Brain Magnetic Resonance Imaging of Intracerebral Hemorrhagic Rats after Alcohol Consumption.

BACKGROUND: Alcoholism is one of the risk factors for cerebrovascular diseases. Our previous study demonstrated that acute alcohol intoxication enhances brain injury and neurological impairment in rats suffering from intracerebral hemorrhage (ICH). We plan to investigate the effect of chronic alcohol consumption (CAC) in rats with ICH by magnetic resonance imaging (MRI). METHODS: Sixteen Sprague-Dawley male rats were divided into 2 groups: CAC group (fed with 10% alcohol drinking water for 4 weeks, n = 8), and Control group (plain drinking water, n = 8). ICH was induced by collagenase infusion into the right striata of all rats. Coronal T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, and diffusion-weighted imaging were generated with a 3.0T MRI scanner to investigate the changes of hemorrhagic volume and edema throughout the injury and recovery stages of ICH in rats. RESULTS: T2-weighted imaging is ideal for monitoring hematoma volume in rats. The hematoma volume was larger in the CAC group than in the control group (P < .001), however, did not correlate to post-ICH progressive edema formation (P > .7), and neurological impairment (P > .28) between the 2 groups, respectively. DISCUSSION: Although our findings indicate that CAC induces larger hematoma in rats with ICH, the underlying mechanism should be studied in the future.

Collagenase-Induced Rat Intra-Striatal Hemorrhage Mimicking Severe Human Intra-Striatal Hemorrhage.

Basal ganglia hemorrhage accounts for approximately 50% of all hemorrhagic strokes. A good rat model that produces severe intrastriatal hemorrhage (ISH) mimicking human severe ISH is lacking. The present study compared the intra-striatal injection of 0.2 U with that of 0.6 U of collagenase in inducing severe ISH in rats. Three-Tesla (3T) magnetic resonance imaging (MRI) was used to evaluate brain injuries in terms of hematoma size (volume), midline shift (MLS), and brain edema. This evaluation was further substantiated by determination of behavior and neurologic functions and mortality over 56 h. The 0.2 U collagenase caused hematoma volume increases for 10.3 to 30.1 mm³, while the 0.6 U caused 36.4 to 114.8 mm³, at post-ISH 1 h to 56 h. The 0.6 U collagenase significantly increased MLS to 1.5-3.0 times greater than the 0.2 U did at all post-intracerebral hemorrhage (ICH) time points. The MLS increased dependently with hematoma expansion with high correlation coefficients, yet no mortality occurred. These two dosages, nevertheless, caused the same pattern and severity in relative apparent diffusion coefficient (rADC) changes for three regions of interest (ROIs). Both ISH models induced consistent behavior deficits. The larger dosage produced severe brain injuries as well as neurological deficits, more closely mimicking severe human ISH. Hematoma volume and MLS can be the most useful parameters for evaluating the ISH severity in the present experimental model. The larger dosage, therefore, would be useful for investigating the pathophysiology of the severer ISH in the striatum. This may be applied for evaluating potential therapeutic strategies and outcomes in the future.

Acute Alcohol Intoxication Aggravates Brain Injury Caused by Intracerebral Hemorrhage in Rats.

OBJECTIVE: Alcohol intoxication is associated with worse intracerebral hemorrhage (ICH) outcome, indicating the important role of alcohol in ICH pathogenesis. We intended to investigate the effects of ethanol pretreatment on the severity of ICH-induced brain injury in rats. METHODS: At 1 hour after intraperitoneal injection of ethanol (3 g/kg), 0.2 U bacterial collagenase was infused into the striatum of male Sprague-Dawley rats to induce ICH. Accumulative mortality rate, body weight changes, and motorsensory and neurological abnormalities were evaluated. The hemorrhagic volume, hematoma expansion, and water content were measured by Drabkin's method, morphometric assay, and dry/wet method, respectively. Blood-brain barrier disruption was assessed using Evans blue assay. Oxidative stress was evaluated by the enzymatic activity of glutathione peroxidase, oxidation of hydroethidine, and the production of malondialdehyde. Cerebral blood flow perfusion volume and hypo-/hyperperfusion neuroimaging were examined by magnetic resonance imaging. RESULTS: Ethanol pretreatment aggravates the hematoma hemolysis, hemorrhagic volume, hematoma expansion, brain edema, blood-brain barrier disruption, microglial activation, elevated oxidative stress, and neuroinflammation in the hemorrhagic striatum. The summation effect of these consequences is the major cause of marked neurological impairment and higher mortality rate (64%) in ethanol-pretreated rats with ICH. CONCLUSION: This is a novel model to evaluate the effects of high-dose alcohol administration on experimental ICH rats. IMPLICATIONS: The present study may provide clues for making novel strategies in the management of patients with ICH who overconsume alcoholic drinks before the attack.

