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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).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.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.Although our findings indicate that CAC induces larger hematoma in rats with ICH, the underlying mechanism should be studied in the future.

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.

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.

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.

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.

Object Intracerebral hemorrhage (ICH) accounts for about 15% of all deaths due to stroke. It frequently causes brain edema, leading to an expansion of brain volume that exerts a negative impact on ICH outcomes. The ICH-induced brain edema involves inflammatory mechanisms. The authors' in vitro study shows that urocortin (UCN) exhibits antiinflammatory and neuroprotective effects. Therefore, the neuroprotective effect of UCN on ICH in rats was investigated. Methods Intracerebral hemorrhage was induced by an infusion of bacteria collagenase type VII-S or autologous blood into the unilateral striatum of anesthetized rats. At 1 hour after the induction of ICH, UCN (0.05, 0.5, and 5 μg) was infused into the lateral ventricle on the ipsilateral side. The authors examined the injury area, brain water content, blood-brain barrier permeability, and neurological function. Results The UCN, administered in the ipsilateral lateral ventricle, was able to penetrate into the injured striatum. Posttreatment with UCN reduced the injury area, brain edema, and blood-brain barrier permeability and improved neurological deficits of rats with ICH. Conclusions Posttreatment with UCN through improving neurological deficits of rats with ICH dose dependently provided a potential therapeutic agent for patients with ICH or other brain injuries.

Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that regulates stress responses. Although UCN is principally expressed in dopaminergic neurons in rat substantia nigra (SN), the function of UCN in modulating dopaminergic neuronal survival remains unclear. Using primary mesencephalic cultures, we demonstrated that dopaminergic neurons underwent spontaneous cell death when their age increased in culture. Treatment of mesencephalic cultures with UCN markedly prolonged the survival of dopaminergic neurons, whereas neutralization of UCN with anti-UCN antibody accelerated dopaminergic neurons degeneration. UCN increased intracellular cAMP levels followed by phosphorylating glycogen synthase kinase-3β (GSK-3β) on Ser9. Moreover, UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological approaches, we further demonstrated that inhibition of GSK-3β and HDAC contributes to UCN-mediated neuroprotection. These results suggest that dopaminergic neurons-derived UCN might be involved in an autocrine protective signaling mechanism.

Noradrenaline (NE) and acetylcholine (ACh) released from the sympathetic and parasympathetic neurones in cerebral blood vessels were suggested initially to be the respective vasoconstricting and dilating transmitters. Both substances, however, are extremely weak post-synaptic transmitters. Compelling evidence indicates that nitric oxide (NO) which is co-released with ACh from same parasympathetic nerves is the major transmitter for cerebral vasodilation, and its release is inhibited by ACh. NE released from the sympathetic nerve, acting on presynaptic β2-adrenoceptors located on the neighbouring parasympathetic nitrergic nerves, however, facilitates NO release with enhanced vasodilation. This axo-axonal interaction mediating NE transmission is supported by close apposition between sympathetic and parasympathetic nerve terminals, and has been shown in vivo at the base of the brain and the cortical cerebral circulation. This result reveals the physiological need for increased regional cerebral blood flow in ‘fight-or-flight response’ during acute stress. Furthermore, α7- and α3β2-nicotinic ACh receptors (nAChRs) on sympathetic nerve terminals mediate release of NE, leading to cerebral nitrergic vasodilation. α7-nAChR-mediated but not α3β2-nAChR-mediated cerebral nitrergic vasodilation is blocked by β-amyloid peptides (Aβs). This may provide an explanation for cerebral hypoperfusion seen in patients with Alzheimer’s disease. α7- and α3β2-nAChR-mediated nitrergic vasodilation is blocked by cholinesterase inhibitors (ChEIs) which are widely used for treating Alzheimer’s disease, leading to possible cerebral hypoperfusion. This may contribute to the limitation of clinical use of ChEIs. ChEI blockade of nAChR-mediated dilation like that by Aβs is prevented by statins pretreatment, suggesting that efficacy of ChEIs may be improved by concurrent use of statins.

The α7-nicotinic ACh receptor (α7-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed bred between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, β-amyloid (Aβ)-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 Aβ 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 Aβ, α-BGTX, and α-conotoxin IMI (α7-nAChR antagonists), or α-conotoxin AuIB (α3β4-nAChR antagonist) but was inhibited by tropinone and tropane (α3-containing nAChR antagonists) and α-conotoxin MII (selective α3β2-nAChR antagonist). Nicotine-induced inward currents in α3β2-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aβ, or α-conotoxin AuIB. mRNAs of α3-, α7-, β2-, and β4-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α3-, β2-, and β4-subunits than that of α7-subunit. The Aβ-insensitive sympathetic α3β2-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aβ-sensitive α7-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aβ in causing diminished cerebral nitrergic vasodilation in diseases involving Aβ.

