Attenuation of temporary focal cerebral ischemic injury in the mouse following transfection with interleukin-1 receptor antagonist.

The proinflammatory cytokine interleukin-1 beta (IL-1beta) is thought to play an important role in the stimulation of the inflammatory response following ischemia and reperfusion. This study investigated the inflammatory effect of IL-1beta during transient focal cerebral ischemia and reperfusion in the mouse transduced with the interleukin-1 receptor antagonist (IL-1ra) gene. An adenoviral vector encoding, either the human IL-1ra gene (AdRSVIL-1ra) or the LacZ gene (AdRSVlacZ) or normal saline, were injected into the right lateral ventricles of adult CD-1 mice (n=96). Five days later, the mice received 1 h temporary middle cerebral artery occlusion (tMACAO) followed by 23 h reperfusion. Cerebral blood flow (CBF), infarct volume, blood-brain barrier (BBB) permeability, and the number of intracellular adhesion molecule-1 positive vessels were measured to determine the effect of IL-1beta during postischemic reperfusion. Infarct volume in the AdRSVIL-1ra-transduced mice was markedly reduced compared to the AdRSVlacZ-transduced and saline-injected mice (36.0+/-5.3 mm(3) vs. 60.0+/-6.2 mm(3), 69. 5+/-6.3 mm(3), after 23 h of reperfusion, n=6-8 per group, p<0.05). BBB disruption and intracellular adhesion molecule-1 expression (135+/-23 vs. 311+/-40 and 357+/-51, n=6-8 per group, p<0.05) in the AdRSVIL-1ra-transduced mice were also less than that of the AdRSVlacZ-transduced and saline-injected mice. Our studies demonstrated that overexpression of IL-1ra in the mouse brain can downregulate intracellular adhesion molecule-1 expression both in the cortex and basal ganglia, which suggests that IL-1beta may play an important role in the activation of the inflammatory response during focal cerebral ischemia by promoting leukocyte adhesion to endothelial cells. The decrease of BBB disruption in AdRSVIL-1ra-transduced mice suggests that the endothelial cells may be a target for IL-1beta during postischemic reperfusion.     

Expression of tumor necrosis factor-alpha and intercellular adhesion molecule-1 after focal cerebral ischemia in interleukin-1beta converting enzyme deficient mice.

Interleukin-1beta (IL-1beta) is expressed after cerebral ischemia and blocking its action reduces subsequent ischemic brain injury. However, the mechanisms by which IL-1beta affects ischemic brain are not understood. To investigate the role of IL- 1beta in regulation of tumor necrosis factor-alpha (TNF-alpha) and intercellular adhesion molecule-1 (ICAM-1) during focal cerebral ischemia, the authors studied mutant mice deficient in the IL-1 converting enzyme (ICE) gene (ICE knockout [KO] mice). Ninety-four adult male ICE KO and wild-type mice underwent 3, 6, 12, and 24 hours of permanent middle cerebral artery occlusion using the suture method. Expression of TNF-alpha and ICAM-1 protein in ischemic brain was examined using immunohistochemistry and Western blot analysis. Neither ICE KO nor wild-type mice had significant differences in CBF and body temperature measurements during the ischemic procedure. TNF-alpha expression increased in the ipsilateral hemisphere after 3 hours of occlusion, peaked at 12 hours and decreased at 24 hours of ischemia in both ICE KO and wild-type mice. ICAM-1 immunohistochemistry showed that the number of ICAM-1-positive vessels in the ischemic hemisphere was reduced in ICE KO mice (P < .05). Western blot analysis showed that ICAM-1 protein expression was significantly attenuated in the ipsilateral hemisphere in the ICE KO mice, which paralleled the immunohistochemistry results. The authors' results indicate that TNF-alpha expression is increased in both ICE KO and wild-type mice suggesting that TNF-alpha expression is not related to or upregulated by IL-1beta . ICAM-1 expression is significantly reduced in the ICE KO mice suggesting that IL-1beta plays an important role in the upregulation of adhesion molecules during focal cerebral ischemia.     

T- and B-cell-deficient mice with experimental stroke have reduced lesion size and inflammation.