Granulocyte-Colony Stimulating Factor Increases Cerebral Blood Flow via a NO Surge Mediated by Akt/eNOS Pathway to Reduce Ischemic Injury.

Granulocyte-colony stimulating factor (G-CSF) protects brain from ischemic/reperfusion (I/R) injury, and inhibition of nitric oxide (NO) synthases partially reduces G-CSF protection. We thus further investigated the effects of G-CSF on ischemia-induced NO production and its consequence on regional cerebral blood flow (rCBF) and neurological deficit. Endothelin-1 (ET-1) microinfused above middle cerebral artery caused a rapid reduction of rCBF (ischemia) which lasted for 30 minutes and was followed by a gradual recovery of blood flow (reperfusion) within the striatal region. Regional NO concentration increased rapidly (NO surge) during ischemia and recovered soon to the baseline. G-CSF increased rCBF resulting in shorter ischemic duration and an earlier onset of reperfusion. The enhancement of the ischemia-induced NO by G-CSF accompanied by elevation of phospho-Akt and phospho-eNOS was noted, suggesting an activation of Akt/eNOS. I/R-induced infarct volume and neurological deficits were also reduced by G-CSF treatment. Inhibition of NO synthesis by L-N(G)-Nitroarginine Methyl Ester (L-NAME) significantly reduced the effects of G-CSF on rCBF, NO surge, infarct volume, and neurological deficits. We conclude that G-CSF increases rCBF through a NO surge mediated by Akt/eNOS, which partially contributes to the beneficial effect of G-CSF on brain I/R injury.

Histone deacetylase inhibition mediates urocortin-induced antiproliferation and neuronal differentiation in neural stem cells.

During cortical development, cell proliferation and cell cycle exit are carefully regulated to ensure that the appropriate numbers of cells are produced. Urocortin (UCN) is a member of the corticotrophin releasing hormone (CRH) family of neuropeptides that regulates stress responses. UCN is widely distributed in adult rat brain. However, the expression and function of UCN in embryonic brain is, as yet, unclear. Here, we show that UCN is endogenously expressed in proliferative zones of the developing cerebral cortex and its receptors are exhibited in neural stem cells (NSCs), thus implicating the neuropeptide in cell cycle regulation. Treatment of cultured NSCs or organotypic slice cultures with UCN markedly reduced cell proliferation. Furthermore, blocking of endogenous UCN/CRHRs system either by treatment with CRHRs antagonists or by neutralization of secreted UCN with anti-UCN antibody increased NSCs proliferation. Cell cycle kinetics analysis demonstrated that UCN lengthened the total cell cycle duration via increasing the G1 phase and accelerated cell cycle exit. UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological and RNA interference approaches, we further demonstrated that antiproliferative action of UCN appeared to be mediated through a HDAC inhibition-induced p21 upregulation. Moreover, UCN treatment in vitro and in vivo led to an increase in neuronal differentiation of NSCs. These findings suggest that UCN might contribute to regulate NSCs proliferation and differentiation during cortical neurogenesis.

Neurochemicals involved in medullary control of common carotid blood flow.