Individual activation of nicotinic acetylcholine receptor (nAChR) or nitric oxide (NO) synthase in the dorsal facial area (DFA) increases blood flow of common carotid artery (CCA) supplying intra- and extra-cranial tissues. We investigated whether the activation of nAChR initiated the activation of NO synthase and guanylyl cyclase to increase CCA blood flow in anesthetized cats. Microinjections of nicotine (a non-selective nAChR agonist), or choline (a selective α7-nAChR agonist) in the DFA produced increases in CCA blood flow ipsilaterally. These increases were significantly reduced by pretreatment with NG-nitro-arginine methyl ester (l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with N5-(1-iminoethyl)-l-ornithine (l-NIO, a potent endothelial NO synthase inhibitor). Control microinjection with d-NAME (an isomer of l-NAME), artificial cerebrospinal fluid or DMSO (a solvent for 7-NI) did not affect resting CCA blood flow, nor did they affect nicotine- or choline-induced response. In conclusion, activation of nAChR, at least α7-nAChR, led to the activation of neuronal NO synthase and guanylyl cyclase in the DFA, which induced an increase in CCA blood flow.

Intraocular ciliary schwannoma is extremely rare and accurate diagnosis prior to surgery is difficult. In this article, we demonstrate a precise pre-operative diagnosis of ciliary nerve schwannoma by meticulous neurological evaluation, combined with high-resolution magnetic resonance imaging with three-dimensional reconstruction of the lesion and adjacent structures. The tumor was identified to be arising from the ciliary nerve during operation and was fully excised. After 9 months of observation, the function of the third nerve recovered except for the ciliary branch. In conclusion, through high-resolution imaging and scrupulous cranial nerve examination, an accurate preoperative diagnosis of intraocular ciliary nerve schwannoma was achieved. This enabled the design of an appropriate surgical procedure and prediction of the surgical outcome.

Detection of the myocardial damage in the early stage of subarachnoid hemorrhage (SAH) is impossible without a good animal model. We investigated the feasibility of using immunohistochemical cardiac troponin I (cTnI) staining to detect the early myocardial damage in rats subject to SAH by endovascular perforation. SAH induced increases in mean blood pressure, heart rate, intracranial pressure, plasma norepinephrine and cTnI accompanied with electrocardiogram abnormalities. The heart was dilated. The myocardium revealed coagulative myocytolysis and remarkable loss of myocardial tissue cTnI in myocytolytic areas within 3 h after SAH. Thus, we create a rat SAH model that can be applied in studying the mechanisms of the early myocardial damage following SAH and for evaluating therapeutic strategies in SAH patients.

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 NG-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.

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-NG-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.

Background and purpose: Actions of glutamate and serotonin on their respective receptors in the dorsal facial area (DFA) of the medulla are known to regulate common carotid arterial (CCA) blood flow in cats. Less is known about acetylcholine action on its nicotinic receptor (nAChR) subtypes in the DFA for regulation of CCA blood flow and this aspect was investigated. Experimental approach: Nicotinic and muscarinic agonists and antagonists were microinjected into the DFA through a three-barrel tubing in anesthetized cats. Results: CCA blood flow was dose-dependently increased by nicotine (a non-selective nAChR agonist) and choline (a selective α7-nAChR agonist). These effects of nicotine were attenuated by α-bungarotoxin (an α7-nAChR antagonist), methyllycaconitine (an α7-nAChR antagonist), mecamylamine (a relatively selective α3β4-nAChR antagonist) and dihydro-β-erythroidine (a relatively selective α4β2-nAChR antagonist). The choline-induced flow increase was attenuated by α-bungarotoxin and mecamylamine, but not by dihydro-β-erythroidine. Muscarinic agonists (muscarine and methacholine) and antagonist (atropine) affected neither the basal nor the nicotine-induced increase in the CCA blood flow. Conclusions and implications: Functional α7, α4β2, and α3β4 subunits of the nAChR appear to be present on the DFA neurons. Activations of these receptors increase the CCA blood flow. The present findings do not preclude the presence of other nAChRs subunits. Muscarinic receptors, if any, on the DFA are not involved in regulation of the CCA blood flow. Various subtypes of nAChRs in the DFA may mediate regulation of the CCA and cerebral blood flows. British Journal of Pharmacology (2006) 149, 206–214; doi:10.1038/sj.bjp.0706844

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; P0.001). 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.

Recent evidence has demonstrated that hyaluronan synthase 2 mRNA is up-regulated after brain ischemia. After a cerebral ischemic event, microglia and macrophages are the major inflammatory cells and are activated by hyaluronan (HA). However, it is unclear how these cells compare with regard to HA responsiveness. We show here that peritoneal macrophages and RAW 264.7 macrophages produced more than five- and 10-fold more tumor necrosis factor-alpha (TNF-alpha) than primary microglia and BV-2 microglia, respectively. Antibody blockade study showed that CD44, Toll-like receptor-4 receptor and the receptor for HA-mediated motility were responsible for HA-induced TNF-alpha release. Furthermore, HA induced higher levels of phosphorylated MAPK in RAW 264.7 cells when compared with BV-2 cells. HA-mediated TNF-alpha production required p38 MAPK, extracellular-regulated kinase and c-Jun N-terminal kinase phosphorylation in both cell types. The levels of HA-induced TNF-alpha mRNA expression in BV-2 cells were only twofold lower compared with RAW 264.7 cells, suggesting that a translational event is involved in the differential production of TNF-alpha. Western blot analysis revealed that HA treatment resulted in more rapid phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and more effective dissociation of 4E-BP1 from eukaryotic initiation factor 4E in RAW 264.7 cells than in BV-2 cells. Additionally, HA-induced phosphorylation of 4E-BP1 was dependent on MAPK signaling, indicating that RAW 264.7 cells exhibited higher levels of hyperphosphorylated 4E-BP1 possibly due to the overactivation of MAPK. The results suggest that resident microglia and blood-derived monocytes/macrophages exhibit differential sensitivities in response to extracellular mediators after brain ischemia.

