ASK family kinases mediate cellular stress and redox signaling to circadian clock.

Daily rhythms of behaviors and physiologies are generated by the circadian clock, which is composed of clock genes and the encoded proteins forming transcriptional/translational feedback loops (TTFLs). The circadian clock is a self-sustained oscillator and flexibly responds to various time cues to synchronize with environmental 24-h cycles. However, the key molecule that transmits cellular stress to the circadian clockwork is unknown. Here we identified apoptosis signal-regulating kinase (ASK), a member of the MAPKKK family, as an essential mediator determining the circadian period and phase of cultured cells in response to osmotic changes of the medium. The physiological impact of ASK signaling was demonstrated by a response of the clock to changes in intracellular redox states. Intriguingly, the TTFLs drive rhythmic expression of Ask genes, indicating ASK-mediated association of the TTFLs with intracellular redox. In behavioral analysis, Ask1, Ask2, and Ask3 triple-KO mice exhibited compromised light responses of the circadian period and phase in their activity rhythms. LC-MS/MS-based proteomic analysis identified a series of ASK-dependent and osmotic stress-responsive phosphorylations of proteins, among which CLOCK, a key component of the molecular clockwork, was phosphorylated at Thr843 or Ser845 in the carboxyl-terminal region. These findings reveal the ASK-dependent stress response as an underlying mechanism of circadian clock flexibility.

Matrix metalloproteinase inhibition attenuates brain edema after surgical brain injury.

BACKGROUND: Neurosurgical operations can result in inevitable brain injury due to the procedure itself. This surgical brain injury (SBI) can cause post-operative complications such as brain edema following blood-brain barrier (BBB) disruption leading to neurological deficits. METHODS: We tested whether inhibition of matrix metalloproteinases (MMPs) 9 and 2 provided neuroprotection against SBI. A rodent SBI model, which involves a partial frontal lobe resection, was used to evaluate two treatment regimens of MMP inhibitor-1 (inhibitor of MMP-9 and MMP-2); a single dose (5 mg/kg, pretreatment) and daily dose treatment (5 mg/kg x 3, pre- and post-treatment). Postoperative assessment at different time periods included brain water content (brain edema), immunohistochemical analysis, zymography for MMP enzymatic activity, and neurological assessment. FINDINGS: The results indicate that SBI caused localized edema around the site of surgical resection with concomitant increase in MMP-9 and MMP-2 activity. Both treatment regimens with MMP inhibitor-1 decreased brain edema and attenuated the rise in MMP-9 and MMP-2 activity. An increased expression of MMP-9 was also seen in the neurons and neutrophils in the affected brain tissue at the periphery of surgical resection. CONCLUSIONS: The study suggests a potential role for MMP inhibition as preoperative therapy before neurosurgical procedures.

Role of c-Jun N-terminal kinase in cerebral vasospasm after experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Inflammation could play a role in cerebral vasospasm after subarachnoid hemorrhage (SAH). SP600125 a c-Jun N-terminal kinase (JNK) inhibitor reduces inflammation. The present study examined if SP600125 could reduce cerebral vasospasm. METHODS: Twenty-seven dogs were assigned to 5 groups: control, SAH, SAH plus dimethyl sulfoxide (DMSO), SAH plus SP600125 (10 micromol/L), and SAH plus SP600125 (30 micromol/L). SAH was induced by the injection of autologous blood into the cisterna magna on day 0 and day 2. Angiograms were evaluated on day 0 and day 7. The behavior of the dogs was evaluated daily. The activation of the JNK pathway, the infiltration of leukocytes, and the production of cytokines were also evaluated. RESULTS: Severe vasospasm was observed in the basilar artery of SAH and DMSO dogs. The JNK signaling pathway was activated in the basilar artery after SAH and SP600125 reduced angiographic and morphological vasospasm and improved behavior scores with a concomitant reduction of infiltrated leukocytes and IL-6 production. CONCLUSIONS: These results demonstrate that SP600125 attenuated cerebral vasospasm through a suppressed inflammatory response, which may provide a novel therapeutic target for cerebral vasospasm.

One-stage anterior approach for four-vessel occlusion in rat.

BACKGROUND AND PURPOSE: We report a modified 4-vessel occlusion (4VO) rat model. METHOD: We used a 1-stage anterior approach for making bilateral hemispheric ischemia. RESULTS: Modified 4VO method decreased cerebral blood flow to 12% to 14% of baseline levels. CONCLUSIONS: This modified 4VO method is a minimally invasive, quick, reliable procedure for producing ischemic changes.