The common carotid artery (CCA) supplies intra- and extra-cranial vascular beds. An area in the medulla controlling CCA blood flow is defined as the dorsal facial area (DFA) by Kuo et al. in 1987. In the DFA, presynaptic nitrergic and/or glutamatergic fibers innervate preganglionic nitrergic and/or cholinergic neurons which give rise to the preganglionic fibers of the parasympathetic 7th and 9th cranial nerves. Released glutamate from presynaptic nitrergic and/or glutamatergic fibers can activate N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors on preganglionic nitrergic and/or cholinergic neurons. By modulating this glutamate release, several neurochemicals including serotonin, arginine, nitric oxide, nicotine, choline and ATP in the DFA regulate CCA blood flow. Understanding the neurochemical regulatory mechanisms can provide important insights of the physiological roles of the DFA, and may help develop therapeutic strategies for diseases involving CCA blood flow, such as migraine, hypertensive disease, Alzheimer's disease and cerebral ischemic stroke.

Reduction of motor disorder in 6-OHDA-induced severe parkinsonism rats by post treatment with granulocyte-colony stimulating factor.

Granulocyte-colony stimulating factor (G-CSF) induced regeneration of dopaminergic neurons and improved behavior deficit in moderate Parkinson's disease (PD) model mice. Post treatment of G-CSF in severe PD model has not been addressed. A very severe PD model in rats was induced by a high dose 6-hydroxydopamine (6-OHDA) injected into the right medial forebrain bundle to evaluate therapeutic effects of G-CSF. G-CSF (50 microg/kg/day for five days) was given on the 9th day after the 6-OHDA injection. Rotational behavior was examined on the 9th and 28th days. Rats were killed on the 28th day and survival dopaminergic neurons in the substantia nigra, dopaminergic axons and dopaminergic receptor 2 in the striatum were examined. We, for the first time, demonstrated that post treatment with G-CSF reduced abnormal rotational behavior and increased the lesion to non-lesion ratio of dopaminergic fibers in the striatum, but the treatment promoted neither the increase in survival dopaminergic neurons nor the increase in dopaminergic receptor 2 expression. We conclude that post treatment with G-CSF can reduce the abnormal rotational behavior of severe PD rats primarily through relative increases in dopaminergic fibers of the lesion side in the striatum. Results of our study suggest therapeutic potentials of G-CSF for treating severe PD patients.

Role of perivascular adipose tissue-derived methyl palmitate in vascular tone regulation and pathogenesis of hypertension.

BACKGROUND: Perivascular adipose tissue (PVAT)-derived relaxing factor (PVATRF) significantly regulates vascular tone. Its chemical nature remains unknown. We determined whether palmitic acid methyl ester (PAME) was the PVATRF and whether its release and/or vasorelaxing activity decreased in hypertension. METHODS AND RESULTS: Using superfusion bioassay cascade technique, tissue bath myography, and gas chromatography/mass spectrometry, we determined PVATRF and PAME release from aortic PVAT preparations of Wistar Kyoto rats and spontaneously hypertensive rats. The PVAT of Wistar Kyoto rats spontaneously and calcium dependently released PVATRF and PAME. Both induced aortic vasorelaxations, which were inhibited by 4-aminopyridine (2 mmol/L) and tetraethylammonium 5 and 10 mmol/L but were not affected by tetraethylammonium 1 or 3 mmol/L, glibenclamide (3 µmol/L), or iberiotoxin (100 nmol/L). Aortic vasorelaxations induced by PVATRF- and PAME-containing Krebs solutions were not affected after heating at 70°C but were equally attenuated after hexane extractions. Culture mediums of differentiated adipocytes, but not those of fibroblasts, contained significant PAME and caused aortic vasorelaxation. The PVAT of spontaneously hypertensive rats released significantly less PVATRF and PAME with an increased release of angiotensin II. In addition, PAME-induced relaxation of spontaneously hypertensive rats aortic smooth muscle diminished drastically, which was ameliorated significantly by losartan. CONCLUSIONS: We found that PAME is the PVATRF, causing vasorelaxation by opening voltage-dependent K+ channels on smooth muscle cells. Diminished PAME release and its vasorelaxing activity and increased release of angiotensin II in the PVAT suggest a noble role of PVAT in pathogenesis of hypertension. The antihypertensive effect of losartan is attributed partly to its reversing diminished PAME-induced vasorelaxation.

Sympathetic activation increases basilar arterial blood flow in normotensive but not hypertensive rats.