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.

Background: The reliability of critical closing pressure (CrCP) estimates derived from peripheral blood pressure (BP) measurements is unclear. We attempted to evaluate the influences of peripheral circulation on determining CrCP. Methods: Twenty-five young healthy volunteers were studied. BP waves were obtained with plethysmography (Portapres) and carotid applanatory tonometry, respectively, for analysis. Transcranial Doppler was used to monitor cerebral flow velocity. Using linear regression analysis, beat-to-beat CrCP was calculated at rest, during voluntary hyperventilation and during 5% CO2 inhalation. Results: Twenty of 25 participants demonstrating satisfactory tonometric tracings for both tests were included in the analysis. The systolic BP measured using plethysmography was higher than that derived from tonometry (139.4 ± 24.7 vs. 105.5 ± 29.6, p < 0.001). CrCP values derived from tonometry were all positive and higher than CrCP values derived from plethysmography (62.9 ± 19.9 vs. 11.1 ± 17.8, p < 0.001). The changes in CrCP induced by 5% CO2 inhalation and hyperventilation had a correlation between two BP monitoring methods (r = 0.52, p = 0.001). Conclusions: Pressure waveform is an important determinant in calculating CrCP by linear regression analysis. The relative changes in CrCP induced by hemodynamic challenges remained a relevant indicator of cerebrovascular regulation regardless of the methods used for non-invasive BP recording.

The role of serotonin and glutamate release in dorsal medulla (DM) for regulation of systemic arterial pressure (SAP) was examined with microdialysis and high performance liquid chromatograph in anesthetized cats. KCl-perfusion in DM increased serotonin and glutamate concentrations in DM. Perfusion of serotonin resulted in decreases in glutamate concentration and SAP. Perfusion of alaproclate, a serotonin reuptake inhibitor that produced an increase in serotonin concentration in DM, had the same results as perfusion of serotonin. In conclusion, serotonin and glutamate appeared to be tonically and endogenously released from nerve terminals in DM, and the decrease in SAP could be attributed to the decreased glutamate release resulting from inhibitory action of serotonin in DM. The putative roles of serotonin and glutamate in DM may be important in SAP regulation.

The interrelationship between the hypothalamic-pituitary-adrenal (HPA) activity and expression of central opioids is determined in ischemic Sprague-Dawley rats to support the therapeutic role of naloxone against cerebral ischemia. Two-month old rats received bilateral common carotid artery occlusion plus unilateral (right side) middle cerebral artery occlusion for 90 min under the anesthesia, and followed by reperfusion for various times. The plasma contents of adrenocorticotropin (ACTH) and RNA expression levels of proopiomelanocortin (POMC) were then determined in ischemic rats with or without naloxone treatment. Results showed that ischemia stimulates but reperfusion suppresses the activity of HPA axis. The induced expression of POMC at striatum and cortex areas appears to suppress the release of ACTH from the HPA axis. The suppression on the other hand is prevented by naloxone.

Elevated extracellular glutamate levels can increase malondialdehyde production in the brains of anesthetized rats. Thus, we investigated whether ionotropic glutamate receptors are involved in glutamate-induced malondialdehyde production. A microdialysis probe was implanted in the brain cortex of anesthetized rats. The malondialdehyde level in microdialysates was analyzed using an HPLC system. Three different ionotropic glutamate receptor agonists were used. At a concentration of 1.5 mM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA, a selective AMPA receptor agonist) induced a dramatic increase in extracellular malondialdehyde production (as much as 14-fold relative to the basal value). N-Methyl-D-aspartic acid (NMDA, a selective NMDA receptor agonist) also induced an increase in extracellular malondialdehyde production; however, the increase was not as much as that observed in the perfusion of AMPA receptor agonist. Kainic acid (a selective kainate receptor agonist) did not significantly increase malondialdehyde production. When co-perfused with L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 31.4 mM), a glutamate uptake transport inhibitor that can increase the extracellular glutamate levels, AMPA receptor antagonist [1-(4-aminophenyl)4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride, 1.0 mM] can significantly reduce PDC-induced malondialdehyde production. Although NMDA receptor antagonist [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate, MK801] also can decrease the PDC-induced malondialdehyde production, it was not as effective as the AMPA receptor antagonist. These results suggest that ionotropic receptors are involved in the glutamate-induced increase in malondialdehdye production. Specifically, AMPA receptor seems to be predominant in the glutamate-induced malondialdehdye production in anesthetized rat brain cortex.