Stroke induction in immunologically competent mice not only produces local ischemia and brain damage, but also induces early inflammatory changes in brain and peripheral immune responses. Although immune elements clearly are activated after brain vascular occlusion, the relative contribution of T and B lymphocytes to the developing lesion has not been quantified. We evaluated effects 22 h after middle cerebral artery occlusion (90 mins) on histologic injury and peripheral immune activation in severe combined immunodeficient (SCID) mice lacking T and B cells. Cortical and total infarct volumes were strikingly reduced in male SCID mice (n=14, 33+/-4% of contralateral cortex, n=10, 52+/-3% of contralateral hemisphere) versus immunologically intact C57BL/6 mice (wild type, n=9, 57+/-5% of contralateral cortex, 57+/-4% of contralateral hemisphere) (P<0.01). Striatal infarction was not altered (77+/-7% of contralateral striatum in SCID, 84+/-7% in wild type), suggesting that the core of the evolving ischemic lesion was not impacted by lack of T and B cells. As expected, inflammatory factors from immune cells in ischemic SCID brains were essentially absent, with the exception of interleukin-1beta increase in both SCID and wild type tissue. Spleen cell numbers were low in SCID mice, but were further reduced 22 h after stroke, with substantial reduction in most inflammatory factors except for increased expression of interferon-gamma and macrophage inflammatory protein (MIP)-2. These data quantify the damaging effect of T and B lymphocytes on early, evolving ischemic brain injury, and further implicate interleukin-1beta in brain and interferon-gamma and MIP-2 in spleen as inflammatory factors produced by cells other than T and B cells.     

Early NFkappaB activation is inhibited during focal cerebral ischemia in interleukin-1beta-converting enzyme deficient mice

Our previous study demonstrated that the inhibition of interleukin-1beta (IL-1beta) reduces ischemic brain injury; however, the molecular mechanism of the action of IL-1 in cerebral ischemia is unclear. We are investigating currently the role of NFkappaB during focal cerebral ischemia, using mutant mice deficient in the interleukin-1 converting enzyme gene (ICE KO) in a middle cerebral artery occlusion (MCAO) model. Adult male ICE KO and wild-type mice (n = 120) underwent up to 24 hr of permanent MCAO. Cytoplasmic phospho-NFkappaB/p65 expression in ischemic brain was examined using Western blot analysis and immunohistochemistry. NFkappaB DNA-binding activity was detected using electrophoretic mobility shift assay (EMSA). Furthermore, ICAM-1 expression was examined in both the ICE KO and wild-type mice (WT). Western blot analysis and immunostaining showed that the level of cytosolic phosphorylated NFkappaB/p65 increased after 2 and 4 hr of MCAO in WT mice; however, NFkappaB/p65 was significantly reduced after MCAO in the ICE KO mice (P < 0.05). EMSA showed that NFkappaB DNA-binding activity increased after MCAO in WT mice; but this effect was reduced in the ICE KO mice. The number of ICAM-1-positive vessels in the ischemic hemisphere was greatly attenuated in the ICE KO mice (P < 0.05), which paralleled the results of immunohistochemistry. Our results demonstrate that NFkappaB phosphorylation is reduced in ICE KO mice, suggesting that ICE or IL-1 are involved in early NFkappaB phosphorylation. Because cerebral ischemia induced infarction is significantly reduced in ICE KO mice, we conclude that early NFkappaB phosphorylation plays a disruptive role in the ischemic process.     

A novel role of interleukin-1-converting enzyme in cytokine-mediated inducible nitric oxide synthase gene expression: Implications for neuroinflammatory diseases

Inducible nitric oxide synthase (iNOS)-derived NO plays an important role in several neurological disorders. Understanding of mechanisms involved in the regulation of iNOS induction is of particular interest. Here, we investigated mechanisms of iNOS induction in rat astrocytes (AC) and in brain endothelial cells (BEC). We find that activation of AC or BEC with pro-inflammatory cytokines reveals a different cell-specific activation pattern for iNOS expression. Despite these differences, in both cell types iNOS expression and activity exclusively depends on the endogenous availability of bioactive IL-1beta as inhibition of ICE activity significantly decreases iNOS promoter activity, iNOS expression and enzyme activity. In summary, we here provide evidence that ICE represents a target for modulating iNOS expression and high-output NO formation in AC and BEC, to our knowledge the first report of a role of ICE in iNOS expression and the advantage of ICE inhibition in attenuating NO mediated inflammation and pathology.     