The close apposition between sympathetic and parasympathetic nerve terminals in the adventitia of cerebral arteries provides morphological evidence that sympathetic nerve activation causes parasympathetic nitrergic vasodilation via a sympathetic-parasympathetic interaction mechanism. The decreased parasympathetic nerve terminals in basilar arteries (BA) of spontaneously hypertensive rat (SHR) and renovascular hypertensive rats (RHR) compared with Wistar-Kyoto rats (WKY), therefore, would diminish this axo-axonal interaction-mediated neurogenic vasodilation in hypertension. Increased basilar arterial blood flow (BABF) via axo-axonal interaction during sympathetic activation was, therefore, examined in anesthetized rats by laser-Doppler flowmetry. Electrical stimulation (ES) of sympathetic nerves originating in superior cervical ganglion (SCG) and topical nicotine (10-30 µM) onto BA of WKY significantly increased BABF. Both increases were inhibited by tetrodotoxin, 7-nitroindazole (neuronal nitric oxide synthase inhibitor), and ICI-118,551 (ß(2)-adrenoceptor antagonist), but not by atenolol (ß(1)-adrenoceptor antagonist). Topical norepinephrine onto BA also increased BABF, which was abolished by atenolol combined with 7-nitroindazole or ICI-118,551. Similar results were found in prehypertensive SHR. However, in adult SHR and RHR, ES of sympathetic nerves or topical nicotine caused minimum or no increase of BABF. It is concluded that excitation of sympathetic nerves to BA in WKY causes parasympathetic nitrergic vasodilation with increased BABF. This finding indicates an endowed functional neurogenic mechanism for increasing the BABF or brain stem blood flow in coping with increased local sympathetic activities in acutely stressful situations such as the "fight-or-flight response." This increased blood flow in defensive mechanism diminishes in genetic and nongenetic hypertensive rats due most likely to decreased parasympathetic nitrergic nerve terminals.

Systemic administration of urocortin after intracerebral hemorrhage reduces neurological deficits and neuroinflammation in rats.

BACKGROUND: Intracerebral hemorrhage (ICH) remains a serious clinical problem lacking effective treatment. Urocortin (UCN), a novel anti-inflammatory neuropeptide, protects injured cardiomyocytes and dopaminergic neurons. Our preliminary studies indicate UCN alleviates ICH-induced brain injury when administered intracerebroventricularly (ICV). The present study examines the therapeutic effect of UCN on ICH-induced neurological deficits and neuroinflammation when administered by the more convenient intraperitoneal (i.p.) route. METHODS: ICH was induced in male Sprague-Dawley rats by intrastriatal infusion of bacterial collagenase VII-S or autologous blood. UCN (2.5 or 25 µg/kg) was administered i.p. at 60 minutes post-ICH. Penetration of i.p. administered fluorescently labeled UCN into the striatum was examined by fluorescence microscopy. Neurological deficits were evaluated by modified neurological severity score (mNSS). Brain edema was assessed using the dry/wet method. Blood-brain barrier (BBB) disruption was assessed using the Evans blue assay. Hemorrhagic volume and lesion volume were assessed by Drabkin's method and morphometric assay, respectively. Pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) expression was evaluated by enzyme-linked immunosorbent assay (ELISA). Microglial activation and neuronal loss were evaluated by immunohistochemistry. RESULTS: Administration of UCN reduced neurological deficits from 1 to 7 days post-ICH. Surprisingly, although a higher dose (25 µg/kg, i.p.) also reduced the functional deficits associated with ICH, it is significantly less effective than the lower dose (2.5 µg/kg, i.p.). Beneficial results with the low dose of UCN included a reduction in neurological deficits from 1 to 7 days post-ICH, as well as a reduction in brain edema, BBB disruption, lesion volume, microglial activation and neuronal loss 3 days post-ICH, and suppression of TNF-α, IL-1ß, and IL-6 production 1, 3 and 7 days post-ICH. CONCLUSION: Systemic post-ICH treatment with UCN reduces striatal injury and neurological deficits, likely via suppression of microglial activation and inflammatory cytokine production. The low dose of UCN necessary and the clinically amenable peripheral route make UCN a potential candidate for development into a clinical treatment regimen.

Oroxylin a, but not vasopressin, ameliorates cardiac dysfunction of endotoxemic rats.