An inflammatory response in the central nervous system mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Silymarin is a polyphenolic flavanoid derived from milk thistle that has anti-inflammatory, cytoprotective and anticarcinogenic effects. In this study, we first investigated the neuroprotective effect of silymarin against lipopolysaccharide (LPS)-induced neurotoxicity in mesencephalic mixed neuron-glia cultures. The results showed that silymarin significantly inhibited the LPS-induced activation of microglia and the production of inflammatory mediators, such as tumour necrosis factor-alpha and nitric oxide (NO), and reduced the damage to dopaminergic neurons. Therefore, the inhibitory mechanisms of silymarin on microglia activation were studied further. The production of inducible nitric oxide synthase (iNOS) was studied in LPS-stimulated BV-2 cells as a model of microglia activation. Silymarin significantly reduced the LPS-induced nitrite, iNOS mRNA and protein levels in a dose-dependent manner. Moreover, LPS could induce the activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase but not extracellular signal-regulated kinase. The LPS-induced production of NO was inhibited by the selective p38 MAPK inhibitor SB203580. These results indicated that the p38 MAPK signalling pathway was involved in the LPS-induced NO production. However, the activation of p38 MAPK was not inhibited by silymarin. Nevertheless, silymarin could effectively reduce LPS-induced superoxide generation and nuclear factor kappaB (NF-kappaB) activation. It suggests that the inhibitory effect of silymarin on microglia activation is mediated through the inhibition of NF-kappaB activation.

In this study, we investigated the dose-effect relationship and safety of tissue plasminogen activator (tPA) for the treatment of intraventricular hemorrhage/hematoma (IVH) in rats. Adult male Sprague-Dawley rats were injected with autologous blood into the left lateral ventricle to establish IVH. Two hours later, Ringer's saline or 0.25-2 microg of tPA were administered directly to the IVH over 3 h. The regional cerebral blood flow (rCBF) on the surface of the left parietal cortex was measured with laser Doppler flowmetry. Twenty-four hours after the build-up of IVH, the brains were removed for morphometrical and histological studies. A dose of 0.5-2 microg tPA significantly diminished the IVH in a dose-dependent manner (p0.001). However, only the dose of 0.5 microg tPA significantly ameliorated the reduction of rCBF 24 h after IVH (p0.01). TPA did not improve the ventricular dilatation on the side with IVH. Instead, 1-2 microg of tPA caused additional injuries, including intraventricular leukocytosis and edema of periventricular tissues and choroid plexus on both hemispheres. These results indicate that higher doses of tPA may have detrimental effects on the brain. The dosage rate of 0.5 microg seems beneficial to treat 5 microl of IVH (equals to a dose of 0.1 mg/ml blood) in our model in terms of the satisfactory fibrinolysis and less damage to the brain.

This study characterizes and examines the P2 receptor-mediated signal transduction pathway of a rat brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca2+ influx and activated phospholipase D (PLD). The ATP-stimulated Ca2+ influx was inhibited by pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), in a concentration-dependent manner. The agonist 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) stimulated the largest increases in intracellular Ca2+ concentrations ([Ca2+]i); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATPgammaS were much less effective, whereas UTP, ADP, alpha,beta-methylene-ATP, and beta,gamma-methylene-ATP were ineffective. Furthermore, removal of extracellular Mg2+ enhanced the ATP- and BzATP-stimulated increases in [Ca2+]i. BzATP stimulated PLD in a concentration- and time-dependent manner that could be abolished by removal of extracellular Ca2+ and was inhibited by suramin, PPADS, and oxidized ATP. In addition, PLD activities were activated by the Ca2+ mobilization agent, ionomycin, in an extracellular Ca2+ concentration-dependent manner. Both staurosporine and prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate that activation of the P2X7 receptors induces Ca2+ influx and stimulates a Ca2+-dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD.

Because of the well-documented importance of glutamate clearance by astrocytes in protecting neurons against excitotoxicity, it was interesting to examine whether L-glutamate exerts a toxic action on cultured astrocytes. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Exposure of astrocyte cultures of the neonatal rat cerebral cortex to L-glutamate resulted in a concentration- and time-dependent increase in the release of LDH. L-Glutamate-induced gliotoxicity appeared to be mediated predominantly by the increase of oxidative stress because the reduced glutathione content and its effects were almost completely blocked by vitamin E and pyrrolidinedithiocarbamate. To support this notion further, the supplementation or depletion of intracellular reduced glutathione content attenuated or worsened L-glutamate toxicity, respectively. Activation of the glutamate transporter mimicked the action of L-glutamate on astrocytes. In addition, degrees of cell damage were not directly correlated to the levels of glutamate uptake. Moreover, the mechanism of this toxicity required energy and macromolecular synthesis. Taken together, brief exposure to L-glutamate resulted in glutamate uptake and cell swelling, whereas sustained exposure injured astrocytes via oxidative stress instead of the excitatory mechanism.

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.