Lack of interleukin-6 expression is not protective against focal central nervous system ischemia

BACKGROUND AND PURPOSE: Interleukin-6 (IL-6) appears to be involved in the inflammatory response associated with central nervous system (CNS) ischemia. Although IL-6 levels increase after stroke, it is not known whether IL-6 directly influences CNS ischemic injury. In this study, we used a focal reversible stroke model to investigate whether mice lacking IL-6 were protected against acute ischemic injury. METHODS: We bred IL-6-deficient C57 black mice (I-129 IL-6 KO back-crossed with C57), including homozygous knockouts (IL-6 -/-), heterozygous littermates (IL-6 +/-), and normal littermates (IL-6 +/+). The status of all animals was confirmed by DNA sampling and polymerase chain reaction analysis. Reversible middle cerebral artery occlusion was produced by advancing a silicone-coated 8-0 filament into the internal carotid artery for 2 hours (experiment 1) or 45 minutes (experiment 2). At 24 hours, animals were evaluated on a 28-point clinical scale, blood and cerebrospinal fluid were obtained, and the brains were evaluated for infarct volume and IL-6 mRNA levels. RESULTS: In experiment 1 (severe ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=15), 57+/-13 mm(3); IL-6 +/- (n=15), 58+/-23 mm(3); and IL-6 +/+ (n=15), 58+/-18 mm(3) (P=NS). ELISA testing confirmed very low to absent levels of IL-6 in the serum and cerebrospinal fluid of knockout animals. Brain mRNA levels of the other proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist, were 50% lower in IL-6-deficient ischemic animals than in normal animals. In experiment 2 (mild ischemia), no differences were seen in lesion size or neurological function between the groups: lesion volume was IL-6 -/- (n=10), 16+/-8 mm(3); IL-6 +/- (n=10), 14+/-4 mm(3); and IL-6 +/+ (n=10), 19+/-12 mm(3) (P=NS). CONCLUSIONS: In this study, infarct size and neurological function at 24 hours were not different in animals deficient in IL-6 after transient CNS ischemia. This suggests that IL-6 does not have a direct influence on acute ischemic injury. Further study investigating the role of IL-6 on long-term recovery after stroke is in progress.     

Inhibition of nNOS and iNOS following hypoxia-ischaemia improves long-term outcome but does not influence the inflammatory response in the neonatal rat brain

In this study, we tested the hypothesis that combined inhibition of nNOS and iNOS will reduce neuronal damage and the inflammatory response induced by perinatal hypoxia-ischaemia (HI). In 12-day-old rats, HI was induced by right carotid artery occlusion followed by 90 min of 8% O2. Immediately upon reoxygenation, the rats were treated with NOS inhibitors (n = 24) or placebo (n = 24). Neuropathology was scored at 6 weeks after HI on a 4-point scale (n = 12 per group). The expression of heat shock protein 70 (HSP70) and mRNA expression for cytokines were measured 12 h after HI (n = 12 per group). Histopathological analysis showed that the ipsilateral hemisphere in the NOS inhibition group was less damaged than in the placebo group (p < 0.05). HI induced a significant increase in HSP70 levels (p < 0.05) in the ipsilateral hemispheres, which tended to be lower in the NOS inhibition group (p = 0.07). HI induced an increase in mRNA expression for IL-1beta, TNF-alpha and TNF-beta, but there was no difference between the ipsi- and contralateral hemispheres. Combined inhibition of nNOS and iNOS did not induce any change in cytokine expression. We conclude that the long-term neuroprotective effects of combined nNOS and iNOS inhibition were not achieved by an altered cytokine response.     