The mortality in septic patients with myocardial dysfunction is higher than those without it. Beneficial effects of flavonoid oroxylin A (Oro-A) on endotoxemic hearts were evaluated and compared with that of arginine vasopressin (AVP) which is used to reverse hypotension in septic patients. Endotoxemia in rats was induced by one-injection of lipopolysaccharides (LPS, 10 mg/kg, i.p.), and hearts were isolated 5-hrs or 16-hrs later. Isolated hearts with constant-pressure or constant-flow mode were examined by Langendorff technique. Rate and force of contractions of isolated atrial and ventricular strips were examined by tissue myography. Isolated endotoxemic hearts were characterized by decreased or increased coronary flow (CF) in LPS-treated-for-5hr and LPS-treated-for-16-hr groups, respectively, with decreased inotropy in both groups. Oro-A-perfusion ameliorated while AVP-perfusion worsened the decreased CF and inotropy in both preparations. Oro-A and AVP, however, did not affect diminished force or rate of contraction of atrial and ventricular strips of endotoxemic hearts. Oro-A-induced CF increase was not affected following coronary endothelium-denudation with saponin. These results suggest that Oro-A ameliorates LPS-depressed cardiac functions by increasing CF, leading to positive inotropy. In contrast, AVP aggravates cardiac dysfunction by decreasing CF. Oro-A is a potentially useful candidate for treating endotoxemia complicated with myocardial dysfunction.

Sympathetic α3ß2-nAChRs mediate cerebral neurogenic nitrergic vasodilation in the swine.

The α(7)-nicotinic ACh receptor (α(7)-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed bred between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, ß-amyloid (Aß)-sensitive nitrergic neurogenic vasodilation. Preliminary studies, however, demonstrated that nicotine-induced cerebral vasodilation in pigs crossbred among Landrace, Yorkshire, and Duroc (LYD) was insensitive to Aß and α-bungarotoxin (α-BGTX). We investigated nAChR subtype on sympathetic neurons innervating LYD basilar arteries. Nicotine-induced relaxation of porcine isolated basilar arteries was examined by tissue bath myography, inward currents on nAChR-expressing oocytes by two-electrode voltage recording, and mRNA and protein expression in the superior cervical ganglion (SCG) and middle cervical ganglion (MCG) by reverse transcription PCR and Western blotting. Nicotine-induced basilar arterial relaxation was not affected by Aß, α-BGTX, and α-conotoxin IMI (α(7)-nAChR antagonists), or α-conotoxin AuIB (α(3)ß(4)-nAChR antagonist) but was inhibited by tropinone and tropane (α(3)-containing nAChR antagonists) and α-conotoxin MII (selective α(3)ß(2)-nAChR antagonist). Nicotine-induced inward currents in α(3)ß(2)-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aß, or α-conotoxin AuIB. mRNAs of α(3)-, α(7)-, ß(2)-, and ß(4)-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α(3)-, ß(2)-, and ß(4)-subunits than that of α(7)-subunit. The Aß-insensitive sympathetic α(3)ß(2)-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aß-sensitive α(7)-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aß in causing diminished cerebral nitrergic vasodilation in diseases involving Aß.

Glycogen synthase kinase-3ß inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades.