In the dorsal facial area (DFA) of the medulla, an activation of either P2 purinergic receptor or nitric oxide synthase (NOS) results in the release of glutamate, leading to an increase in blood flow of the common carotid artery (CCA). It is not known whether activation of the P2 receptor by ATP may mediate activation of NOS/guanylyl cyclase to cause glutamate release and/or whether L-Arg (nitric oxide (NO) precursor) may also cause ATP release from any other neuron, to cause an increase in CCA flow. We demonstrated that microinjections of P2 receptor agonists (ATP, α,β-methylene ATP) or NO precursor (L-arginine) into the DFA increased CCA blood flow. The P2-induced CCA blood flow increase was dose-dependently reduced by pretreatment with NG-nitro-arginine methyl ester (L-NAME, a non-specific NOS inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NOS inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with D-NAME (an isomer of L-NAME) or N5-(1-iminoethyl)-L-ornithine (L-NIO, a potent endothelial NOS inhibitor). Involvement of glutamate release in these responses were substantiated by microdialysis studies, in which perfusions of ATP into the DFA increased the glutamate concentration in dialysates, but co-perfusion of ATP with L-NAME or 7-NI did not. Nevertheless, the arginine-induced CCA blood flow increase was abolished by combined pretreatment of L-NAME and MB, but not affected by pretreatment with a selective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). In conclusion, ATP activation of the P2 receptor in the DFA induced activation of neuronal NOS/guanylyl cyclase, which causes glutamate release leading to an increase in CCA blood flow. However, arginine activation of neuronal NOS/guanylyl cyclase, which also caused glutamate release and CCA blood flow increase, did not induce activation of P2 receptors. These findings provide important information for drug design and/or developing therapeutic strategies for the diseases associated with CCA blood flow that supplies intra- and extra-cranial tissues.

Urocortin exerts neuroprotective effects in intracerebral hemorrhage (ICH) of rats. For pre-clinical trial, we intended to study the neuroprotective efficacy of human UCN (hUCN)-1, -2 and -3 in treating ICH rats. ICH was induced by infusing bacterial collagenase VII (0.23 U in sterile saline) to the striatum. The hUCN-1, -2, and -3 were administrated (2.5 μg/kg, i.p.) at 1 h after ICH insult, respectively. Neurological deficits were evaluated by modified Neurological Severity Scores. Brain edema and hematoma expansion was evaluated by coronal T2-WI and DWI magnetic resonance imaging on 1, 3, 6, 24, and 56 h after ICH insult. Blood–brain barrier permeability was evaluated by Evans blue assay on day 3 after ICH. Brain lesion volume was evaluated by morphormetric measurement on day 7 after ICH. Our results demonstrated that the hUCN-1 significantly reduced hematoma, blood–brain barrier disruption and neurological deficits on day 3, and brain lesion volume on day 7 after ICH insult. The prediction of secondary structure of the hUCNs clarifies that the percentage of alpha-helix, random coil and extended strand between rat-UCN (rUCN)-1 and hUCN-1 are the same. The structure similarity between human- and rat-UCN-1 may be one of the reasons that both can exert similar therapeutic potential in ICH rats.

Objective: Excised fat tissue (EFT) is an excellent source of adipose-derived stem cells (ADSCs) for autologous cell therapy. However, the current technique for excising fat tissues with conventional surgical scissors and using culture medium containing fetal bovine serum (FBS) may not comply with Good Tissue Practice (GTP) guidelines. We thus developed a semi-automated process complying with the abovementioned guideline to manufacture clinicalgrade ADSCs from small volumes of EFT. Methods: A semi-automated process was developed by using a disposable disperser tube operated on a Tube Disperser Workstation to obtain stromal vascular fraction (SVF) from EFT. A culture medium supplemented with human autologous serum (HAS) and mesenchymal stem cell culture adjuvant (MCA) consisted of basic fibroblast growth factor (bFGF/FGF-2) and antioxidants, was formulated to expand ADSCs from SVF in vitro. Results: The semi-automated process can markedly enhance cell yields and reduce the operation time for mincing tissues from an hour down to a few minutes. The growth of ADSCs was slower in the 10% HAS medium than that in medium supplemented with FBS, but their growth in the 10% HAS medium containing MCA (10% HAS+MCA) was superior to both media. The 10% HAS+MCA medium also promoted adipogenesis, osteogenesis, chondrogenesis and increased the expression of CD44, CD73 and the stemness genes in the ADSCs. Conclusion: Our novel semi-automated process provides an efficient isolation of SVF, and the 10% HAS+MCA medium accelerates the growth of ADSCs from small amounts of EFT without losing their differentiation ability.

Levels of dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) decline during aging and reach even lower levels in Alzheimer's disease (AD). DHEA is known to exhibit a variety of functional activities in the CNS, including an increase of memory and learning, neurotrophic and neuroprotective effects, and the reduction of risk of age-related neurodegenerative disorders. However, the influence of DHEA on the immune functions of glial cells is poorly understood. In this study, we investigated the effect of DHEA on activated glia. The production of inducible nitric oxide synthase (iNOS) was studied in lipopolysaccharide (LPS)-stimulated BV-2 microglia, as a model of glial activation. The results showed that DHEA but not DHEAS significantly inhibited the production of nitrite in the LPS-stimulated BV-2 cell cultures. Pretreatment of BV-2 cells with DHEA reduced the LPS-induced iNOS mRNA and protein levels in a dose-dependent manner. The LPS-induced iNOS activity in BV-2 cells was decreased by the exposure of 100 microM DHEA. Moreover, DHEA suppressed iNOS gene expression in LPS-stimulated BV-2 cells did not require de novo synthesis of new proteins or destabilize of iNOS mRNA. Since DHEA is biosynthesized by astrocytes and neurons, our findings suggest that it might have an important regulatory function on microglia.