脑缺血再灌注损伤后室旁区巢蛋白和干细胞因子表达的研究

目的 研究大鼠缺血性脑损伤后室旁区巢蛋白（nestin）和干细胞因子（SCF）基因表达的变化规律，探讨肌苷治疗中枢神经缺血性损伤的作用机制。方法 成年健康雌性SD大鼠68只，以线栓法建立大脑中动脉缺血再灌注模型，随机分为治疗组（注射肌苷注射液100mg／kg）和对照组（注射相同剂量的生理盐水），各32只，另外4只作假手术组（不插线）。应用原位杂交技术检测脑缺血再灌注后脑组织室旁区nestin和SCF的表达。结果 假手术组室旁区nestin和SCF表达很弱。对照组缺血侧nestin的表达除2h、6h、2d、14d以外各时间点均显著高于假手术组（P〈0．01）。治疗组nestin表达较对照组于各时间点均显著升高（P〈0．01）。对照组缺血侧SCF的表达除2h、2d、14d以外各时间点均显著高于假手术组（P〈0．01）。治疗组SCF表达较对照组于各时间点均显著升高（P〈0．01）。结论 肌苷可以促进大鼠脑缺血再灌注后nestin、SCF的表达，推测具有促进神经干细胞生成的作用。     

Inhibition of MEK/ERK 1/2 pathway reduces pro-inflammatory cytokine interleukin-1 expression in focal cerebral ischemia

It has been proposed that mitogen-activated protein kinase (MAPK) pathways may play a role in the regulation of pro-inflammatory cytokines, such as interlukine-1, during cerebral ischemia. Our previous study showed that extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) were activated during focal cerebral ischemia in mice [J. Cereb. Blood Flow Metab. 20 (2000) 1320]. However, the effect of ERK 1/2 activation in focal cerebral ischemia is still unclear. In this study we reported that in vivo phospho-ERK 1/2 expression increased following 30 min of middle cerebral artery occlusion (MCAO) in the mouse brain in both the ischemic core and perifocal regions. Western blot analysis and immunohistochemistry demonstrated that pro-treatment with 1,4-diamino-2,3-dicyano-1,4-bis butadiene (U0126) [J. Biol. Chem. 273 (1998) 18623] could significantly inhibit mouse brain phospho-MEK 1/2 and phospho-ERK 1/2 expression after 1-2 h of MCAO (p<0.05). Compared to the control group of mice, brain infarct volume was significantly decreased after 24 h of MCAO in the U0126-treated mice (27+/-6 vs. 46+/-9 mm(2), p<0.05). Inhibition of the MEK/ERK 1/2 pathway also prevented downstream kinase Elk-1 phosphorylation, and further reduced cytokine IL-1beta mRNA, but not TNFalpha, IL-1alpha, or chemokine MIP-1alpha mRNA expression. Our data demonstrates that in vivo the close linking of MEK 1/2, ERK 1/2, Elk-1, and IL-1 mRNA expression in the cerebral ischemia animals suggests that ERK 1/2 pathway activation is important in pro-inflammatory cytokine IL-1beta signaling, which induces an inflammatory response and exacerbates ischemic brain injury. Inhibiting the ERK 1/2 pathway may therefore provide a novel approach for the reduction of ischemia-induced IL-1beta overexpression.     

Cardiovascular effects of catecholamines injected into the DBB of rats, influence of urethane anaesthesia and local colchicine

In a previous publication it was shown that 1 microgram colchicine injected into the diagonal band of Broca (DBB) produced a significant decrease in femoral artery blood pressure (and/or volume) measured in urethane-anaesthetised rats. In order to test if the central catecholamines were involved in this effect, guide cannulae were implanted in the DBB and a catheter in the femoral artery. On-line pressure recordings were taken before during and after alpha1, alpha2 and beta adrenoreceptor agonists and antagonists were injected into the region of the DBB of non-anaesthetised and urethane anaesthetised male Wistar rats with and without injection of colchicine. Arterial pressure was significantly increased in the non-anaesthetised rats (114.6+/-2.6 n=11 vs. 149.3+/-3.3 mmHg n=12, p<0.01) yet significantly reduced (82.0+/-3.9 n=11 vs. 63.8+/-4.5 mmHg n=12, p<0.01) in the urethane treated rats by the alpha2 agonist clonidine. The alpha2 antagonist yohimbine blocked these effects in both preparations. In contrast, the beta adrenoreceptor agonist isoprenaline produced a significant decrease in arterial pressure in both preparations (107.7+/-3.9 n=11 vs. 85.9+/-4.0 mmHg n=12, p<0.01) (102.6+/-6.7 n=11 vs. 81.7+/-3.4 mmHg n=12, p<0.01) and this effect was blocked by the beta antagonist propranolol. Colchicine injected into the DBB abolished the effects of the alpha2 agonist and antagonist in the non-anaesthetised but not the anaesthetised rats. The responses to the beta agonist and antagonist were not greatly affected by the colchicine in the non-anaesthetised rats whereas in the anaesthetised rat beta agonist injection tended to totally depress arterial pressure. These results suggest that the sympathetic nervous system in the DBB plays a significant role in the central control of arterial pressure and that the alpha2 component is significantly affected by the state of anaesthesia.     