BACKGROUND: Deciphering the mechanisms that modulate the inflammatory response induced by microglial activation not only improves our insight into neuroinflammation but also provides avenues for designing novel therapies that could halt inflammation-induced neuronal degeneration. Decreasing glycogen synthase kinase-3ß (GSK-3ß) activity has therapeutic benefits in inflammatory diseases. However, the exact molecular mechanisms underlying GSK-3ß inactivation-mediated suppression of the inflammatory response induced by microglial activation have not been completely clarified. Tumor necrosis factor-α (TNF-α) plays a central role in injury caused by neuroinflammation. We investigated the regulatory effect of GSK-3ß on TNF-α production by microglia to discern the molecular mechanisms of this modulation. METHODS: Lipopolysaccharide (LPS) was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Release of TNF-α was measured by ELISA. Signaling molecules were analyzed by western blotting, and activation of NF-κB and AP-1 was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Protein interaction was examined by coimmunoprecipitation. RESULTS: Inhibition of GSK-3ß by selective GSK-3ß inhibitors or by RNA interference attenuated LPS-induced TNF-α production in cultured microglia. Exploration of the mechanisms by which GSK-3ß positively regulates inflammatory response showed that LPS-induced IκB-α degradation, NF-κBp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3ß activity. However, GSK-3ß inactivation inhibited transactivation activity of p65 by deacetylating p65 at lysine 310. Furthermore, we also demonstrated a functional interaction between mixed lineage kinase 3 (MLK3) and GSK-3ß during LPS-induced TNF-α production in microglia. The phosphorylated levels of MLK3, MKK4, and JNK were increased upon LPS treatment. Decreasing GSK-3ß activity blocked MLK3 signaling cascades through disruption of MLK3 dimerization-induced autophosphorylation, ultimately leading to a decrease in TNF-α secretion. CONCLUSION: These results suggest that inactivation of GSK-3ß might represent a potential strategy to downregulate microglia-mediated inflammatory processes.

Nitric oxide and glutamate in the dorsal facial area regulate common carotid blood flow in the cat.

Nitric oxide (NO) or glutamate stimulation of dorsal facial area (DFA) increases blood flow in the common carotid artery (CCA), which supplies intra-and extra-cranial tissues. Nitrergic fibers and neurons as well as preganglionic cholinergic neurons are present in the DFA. We hypothesized the presence of nitrergic-glutamatergic fibers and preganglionic nitrergic-cholinergic neurons in the DFA that are involved in the regulation of CCA blood flow. In microdialysis studies, perfusion of the DFA with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) increased the glutamate concentration in the dialysate. This effect was abolished by co-perfusion of methylene blue (a guanylyl cyclase inhibitor). Intra-DFA injection of l-arginine (an NO precursor) or glutamate increased CCA blood flow. The l-arginine-induced flow increase was reduced by prior administration of NG-nitro-arginine methyl ester (l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor), d-2-amino-5-phosphonopentanoate (d-AP5, a competitive NMDA receptor antagonist), or glutamate diethylester (GDEE, a competitive AMPA receptor antagonist). The glutamate-induced blood flow increase was reduced by prior administration of l-NAME, 7-NI, or methylene blue. The induced increase in CCA blood flow, however, was not affected by endothelial NO synthase inhibitor. The findings indicate that NO-signal transduction within the DFA might cause glutamate release from presynaptic nitrergic-glutamatergic fibres and that the released glutamate activates NMDA/AMPA receptors on preganglionic nitrergic-cholinergic neurons in the nucleus to activate neuronal NO synthase and guanylyl cyclase in the neurons, leading to an increase in CCA blood flow. These findings may be important for developing therapeutic strategies for the diseases associated with CCA blood flow.

Delayed formation of hematomas with ethanol preconditioning in experimental intracerebral hemorrhage rats.

OBJECTIVE: Spontaneous intracerebral hemorrhage (ICH) accounts for 10%-15% of all strokes and causes high mortality and morbidity. In the previous study, we demonstrated that ethanol could aggravate the severity of brain injury after ICH by increasing neuroinflammation and oxidative stress. In this study, we further investigate the acute effects of ethanol on brain injury within 24 h after ICH. MATERIALS AND METHODS: Totally, 66 male Sprague-Dawley rats were assigned randomly into two groups: saline pretreatment before ICH (saline + ICH), and ethanol pretreatment before ICH (ethanol + ICH). Normal saline (10 mL/kg) or ethanol (3 g/kg, in 10 mL/kg normal saline) was administered intraperitoneally 1 h before induction of experimental ICH. Bacterial collagenase VII-S (0.23 U in 1.0 µL sterile saline) was injected into the right striatum to induce ICH in the rats. We evaluated the hematoma expansion, hemodynamic parameters (heart rate and blood pressure), activated partial thromboplastin time (aPTT), prothrombin time (PT), and striatal matrix metallopeptidase 9 (MMP-9) expressions at 3, 6, 9, and 24 h after ICH. RESULTS: The ethanol + ICH group exhibited decreased hematoma at 3 h after ICH; nevertheless, there was a larger hematoma compared with the saline + ICH group at 9 and 24 h after ICH. The ethanol + ICH group had lower blood pressure at 3, 6, and 9 h post-ICH, but both groups maintained similar heart rates after ICH. There was no significant difference in the aPTT and PT between the two groups. Incremental ethanol concentrations had no influence on collagenase VII-S activity at 120 min in vitro. MMP-9 expression was upregulated in the right striata of the ethanol + ICH group, especially at 3 and 9 h after ICH. CONCLUSION: Ethanol delayed hematoma formation in the first 3 h due to a hypotensive effect; however, the accelerated growth of hematomas after 9 h may be a sequela of ethanol-induced MMP-9 activation.