The molecular mechanisms responsible for the conditioned enhancement of natural killer (NK) cell activity were investigated. The primary goal of the study was to examine the roles of glutamate and gamma-aminobutyric acid (GABA) in recall of the conditioned NK cell response. Both neurochemical blocking assay and high performance liquid chromatography (HPLC) technique were used in the study. Results from the neurochemical blocking assay demonstrated that glutamate but not GABA was required in recall of the conditioned NK cell response. NMDA but not the kainate/AMPA receptors, are believed to be involved. The levels of glutamate that were released and/or taken up also appeared to be critical in that interruption of glutamate release and/or uptake blocked the conditioned NK cell response. Results from the HPLC analysis, however, did not show any significant difference in the glutamate content between the conditioned and control brains.

In 20 urethane-anaesthetised cats, microinjection of N -methyl- d -aspartate (NMDA) into the intermediate-lateral (IL-) or the dorsolateral (DL-) periaqueductal grey areas (PAG) of the midbrain elicited similar patterns of cardiovascular responses: increases in mean systemic arterial pressure (MSAP), heart rate (HR) and mean blood flows (F) of the common carotid and femoral arteries, accompanied with a ‘hissing–howling’ response. Similar increases in MSAP and Fs were induced by microinjections of S-nitroso- N -acetylpenicillamine (SNAP), a potent nitric oxide (NO) donor, on the IL-PAG (A2.5–A0.5). In contrast, microinjections of N G -nitro- l -arginine methyl ester ( l -NAME), a NO synthase inhibitor, in the same area elicited a decrease in MSAP and Fs. On the other hand, microinjections of either SNAP or l -NAME reversed the original IL-PAG responses when injected into the DL-PAG. Pretreatments with SNAP significantly inhibited NMDA-induced responses in the DL-PAG but potentiated such responses in the IL-PAG. In contrast, pretreatments with l -NAME potentiated the NMDA-induced responses in the DL-PAG but inhibited such responses in the IL-PAG. These data suggest that NO may be a neurotransmitter or neuromodulator that exerts differential function in different defense areas, namely the IL- and the DL-PAG.

A dual-probe microdialysis technique was developed for the simultaneous monitoring of glucose and related metabolite levels during testicular ischemia in rats. The determinations of lactate and pyruvate were achieved by liquid chromatography within 5 min. Glucose was determined by a microdialysis analyzer. A unilateral ligation was produced by occlusion of the right artery for 2 or 4 h in anesthetized rats. Microdialysis probes were inserted in both sides of the testis to simultaneously monitor glucose, lactate and pyruvate during basal, ischemia and reperfusion periods. Dynamic and comparative changes in these analytes in ipsilateral and contralateral testes were demonstrated. The present technique can be used as a tool for exploring energy related metabolites and their relationships in testicular ischemia.

Inflammation plays an important role in the pathogenesis of neurodegenerative diseases including ischemia. Occlusion of common carotid artery and middle cerebral artery has been used to produce focal ischemic lesions in the rat. Here, we examined the associations between immune reactions and postischemic brain infarction. Ischemia/reperfusion time-dependently caused brain infarction. The kinetics of inflammatory reactions in rat brain including inflammatory cell infiltration, edema formation, cytokines/chemokines and adhesion molecules production and matrix metalloproteinase activation were relevant to the progression of ischemic infarction. Differential induction profile after ischemia suggests that this activation might contribute to secondary brain damage in ischemic tissues. On the other hand, another possibility of this response is to trigger processes that mediate the neural regeneration after ischemic injury.

An assay for in vivo, continuous and automatic monitoring of extracellular malondialdehyde concentrations in anesthetized rat brain cortex was developed. This method involved the use of microdialysis perfusion, on-line derivatization and on-line high-performance liquid chromatographic analysis. Microdialysate from an implanted microdialysis probe was on-line reacted with thiobarbituric acid at 80°C for 10 min prior to on-line collection and automatic injection into a HPLC system equipped with a fluorescence detector. This method gave a linear response between the concentrations of the malondialdehyde in the microdialysates and the TEP solution where the microdialysis probe was placed. This method was used to observe the increased extracellular malondialdehyde production following elevated extracellular glutamate levels, which were achieved by perfusion of l -trans-pyrrolidine-2,4-dicarboxylate, a competitive inhibitor of glutamate uptake transporter.

We investigated the responses of systemic arterial pressure and vertebral sympathetic nerve activity to glutamate microinjections (0.1 M, 70 nl) in the dorsomedial (DM) and the rostral ventrolateral medulla (RVLM) before hypoxia and after reoxygenation (posthypoxia) after various degrees of hypoxia in anesthetized cats. Hypoxia was produced by ventilating 5% O2 and 95% N2 for different durations (hypoxia I-III). In intact cats, the glutamate-induced systemic arterial pressure and vertebral nerve activity responses of the DM were depressed after all degrees of hypoxia. Posthypoxic depression in the RVLM, however, was not observed until hypoxia II and III. Precollicular decerebration prevented depression in the RVLM, but, for the DM, it was effective only for hypoxia I. Baro- and chemoreceptor denervation abolished all posthypoxic depression in both the DM and the RVLM. Pressor responses to tyramine (100–400 μg/kg iv) remained unchanged after all degrees of hypoxia. These results suggest that the DM is more susceptible to hypoxia than the RVLM. The peripheral baro- and chemoreceptors and the suprapontine structures apparently play an important role in posthypoxic depression. Moreover, the depression is not due to the postganglionic norepinephrine depletion.