MRI of combination treatment of embolic stroke in rat with rtPA and atorvastatin

To test the hypothesis that combination treatment of embolic stroke with rtPA and statins improves the efficacy of thrombolytic therapy in rats. Rats subjected to embolic MCA occlusion (MCAo) were randomized into control (n = 10) and treatment (n = 9) groups. Four hours after MCAo, a combination of rtPA and atorvastatin (treatment) or saline (control) was administered. MRI measurements were performed on all animals at 2 h, 24 h and 48 h after MCAo. The patency of cerebral microvessels was examined using fluorescent microscopy. MRI images showed complete blockage of the right MCA and a reduction of CBF in the territory supplied by the MCA 2 h after MCAo for all animals. By 48 h after stroke, MRI showed that the decreased lesion size, elevated CBF and increased incidence of recanalization were found in treated rats compared with the control rats. The combination treatment significantly increased microvascular patency (16.3 +/- 5.5% vs. 12.4 +/- 3.5%, of field-of-view) and reduced the infarct volume (23.1 +/- 9.6% vs. 38.8 +/- 13.3%, of hemisphere). These data demonstrate that the co-administration of rtPA and atorvastatin 4 h after ischemia is efficacious and is reflected by the MRI indices of recanalization of the MCA, reduction of secondary microvascular perfusion deficits and reduction of the ischemic lesion.     

Excitotoxic brain damage in the rat induces interleukin-1beta protein in microglia and astrocytes: correlation with the progression of cell death

Interleukin-1 beta (IL-1beta) has been proposed as a mediator of several forms of brain damage, including that induced by excitotoxins. In vitro studies suggest that glial cells are the effector cells of IL-1beta-mediated neurodegeneration. We have investigated the expression of IL-1beta protein by glial cells in vivo in response to NMDA receptor-mediated excitotoxicity in the rat parietal cortex and striatum. Expression of IL-1beta by glial cells was investigated using immunocytochemistry 30 min to 7 days after infusion of the NMDA agonist cis-2,4-methanoglutamate (MGlu; 10 nmol) into the cortex. Early expression (1-4 h) of IL-1beta by microglia was directly related to lesion development. Later expression by microglia (up to 24 h), and by astrocytes (2-7 days), was widespread compared to the area involved in excitotoxic cell death and co-localised with areas of reactive gliosis. Infusion of MGlu into the striatum induced a similar temporal pattern of IL-1beta expression by microglia and astrocytes. However, IL-1beta-expressing glial cells were localised strictly to the area of striatal cell death. Infusion of PBS or a subtoxic dose of MGlu into the cortex or striatum induced only limited neuronal death and negligible glial IL-1beta expression. These studies reveal that IL-1beta is expressed specifically by microglia during the early response to excitotoxicity in the adult rat cortex and striatum. However, the widespread and delayed IL-1beta expression by astrocytes suggests diverse roles for IL-1beta in response to excitotoxicity.     

Increased expression of mRNA encoding interleukin-1beta and caspase-1, and the secreted isoform of interleukin-1 receptor antagonist in the rat brain following systemic kainic acid administration