Regulation of common carotid arterial blood flow by nitrergic neurons in the medulla of cats.

Glutamate stimulation of the dorsal facial area, an area located dorsal to the facial nucleus, increases common carotid arterial blood flow. Nitrergic neurons are important in cardiovascular regulatory areas. We investigated whether the nitrergic neurons might be present and play a role in the dorsal facial area to regulate the arterial blood flow. Injections of L-arginine (an NO precursor) and sodium nitroprusside (an NO donor) into the area caused dose-dependent increases in the arterial blood flow. Injection of N(G)-nitro-arginine methyl ester (L-NAME, an NO synthase inhibitor) or methylene blue (a guanylate cyclase inhibitor) decreased the arterial blood flow. Nitrergic neurons and fibers were found in the dorsal facial area by histochemical staining of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase, a maker of NO synthase. In conclusion, nitrergic neurons are present in the dorsal facial area and appear to release NO tonically in stimulating the area to cause increase in common carotid arterial blood flow.

Neuroprotection with methylaminochroman and lazaroid of embryonic ventral mesencephalic tegmental dopaminergic neurons in cold storage.

Embryonic ventral mesencephalic tegmental (EVMT) neurons die off over time in cold storage at 4 degrees C in hibernation buffers (HB). Manipulation of HB conditions may improve the survival of neurons in cold storage. We examined the effect of lipid peroxidation inhibitors, a methylaminochroman (U83836E) and a lazaroid (U74389G) on the viability and survival of embryonic dopaminergic neurons in the co-culture system of embryonic striatal target (EST) cells and EVMT neurons that had been stored for 3 days at 4 degrees C in HB with or without U83836E or U74389G. One-way analysis of variance (ANOVA) was used for analysis of data. The density of tyrosine hydroxylase immunoreactive (THIR)-positive neurons was significantly higher in the groups stored in supplemented HB than in the control (HB alone; P < 0.001). The neuroprotective effect was concentration-dependent. We conclude that either U83836E or U74389G-conditioned HB exerted a concentration-dependent neuroprotective effect on embryonic dopaminergic neurons in cold storage for 3 days. Supplementation of U83836E and U74389G or other methylaminochromans and lazaroids in HB may be important for cold storage of donor neuronal cells.

Sympathetic and parasympathetic activities evaluated by heart-rate variability in head injury of various severities.

OBJECTIVE: To investigate the autonomic function in patients with brain damage of various extents. The purposes were to correlate the parameters derived from spectral analysis of the heart-rate variability (HRV) with the classic Glasgow coma scale (GCS), and to evaluate the possible clinical application of HRV in the autonomic functions in patients with various severities of brain-stem injury. METHODS: A total of 90 patients was divided into 5 groups based on the GCS: I: 15, II: 9-14, III: 4-8, no pupil dilatation, IV: 4-8, pupil dilatation, and V: 3, brain death. Electrocardiogram was recorded for frequency-domain analysis of RR intervals. HRV were categorized into the low-frequency (LF, 0.04-0.15Hz) and high-frequency power (HF, 0.15-0.40Hz), LF to HF power ratio (LF/HF), normalized powers (LF and HF%). These HRV parameters were correlated with the severity of brain damage. RESULTS: The LF, HF, LF%, and LF/HF in Group I were essentially similar to those in the normal subjects. LF and HF decreased from Group I to IV. All parameters were nearly absent in Group V. CONCLUSIONS: The increases in LF% and LF/HF with the decrease in HF indicate augmented sympathetic and attenuated parasympathetic drive. These changes were related to the severity of brain-stem damage. Both LF and HF were nearly abolished in brain death. SIGNIFICANCE: Our analysis indicates that HRV may be an useful tool for evaluating the autonomic functions in patients with brain damage of various degrees.

