Koji Osuka

Interleukin-6 and Development of Vasospasm after Subarachnoid Haemorrhage

Summaryu2003The authors characterized the role of interleukins in the cerebrospinal fluid (CSF) in the development of vasospasm after subarachnoid haemorrhage (SAH), particularly interleukin-6 (IL-6).u2003Concentrations of interleukin-1β (IL-1 β), IL-6, and interleukin-8 (IL-8) were measured serially in CSF of 24 patients and in serum of 9 patients with SAH and correlated clinically. Additionally, the effects of the same cytokines on the cerebral arteries of dogs were analyzed on angiograms after intracisternal injection. Changes in levels of eicosanoids, angiogenic factors, and soluble cell adhesion molecules were investigated in the CSF of injected dogs.u2003CSF concentrations of IL-6 and IL-8 were elevated significantly above control levels from the acute stage of SAH until the chronic stage. Patients with symptomatic vasospasm had significantly higher levels of IL-6 as well as IL-8 in CSF on days 5 and 7. Intracisternal injection of IL-6 induced long-lasting vasoconstriction in five out of eight dogs, while IL-8 did not. The diameter of canine basilar artery after IL-6 was reduced 29±5% from pretreatment diameter at 8 hours. Prostaglandins E2 and I2 were elevated in CSF for the first 4.5 hour of this IL-6-induced vasospasm. Neither angioenic factors such as platelet-derived growth factor-AB and vascular endothelial growth factor nor soluble cell adhesion molecules were significantly elevated in CSF.u2003IL-6, which increases to very high concentrations in CSF after SAH, may be important in inducing vasospasm, as IL-6 produced long-lasting vasoconstriction in the canine cerebral artery, which may be partly related to activation of the prostaglandin cascade.

Intravenous infusion of adrenomedullin and increase in regional cerebral blood flow and prevention of ischemic brain injury after middle cerebral artery occlusion in rats.

The intravenous infusion of rat adrenomedullin, at concentrations ranging from 0.1 to 1.0 μg/kg/min, for 60 min increased the regional cerebral blood flow (rCBF) in a dose-dependent manner in rats. rCBF was measured using a laser Doppler flowmetry device placed on the surface of the parietal cortex. The increase in rCBF induced by 1.0 μg/kg/min of adrenomedullin was up to 145 ± 10.8% of controls at 60 min (n = 5, p < 0.001). These concentrations of adrenomedullin did not affect systemic blood pressure or other physiologic parameters, including pH, PaCO2, PaO2, hemoglobin, and blood glucose. Repeated infusion of 1.0 μg/kg/min of adrenomedullin at 2-h intervals caused tachyphylaxis (n = 5, p < 0.01). Rat adrenomedullin (1.0 μg/kg/min) demonstrated a more potent effect than the same dose of human adrenomedullin. The C-terminal fragment of human adrenomedullin (0.5 and 5.0 μg/kg/min), adrenomedullin22–52, which did not affect rCBF alone, inhibited the effect of rat adrenomedullin (0.5 μg/kg/min) as a receptor antagonist in a dose-dependent manner. In a model of middle cerebral artery (MCA) occlusion in spontaneously hypertensive rats, pre- and postinfusion of 1.0 μg/kg/min of adrenomedullin suppressed the reduction in rCBF following MCA occlusion (control, 29 ± 15.1%; adrenomedullin group, 45 ± 14.4%; not significant) and decreased the volume of ischemic brain injury (control, 288 ± 35 mm3; adrenomedullin group, 232 ± 35 mm3; p < 0.05). These results suggest that adrenomedullin increases rCBF and prevents ischemic brain injury, partly by increasing the collateral circulation.

Activation of JAK/STAT signalling in neurons following spinal cord injury in mice

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway is one of the most important in transducing signals from the cell surface to the nucleus in response to cytokines. In the present study, we investigated chronological alteration and cellular location of JAK1, STAT3, phosphorylated (p)‐Tyr1022/1023‐JAK1, p‐Tyr705‐STAT3, and interleukin‐6 (IL‐6) following spinal cord injury (SCI) in mice. Western blot analysis showed JAK1 to be significantly phosphorylated at Tyr1022/1023 from 6u2003h after SCI, peaking at 12u2003h and gradually decreasing thereafter, accompanied by phosphorylation of STAT3 at Tyr705 with a similar time course. ELISA analysis showed the concentration of IL‐6 in injured spinal cord to also significantly increase from 3u2003h after SCI, peaking at 12u2003h, then gradually decreasing. Immunohistochemistry revealed p‐Tyr1022/1023‐JAK1, p‐Tyr705‐STAT3, and IL‐6 to be mainly expressed in neurons of the anterior horns at 12u2003h after SCI. Pretreatment with a JAK inhibitor, AG‐490, suppressed phosphorylation of JAK1 and STAT3 at 12u2003h after SCI, reducing recovery of motor functions. These findings suggest that SCI at the acute stage produces IL‐6 mainly in neurons of the injured spinal cord, which activates the JAK/STAT pathway, and that this pathway may be involved with neuronal response to SCI.

Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat

Tamoxifen (TAM), a widely used non‐steroidal anti‐estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia‐induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2u2003h rMCAo produced a time‐dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24u2003h. The neuronal (n)NOS inhibitor, 7‐nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC50 values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [3H]arginine to [3H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100u2003µm TAM. These data suggest that TAM is an effective inhibitor of Ca2+/calmodulin‐dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

Nitric oxide metabolites in the cisternal cerebral spinal fluid of patients with subarachnoid hemorrhage.

OBJECTIVEnTo investigate nitric oxide (NO) metabolism after subarachnoid hemorrhage (SAH).nnnMETHODSnWe measured the concentrations of the NO metabolites, nitrite and nitrate, in cerebrospinal fluid (CSF) obtained from the cisternal drainage of patients with SAH. Studies were performed for 31 patients who had undergone surgical obliteration of bleeding aneurysms within 3 days of their hemorrhage. The concentrations of nitrite and nitrate in the CSF were measured for 14 days using a nitrate/nitrite kit and samples that were obtained on a daily basis from the cisternal drainage.nnnRESULTSnCompared with the control values in the CSF (2.6 +/- 0.4 mumol/L, n = 14) obtained from patients with hemifacial spasm, trigeminal neuralgia, or nonruptured aneurysms, the concentrations of nitrite and nitrate in the CSF were significantly elevated in the acute stage of SAH and remained elevated. The concentration of NO metabolites may correlate with the amount of bleeding, inasmuch as the values in patients in Fisher Group 3 (n = 25) were higher than those in patients in Fisher Group 2 (n = 6). The concentration of nitrate was higher than that of nitrite, suggesting that NO in the subarachnoid space is mainly absorbed by hemoglobin and degraded to nitrate. No differences were demonstrated in patients treated with high doses of methylprednisolone (n = 17) compared with those treated with usual-dose steroids (n = 14). Steroids are known to prevent the formation of inducible NO synthase mediated by inflammatory cytokines.nnnCONCLUSIONnNO metabolism in the brain is stimulated after SAH. Nitrate is the dominant NO metabolite in CSF after SAH. The involvement of inducible NO synthase in the pathophysiology of NO metabolism after SAH was not clearly suggested based on the present data.

Phosphorylation of neuronal nitric oxide synthase at Ser847 by CaM-KII in the hippocampus of rat brain after transient forebrain ischemia.

The authors previously demonstrated that Ca 2+/calmodulin (CaM)-dependent protein kinase IIα (CaM-KIIα) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study, they established that forebrain ischemia causes an increase in the phosphorylation of nNOS at Ser847 in the hippocampus. This nNOS phosphorylation appeared to be catalyzed by CaM-KII: (1) it correlated with the autophosphorylation of CaM-KIIα; (2) it was blocked by the CaM-KII inhibitor, KN-93; and (3) nNOS and CaM-KIIα were found to coexist in the hippocampus. Examination of the spatial relation between nNOS and CaM-KIIα in the brain revealed coexistence in the hippocampus but not in the cortex during reperfusion, with a concomitant increase in autophosphorylation of CaM-KIIα. The phosphorylation of nNOS at Ser847 probably takes place in nonpyramidal hippocampal neurons, which increased after 30 minutes of reperfusion in the hippocampus, whereas no significant increase was detected in the cortex. An intraventricular injection of KN-93 significantly decreased the phosphorylation of nNOS in the hippocampus. These results point to CaM-KII as a protein kinase, which by its colocalization may attenuate the activity of nNOS through its Ser847 phosphorylation, and may thus contribute to promotion of tolerance to postischemic damage in hippocampal neurons.

Inducible Cyclooxygenase Expression in Canine Basilar Artery After Experimental Subarachnoid Hemorrhage

BACKGROUND AND PURPOSEnInducible cyclooxygenase (COX-2) has been found to play a pathological role in cerebral insult. We investigated the expression of COX-2 in the basilar artery after experimental subarachnoid hemorrhage (SAH).nnnMETHODSnIn a canine two-hemorrhage model of SAH, the basilar arteries were obtained on day 2 after a cisternal injection of autologous blood or on days 4, 6, 7, or 9 after the second injection. Basilar arteries also were obtained 12 hours after intracisternal injection a cytokine: interleukin (IL)-1 beta (0.03 microgram), IL-6 (3 micrograms), or IL-8 (10 micrograms). Western blotting with a polyclonal anti-COX-2 antibody was performed in these arteries.nnnRESULTSnCOX-2 protein was not demonstrated in the basilar artery in control animals without SAH. However, it was expressed in the basilar artery on days 2, 4, 6, and 7 after blood injection but not on day 9. Intracisternal injection of IL-1 beta, IL-6, or IL-8 also induced COX-2 in the basilar artery.nnnCONCLUSIONSnCOX-2 expression was detected in basilar arterial tissue in both acute and chronic stages after SAH. Elevation of inflammatory cytokines after SAH may be involved in the induction of COX-2, which may produce sufficient quantities of eicosanoids to affect hemodynamics after SAH.