Neural cell adhesion molecule (NCAM) regulates tissue organization during development and in the adult. NCAM upregulation occurs after an injury to brains and sciatic nerves. However, little is known about NCAM expression after spinal cord injury (SCI). By using a complete spinal cord transection with a 5 mm tissue removal, an increase in the NCAM level is detected in spinal cord stumps proximal and distal to the transection site at 1 d and 3 d post injury, while its expression at 8 d is declined to a lower level than that observed in sham-operated spinal cords. The strong NCAM expression is present in motor neurons at 3 d post transection whereas the intensive NCAM immunostaining is localized in dorsal sensory and corticospinal fiber tracts at 8 d following injury. Collectively, NCAM level is elevated and strongly expressed in dorsal fiber tracts after SCI, implying that the endogenous process for spinal cord regeneration may take place after SCI.

Aim Nicotine stimulation of α3β2-nicotinic acetylcholine receptors (α3β2-nAChRs) located on sympathetic nerves innervating basilar arteries causes calcium-dependent noradrenaline release, leading to activation of parasympathetic nitrergic nerves and dilation of basilar arteries. This study aimed to investigate the major subtype of calcium channels located on cerebral peri-vascular sympathetic nerves, which is involved in nicotine-induced α3β2-nAChR-mediated nitrergic vasodilation in basilar arteries. Methods Nicotine- and transmural nerve stimulation (TNS)-induced dilation of isolated porcine basilar arteries was examined using in vitro tissue bath. Nicotine-induced calcium influx, nicotine-induced noradrenaline release and nicotine-induced inward currents were evaluated in rat superior cervical ganglion (SCG) neurones, peri-vascular sympathetic nerves of porcine basilar arteries and α3β2-nAChRs-expressing oocytes respectively. mRNA and protein expression of Cav1.2 and Cav1.3 channels were detected by RT-PCR, Western blotting and immunohistochemistry. Results Nicotine-induced vasodilation was not affected by ω-agatoxin TK (selective P/Q-type calcium channel blocker) or ω-conotoxin GVIA (N-type calcium channel blocker). The vasodilation, however, was inhibited by nicardipine (L-type calcium channel blocker) in concentrations which did not affect TNS-induced vasodilation, suggesting the specific blockade. Nicardipine concentration-dependently inhibited nicotine-induced calcium influx in rat SCG neurones and reduced nicotine-induced noradrenaline release from peri-vascular sympathetic nerves of porcine basilar arteries. Nicardipine (10 μm), which significantly blocked nicotine-induced vasorelaxation by 70%, did not appreciably affect nicotine-induced inward currents in α3β2-nAChRs-expressing oocytes. Furthermore, the mRNAs and proteins of Cav1.2 and Cav1.3 channels were expressed in porcine SCG and peri-vascular nerve terminals. Conclusion The sympathetic neuronal calcium influx through L-type calcium channels is modulated by α3β2-nAChRs. This calcium influx causes noradrenaline release, initiating sympathetic–parasympathetic (axo-axonal) interaction-induced nitrergic dilation of porcine basilar arteries.

Little is known about the response of neural progenitors to inflammation following injuries of the central nervous system. In combination with bromodeoxyuridine (BrdU) intraperitoneally (i.p.) injected, tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine that increased ED1+ activated microglia/macrophage population at injured sites, was administrated into adult rat brains. No difference in the immunostaining for proliferating cell nuclear antigen (PCNA) was observed in the subventricular/ventricular zone (SVZ/VZ) between TNF-alpha injected sites and controls. However, BrdU+ cells were apparently observed in the SVZ/VZ proximal to TNF-alpha injected site, and the number of BrdU+ cells increased at 6 and 24 h post injection. Since cell apoptosis was rarely found in the SVZ/VZ after TNF-alpha injection, these observations suggest that the diffusible TNF-alpha may directly and/or indirectly modulate the proliferation of neural progenitors.

The stimulus-transcriptional coupling during ischemia/hypoxia was examined for ATP-stimulated expression of immediate early genes (IEGs; c-fos, zif268, c-myc and nur77) in a rat brain-derived type 2 astrocyte cell line, RBA-2. Incubation of cells with 1 mM of extracellular ATP stimulated time-dependent expression of c-fos and zif268. ATP induced the largest increases in zif268 mRNA and a lesser one in c-fos mRNA. ATP also induced a slight increase in nur77 mRNA but was ineffective in inducing c-myc expression in these cells. Brief exposure of cells to potassium cyanide to simulate chemical hypoxia induced 9-fold and 7-fold transient increases in c-fos and zif268 expression, respectively, but did not affect c-myc or nur77 expression. When cyanide and ATP were added together, the expression of c-fos and zif268 expression was inhibited, and the effect was mimicked by simulating chemical hypoxia with sodium azide. To elucidate the mechanism involved, the effect of cyanide on ATP-stimulated increases in intracellular Ca2+ concentrations, [Ca2+]i, and phospholipase D (PLD) activities were measured. Cyanide induced an increase in [Ca2p]i and further enhanced the ATP-stimulated increases in [Ca2+]i and PLD activities. Nevertheless, metabolic inhibitor, iodoacetate, blocked the ATP-induced c-fos and partially inhibited zif268 expression, and deprivation of cells with glucose also inhibited the ATP-induced c-fos expression. Taken together, these results demonstrate that both extracellular ATP and chemical hypoxia induce c-fos and zif268 expression in RBA-2 type 2 astrocytes. The chemical hypoxia inhibited ATP-stimulated c-fos and zif268 expression is not due to alterations in Ca2+ and PLD signaling, and is at least partially related to metabolic disturbance in these cells. J. Cell. Biochem. 77:323–332, 2000. © 2000 Wiley-Liss, Inc.

