyzed the expression of Fn14 and its ligand TWEAK in carotid atherosclerotic plaques and its potential modulation by atorvastatin in vivo. Furthermore, we have studied whether proinflammatory cytokines regulate Fn14 expression in human aortic smooth muscle cells (hASMCs) in culture as well as the potential regulation by atorvastatin treatment. Methods— Fn14 and TWEAK expression was analyzed in human carotid atherosclerotic plaques. Furthermore, Fn14 expression was studied in hASMCs in culture. Results— Fn14 and TWEAK are expressed in macrophages and smooth muscle cells in carotid atherosclerotic plaques. Proinflammatory cytokines (interleukin-1β and interferon-γ) upregulate Fn14 expression in hASMCs. This effect was prevented by atorvastatin treatment and reversed by mevalonate and geranylgeranyl pyrophosphate. Geranylgeranyl transferase inhibitor, toxin B (Rac and Rho inhibitor), C3 exoenzyme (Rho inhibitor), and Y-27632 (Rho kinase inhibitor) also decreased Fn14 expression, implicating the Rho/Rho kinase pathway in the regulation of Fn14 expression. Finally, atorvastatin treatment reduced Fn14 expression in vivo. Conclusions— TWEAK and Fn14 are expressed in atherosclerotic plaques and could be novel mediators of atherosclerosis. Atorvastatin diminishes Fn14 expression in vitro and in vivo providing novel information of the beneficial properties of statins.