Kainic acid, an analogue of glutamate, injected systemically to rats evokes seizures that are accompanied by nerve cell damage primarily in the limbic system. In the present study, we have analyzed the temporal profile of the expression of the cytokines interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra), and the related IL-1beta-converting enzyme (ICE/caspase-1), in different regions of the rat brain in response to peripheral kainic acid administration (10 mg/kg, i.p.). In situ hybridization histochemistry experiments revealed that IL-1beta mRNA-expressing cells, morphologically identified as microglial cells, were mainly localized to regions showing pronounced neuronal degeneration; hippocampus, thalamus, amygdala, and certain cortical regions. The strongest expression of IL-1beta mRNA was observed after 12 hr in these regions. A weak induction of the IL-1beta mRNA expression was observed already at 2 hr. Similar results were obtained by RT-PCR analysis, showing a significantly increased expression of IL-1beta mRNA in the hippocampus and amygdala after 12 hr. In addition, RT-PCR analysis revealed that IL-1ra mRNA, and specifically mRNA encoding the secreted isoform of IL-1ra (sIL-1ra), was strongly induced in the hippocampus and amygdala at 12 and 24 hr post-injection. RT-PCR analysis of mRNA encoding caspase-1 showed a significantly increased expression in the amygdala after 12 hr. In conclusion, in response to systemic kainic acid injection IL-1beta mRNA is rapidly induced and followed by induction of IL-1ra mRNA and caspase-1 mRNA, supporting a role of the IL-1 system in the inflammatory response during excitotoxic damage.     

Intrathecal interleukin-1beta administration induces thermal hyperalgesia by activating inducible nitric oxide synthase expression in the rat spinal cord

The effect of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) on the inducible nitric oxide synthase-nitric oxide (iNOS-NO) cascade in nociceptive signal transduction was examined in the intact rat spinal cord. All rats were implanted with an intrathecal (i.t.) catheter; some were also implanted with an i.t. microdialysis probe. The paw withdrawal latency to radiant heat was used to assess thermal hyperalgesia. The iNOS protein expression in the spinal cord dorsal horn was examined by western blot analysis and NOS activity assay. NO production in the CSF dialysate was also measured. IL-1beta i.t. (100 ng) produced thermal hyperalgesia from 4 to 24 h after i.t. injection. The iNOS protein expression was induced at 4 h after i.t. IL-1beta injection, peaked at the 6th hour, and disappeared at 24 h. The iNOS activity showed a similar time-dependent change as the iNOS protein expression. NO release increased by 1.1- to 1.9-fold between 4 and 12 h, also with a peak at the 6th hour, after i.t. IL-1beta administration. Pretreatment with the iNOS inhibitor 1400W (10 microg, i.t.) 1 h before i.t. IL-1beta injection prevented all the responses of IL-1beta. Neither 1400W nor artificial CSF (aCSF) affected the thermal nociceptive threshold and NO production. These results demonstrate that i.t. administration of IL-1beta induced thermal hyperalgesia by activating the iNOS-NO cascade in the rat spinal cord. On the basis of the present findings, we suggest that i.t. administration of iNOS inhibitors may have potential in the treatment of inflammatory and neuropathic pain syndromes.     

Endogenous alpha-MSH modulates the hypothalamic-pituitary-adrenal response to the cytokine interleukin-1beta

We and others have previously shown that exogenous alpha-MSH antagonizes the stimulatory effects of the cytokine interleukin (IL)-1 on the hypothalamic-pituitary-adrenal (HPA) axis. It is currently unknown, however, if endogenous alpha-MSH plays a physiological role in regulating the HPA response to IL-1. We have therefore examined the HPA response to IL-1beta in rats pretreated with an affinity purified alpha-MSH antiserum (AS) infused intracerebroventricularly to neutralize endogenous alpha-MSH within the brain. alpha-MSH AS or a similarly purified fraction of normal rabbit serum (NRS) was injected intracerebroventricularly at 16 h and at 1 h prior to the i.c.v. injection of IL-1beta (2 ng or 20 ng) and blood samples were collected through an indwelling atrial catheter. After 2 ng IL-1beta, the adrenocorticotropic hormone (ACTH) response was significantly greater in the alpha-MSH AS treated rats (n = 7) compared to the NRS treated rats (n = 7) (P <0.01); the mean ACTH level rose to a peak of 594+208 pg/ml in the alpha-MSH AS treated rats vs 274+/-122 pg/ml in the NRS treated rats. The area under the ACTH response curve in the alpha-MSH AS treated animals was 181% of that in the NRS treated animals (P<0.05). A significant effect of alpha-MSH AS on the corticosterone response to i.c.v. IL-1beta was also noted during the first 3 h of the study (P<0.05). The mean area under the corticosterone response curve for the first 3 h in the alpha-MSH AS treated animals was 144% of that in the NRS treated animals (P <0.05). After 20 ng IL-1beta, the ACTH response over time was again significantly greater in the alpha-MSH AS treated rats (n=8) compared to the NRS treated rats (n=9) (P<0.02); the mean ACTH level rose to a peak of 673+/-190 pg/ml after alpha-MSH AS vs 490+/-115 pg/ml after NRS. Corticosterone levels rose to a peak of 42+/-3.9 microg/dl in the alpha-MSH AS treated rats vs 37+/-4.6 microg/dl in the NRS treated rats; this difference was not significant. We conclude that the IL-1beta induced stimulation of ACTH is significantly enhanced by antagonizing the activity of alpha-MSH. These results support a physiological role for endogenous alpha-MSH in limiting the HPA response to this inflammatory cytokine.     