An in vitro microdialysis system was constructed for the measurement of catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2-0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 microl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.

Release of neurotransmitters, including dopamine and glutamate, has been implicated in hypoxia/ischemia-induced alterations in neuronal function and in subsequent tissue damage. Although extensive studies have been done on the mechanism underlying the changes in glutamate release, few have examined the mechanism that is responsible for the changes in catecholamines. Rat pheochromocytoma-12 (PC12) cells synthesize, store, and release catecholamines including DA and NE. Therefore, we used HPLC and ED to evaluate extracellular DA and NE concentrations in a medium during chemical hypoxia in PC12 cells. Chemical hypoxia produced by KCN induced differential release of DA and NE. Under normal glucose conditions, KCN induced release of NE, but not DA. Under glucose-free conditions, KCN-induced release of DA was elevated transiently, whereas the release of NE increased progressively. Under parallel conditions, KCN biphasically elevated the level of cytosolic free calcium ([CA2+]i) in glucose-free DMEM, peaking at 95 ± 18 nM at 1,107 ± 151 s, followed by a new plateau level at 249 ± 24 nM sustained from 4,243 ± 466 to 5,263 ± 440 s. Cell toxicity, as measured by LDH release, was increased significantly by KCN in glucose-free DMEM but was diminished in the presence of glucose, and was correlated with DA release by chemical hypoxia. The protein kinase C (PKC) inhibitor GO6976 or staurosporine inhibited KCN-induced LDH release as well as the release of NE and DA. Taken together, selective activation of DA but not NE was correlated with the LDH release by chemical hypoxia, and was diminished with GO6976 or staurosporine. These results suggest that selective activation of PKC isoforms is involved in the chemical hypoxia-induced DA release, which may lead to neuronal cell toxicity. J. Cell. Biochem. 79:191–201, 2000. © 2000 Wiley-Liss, Inc.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 5,7-dihydroxytryptamine (5,7-DHT) on striatal levels of dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites, as well as on locomotor activity were investigated in C57BL/6 mice. The results showed that MPTP significantly increased locomotor activity and decreased striatal DA levels. However, injection of the serotonergic neurotoxin 5,7-DHT in the striatum, either alone or following high doses of MPTP, significantly decreased locomotor activity, and concomitantly decreased striatal levels of 5-HT and 5-HIAA. This study suggests that the increased locomotor activity may be due to increased striatal serotonergic activity which overcompensates for the DA deficiency. The locomotor hypoactivity, induced by 5,7-DHT, might be due to the decreased striatal levels of 5-HT and 5-HIAA.

To mimic chronic exposure to neurotoxins in inducing dopaminergic cell damage, multiple doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were injected in C57BL/6 mice. Effects of pre- and post-treatment with the glial cell line-derived neurotrophic factor (GDNF) by injections into the striatum were investigated. GDNF exerts protective and reverse effects on the dopaminergic damage, supporting the potential application of GDNF in prevention and treatment of Parkinson's disease.

Ciliary neurotrophic factor (CNTF) is a member of cytokines, with trophic effects on ciliary, motor sympathetic, sensory, retinal and hippocampal neurons. In the present study, we examined the temporal and spatial expression profiles of CNTF and CNTF receptor alpha (CNTFR alpha) mRNAs in a focal cerebral ischemia model induced by transient occlusion of the right middle cerebral artery and both common carotid arteries. Northern blot analysis showed a slow and sustained increase in the 1.2 kb transcript of CNTF mRNA in the ischemic cortex of rats subjected to a transient 60 min ischemic insult. A delayed decrease in the 2.1 kb transcript of CNTFR alpha mRNA in the ischemic cortex was observed in rats subjected to 60 min ischemia followed by 72 h of reperfusion. In situ hybridization studies revealed constitutive expression of CNTFR alpha mRNA in the majority of neurons in the brain. Following 4 h of reperfusion, increased expression of CNTFR alpha mRNA was observed in the ipsilateral dentate gyrus, which is opposite to the down-regulation noted in the ischemic cortex. Within the infarct area CNTFR alpha mRNA had a marked increase in cortical layer II but a decrease in cortical layer V following 1 day of reperfusion. No signal of CNTFR alpha mRNA was detected within the infarct region following 3 days of reperfusion. Following 1 week of reperfusion, although no marked changes was observed in the level of CNTFR alpha mRNA in the area immediately surrounding the necrosis region where the reactive astrocytes were noted, a striking increase in the CNTF mRNA signal was noted. In summary, differential regulation of CNTF and CNTFR alpha mRNAs was noted in the ischemic cortex. Regional differences in CNTF receptor expression were noted between the ischemic cortex and ipsilateral dentate gyrus as well as between cortical layer II and V within the infarct region. CNTF mRNA, but not CNTFR alpha mRNA, had a marked increase in the area immediately adjacent to the necrosis. The mechanisms and patho-physiological significance for these differential regulation remain to be studied.