Activation of the JAK-STAT signaling pathway in the rat basilar artery after subarachnoid hemorrhage.

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) is one of the most important signaling pathways transducing signals from the cell surface in response to cytokines. Subarachnoid hemorrhage (SAH) produces cytokines in the CSF. We investigated whether this signaling pathway is activated in the rat basilar artery after SAH by cytokines. In a rat single-hemorrhage model of SAH, basilar arteries and CSF were obtained until 7 days after SAH. The concentration of interleukin-6 (IL-6) in CSF was measured by ELISA. Western blot analysis with JAK1, phosphospecific-JAK1, STAT3, phosphospecific STAT3 at Tyr705 and Ser727, cyclooxygenase-2 (COX-2), and actin antibodies was performed in basilar artery. The expressions of STAT3, phosphospecific STAT3 at Tyr705 and Ser727, and COX-2 in basilar artery were examined by immunohistochemical studies. The concentration of IL-6 immediately increased after SAH and Western blot analysis revealed that JAK1 was phosphorylated within 2 h, accompanied by phosphorylation of STAT3 at Tyr705, extending to Ser727 at days 1-2. Immunohistochemistry revealed phosphorylation of STAT3 to occur in endothelial and smooth muscle cells of the basilar artery. In addition, intracisternal injection of IL-6 by itself significantly increased phosphorylation of STAT3 at Tyr705 and Ser727. Expression of COX-2 was also upregulated in endothelial cells of the basilar artery. These results indicate that SAH produces the proinflammatory cytokine IL-6 in the CSF, which activates the JAK-STAT signaling pathway in the basilar artery and induces transcription of immediate early genes.

Vasodilator Effects on Canine Basilar Artery Induced by Intracisternal Interleukin-1β

The effect of interleukin-1β (IL-1β) on a cerebral artery was investigated in anesthetized dogs. Intracisternal administration of IL-1β (0.03 and 0.3 μg) dilated the canine basilar artery in a dose-dependent manner, without affecting systemic blood pressure or heart rate. The increase in diameter induced by 0.3 μg of IL-1β was 28.4% ± 13.4% of control at 2 hours and was inhibited by 30 μg of the IL-1β receptor antagonist, zinc protoporphyrin (4.5% ± 13.5%, P < 0.05). Interleukin-1β did not affect the concentration of nitric oxide metabolites in CSF. However, there was an increase in the concentration of eicosanoids in CSF, and the elevation of 6-keto-PGF1α paralleled the vasodilation. Pretreatment with 30 μg of the selective inducible cyclooxygenase (COX-2) inhibitor NS-398 also inhibited the IL-1β-induced vasodilation significantly (5.9% ± 9.4% at 2 hours, P < 0.01). Western blot analysis revealed the expression of a 68-kD COX-2-like protein in basilar artery extracts. These findings suggest that the IL-1β-induced vasodilator effect is linked to the prostaglandin cascade, predominantly to prostaglandin I2, by induction of COX-2, but not to the stimulation of nitric oxide metabolism.

Changes in serum cytokine concentrations after neurosurgical procedures

SummaryCytokine responses to surgical trauma have been studied in 70 patients undergoing various neurosurgical procedures. Serum concentrations of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8, (IL-8), tumour necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were measured before and after surgery using enzyme-linked immunosorbent assays.The serum concentrations of IL-1ß, IL-8, TNF-α, and IFN-γ did not change significantly following neurosurgical operations. In contrast, serum IL-6 levels were significantly elevated following surgery, peaking at postoperative day 1 and then gradually decreasing. Maximum IL-6 concentrations were considerably higher in patients who underwent surgery for brain tumours or aneurysms as compared with patients who had a ventriculoperitoneal shunt, neurovascular decompression or transsphenoidal adenomectomy. Intra-operative use of methylprednisolone, which is known to block the production and action of cytokines, suppressed the increase in IL-6 levels after surgery. There was a statistically significant correlation between the IL-6 peak concentration and the length of surgery in patients not treated with steroids, but not in patients treated with steroids. Additionally patients who underwent supratentorial surgery had higher peak concentrations of IL-6 than those who underwent infratentorial surgery. These results suggest that IL-6 is an early marker of tissue damage and may be useful in assessing the extent of postoperative stress.