Preganglionic neurons of the sphenopalatine ganglia reside in the dorsal facial area of the medulla in cats.

Stimulation of the sphenopalatine ganglion (SPG), a parasympathetic ganglion of the facial nerve, or the dorsal facial area (DFA), an area in the lateral tegmental field just dorsal to the facial nucleus, induces an increase in blood flow of the common carotid artery (CCA). This study attempted to clarify the anatomical and functional relationships between the SPG and the DFA, and to demonstrate putative serotonergic (5-HT) and substance P (SP) innervations to the neurons of the DFA in regulation of the CCA blood flow in cats. Horseradish peroxidase (HRP), a retrograde tracer, was injected in the SPG. All HRP-labeled neurons were distributed in the reticular areas dorsal and lateral to the superior olivary nucleus and the facial nucleus, extending from the caudal half of the superior olivary nucleus to the rostral 3/4 of the facial nucleus on the HRP-injected side. They were grouped into five clusters, namely lateral circumference of the superior olivary nucleus, dorsal circumference of the superior olivary nucleus, lateral circumference of the facial nucleus, dorsal circumference of the facial nucleus, and the DFA. The percentage of HRP-neurons in each cluster was 0.5 +/- 0.1% (mean +/- S.E., n=6), 15.2 +/- 1.9%, 23.7 +/- 0.9%, 52.5 +/- 1.7%, and 8.3 +/- 0.7%, respectively. Glutamate stimulation of the DFA (at 5.0 to 7.0 mm rostral to the obex, 2.8 to 4.0 mm lateral to the midline, and 2.5 to 3.5 mm ventral to the dorsal surface of the medulla), but not other areas, resulted in the increased CCA blood flow. The 5HT- and SP-immunoreactive nerve terminals abutted on the ChAT-immunoreactive cell body (preganglionic neurons) in the DFA. In conclusion, parasympathetic preganglionic neurons in the DFA project fibers to the SPG, are innervated by 5HT- and SP-like nerve terminals, and are responsible for regulation of the CCA blood flow. They may be also important in regulation of the cerebral blood flow.

Microglia, apoptosis and interleukin-1beta expression in the effect of sophora japonica l. on cerebral infarct induced by ischemia-reperfusion in rats.

Sophora Japonica L. (SJ) is a traditional Chinese herb used to cool blood, stop bleeding and to treat hemorrhoids with bleeding. Although several recent studies found that both SJ and Ginkgo biloba have the same components of quercetin and rutin, only Ginkgo biloba has been widely used to treat cerebrovascular disorders and dementia in humans. This study investigated the effect of SJ on cerebral infarct in rats. A total of 66 Sprague-Dawley (SD) rats were studied. Focal cerebral infarct was established by occluding the bilateral common carotid arteries and the right middle cerebral artery for 90 minutes. After 24 hours of reperfusion, the neurological status was evaluated. The rats were then killed, and brain tissue was stained with 2,3,5-triphenyl-tetrazolium chloride. The grading scale of neurological deficit and the ratio of cerebral infarction area were used as an index to evaluate the effect of SJ on cerebral infarct. In addition, the number of ED1 and interleukin-1beta immunostaining positive cells, and apoptotic cells were measured in the cerebral infarction zone. The results indicated that pre-treatment with 100 or 200 mg/kg SJ and post-treatment with 200 mg/kg SJ significantly reduced the grade of neurological deficit and the ratio of cerebral infarction area. In addition, pre-treatment with 200 mg/kg SJ also significantly reduced ED1 and interleukin-1beta immunostaining positive cells, and apoptotic cells in ischemia-reperfusion cerebral infarct rats. This study demonstrated that SJ could reduce the cerebral infarction area and neurological deficit induced by ischemia-reperfusion in rats, suggesting its potential as a treatment for cerebral infarct in humans. This effect of SJ involves its suppressive action of microglia, interleukin-1beta and apoptosis.