IL-1beta, an immediate early protein secreted by activated microglia, induces iNOS/NO in C6 astrocytoma cells through p38 MAPK and NF-kappaB pathways

In the present study we sought to examine cell-cell interactions by investigating the effects of factors released by stimulated microglia on inducible nitric oxide (NO) synthase (iNOS) induction in astrocytoma cells. After examining the temporal profiles of proinflammatory molecules induced by lipopolysaccharide (LPS) stimulation in BV2 microglial cells, iNOS and IL-1beta were observed to be the first immediate-response molecules. Removal of LPS after 3 hr stimulation abrogated NO release, whereas a full induction of IL-1beta was retained in BV2 cells. We observed consistently that conditioned medium (CM) from activated microglia resulted in the induction of iNOS in C6 cells, and IL-1beta was shown to be a key regulator of iNOS induction. An IL-1beta-neutralizing antibody diminished NO induction. Incubation with recombinant IL-1beta stimulated NO release to a lesser extent compared to microglial CM; co-treatment of LPS and IL-1beta had a potent, synergistic effect on NO release from C6 cells. Transient transfection with MEK kinase 1 (MEKK1) or nuclear factor-kappa B (NF-kappaB) expression plasmids induced iNOS, and IL-1beta further enhanced the MEKK1 response. Furthermore, IL-1beta-mediated NO release from C6 cells was significantly suppressed by inhibition of p38 mitogen activated protein kinase (MAPK) or NF-kappaB by specific chemical inhibitors. Both IL-1beta and MEKK1 stimulated p38 and JNK MAPKs, as well as the NF-kappaB pathway, to induce iNOS in C6 cells. Microglia may represent an anti-tumor response in the central nervous system, which is potentiated by the local secretion of immunomodulatory factors that in turn affects astrocytoma (glioma) cells. A better understanding of microglia-glioma or microglia-astrocyte interactions will help in the design of novel immune-based therapies for brain tumors or neuronal diseases.     

Hyperbaric oxygen induces rapid protection against focal cerebral ischemia

BACKGROUND AND PURPOSE: The timing and mechanisms of protection by hyperbaric oxygen (HBO) in cerebral ischemia have only been partially elucidated. We monitored the early in vivo effects of HBO after 2 h transient focal ischemia using repetitive MRI. METHODS: Wistar rats underwent filament occlusion of the middle cerebral artery (MCAO). 40 min after MCAO, rats were placed in a HBO chamber and breathed either 100% O(2) at 3.0 atmospheres absolute (ata; n = 24) or at 1.0 ata (control; n = 24) for 1 h. Diffusion, perfusion and T2-weighted MR-images were obtained after 15 min and 3, 6 and 24 h of reperfusion. In 6 axial MR slices, volume of abnormal diffusion and T2w signals were measured in the ischemic hemisphere. Furthermore, hemispheric mean apparent diffusion coefficient- (ADC) and T2 values were calculated for statistical analysis. RESULTS: HBO significantly reduced volume of abnormal DWI signal beginning immediately after reperfusion (control: 92 +/- 28 mm(3); HBO: 64 +/- 17) and lesion size on T2w (control: 375 +/- 91 mm(3); HBO: 225 +/- 39) after 24 h. Correspondingly, mean ADC levels were lower and T2 values higher in the ischemic hemisphere in the control group. HBO reduced histological infarct size at 24 h. CONCLUSION: High-dose intraischemic HBO therapy has an immediate protective on the brain which is superior to normobaric oxygen.