Nicotine exposure, mimicked smoking, directly and indirectly enhanced protein kinase C activity in isolated canine basilar artery, resulting in enhancement of arterial contraction.

Cigarette smoking is a significant risk factor in the incidence of cerebrovascular disorders. Among the many compounds in cigarette smoke, nicotine is considered to most significantly affect cerebral arterial tone. The purpose of this study is to investigate precise pharmacological effects of nicotine on the regulation of cerebral arterial tone. To mimic smoking, a low concentration of nicotine (10(-6) mol/L), which is equivalent to the serum level of habitual smokers, was treated for 1 hour in an isometric tension study and for 24 hours in a study using cultured vascular endothelial cells (VECs). Using the canine basilar artery, the effect of nicotine on uridine 5'-triphosphate (UTP)-induced vasoconstriction was examined in the isometric tension study. Protein kinase C (PKC) activity in the canine basilar artery was measured by enzyme immunoassay. Endothelial function was assessed by endothelium-dependent vasodilatation and endogenous nitric oxide (NO) synthesis in VECs using a fluorescent indicator, diaminofluorescein-FM diacetate (DAF-FM/DA). Nicotine significantly enhanced UTP-induced contraction and PKC activity in the artery, and attenuated endothelium-dependent vasodilatation and NO synthesis in VECs. Because PKC activity was increased by de-endothelialization itself, endothelial dysfunction by nicotine enhances PKC activity. Because PKC was further activated by nicotine even in the de-endothelialized artery, nicotine directly affects PKC activities in smooth muscle. These results indicate that nicotine potentiates contractile response through direct and indirect PKC activation in the canine basilar artery.

Role of p53 and apoptosis in cerebral vasospasm after experimental subarachnoid hemorrhage.

Our previous studies indicate that apoptosis in endothelial cells of major cerebral arteries contributes to cerebral vasospasm after subarachnoid hemorrhage (SAH). This study examined the pathologic roles of tumor suppressor p-53 in cerebral vasospasm using an established dog double-hemorrhage model. Twenty mongrel dogs were divided into four groups: (1) control, (2) SAH, (3) SAH+DMSO (vehicle), and (4) SAH+pifithrin-alpha (PFT) (p53 inhibitor). The p53 inhibitor (200 nmol/L) was injected into the cisterna magna daily from Day 0 through Day 3. Angiogram was performed on Day 0 and Day 7. Western blot, cell proliferation assay, histology, and TUNEL staining were conducted on the basilar arteries collected on Day 7 after SAH. The arterial diameter on Day 7 was 42%+/-4%, 40%+/-5%, and 59%+/-4% for SAH, SAH+DMSO, and SAH+PFT, respectively. In addition, positive staining of TUNEL and increased protein expression of p53, Bax, and PCNA in the basilar artery were observed on Day 7. PFT suppressed apoptosis in endothelial cells and proliferation in smooth muscle cells, and attenuated angiographic vasospasm. In conclusion, p53 may be a key factor in endothelial apoptosis and smooth muscle proliferation after SAH. Inhibition of p53 may potentially reduce or even prevent cerebral vasospasm.

Ras protein contributes to cerebral vasospasm in a canine double-hemorrhage model.

BACKGROUND AND PURPOSE: Mitogen-activated protein kinase (MAPK) has been shown to be involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). In the present study we examined the role of Ras protein, an upstream regulator of MAPK, and the effects of the inhibitors of Ras farnesyltransferase (FTase), FTI-277 and FTase inhibitor I, on angiographic vasospasm and clinical evaluations. METHODS: Twenty-five dogs were randomly divided into 5 groups: control, SAH, SAH+dimethyl sulfoxide, SAH+FTI-277, and SAH+FTase inhibitor I. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days 0 and 2. Angiography was performed at days 0 and 7. Clinical behavior and the activation of Ras (GTP-Ras) and phosphorylated ERK1/2 of MAPK in the basilar arteries were examined. RESULTS: Severe vasospasm was obtained in the SAH and SAH+dimethyl sulfoxide dogs (42.5+/-2.5% and 38.9+/-2.4%, respectively). Enhanced GTP-Ras and phosphorylated ERK1/2 were observed in the spastic basilar arteries (P<0.05). Inhibitors of Ras FTase decreased GTP-Ras and phosphorylated ERK1/2, attenuated angiographic vasospasm, and improved appetite and activity scores. CONCLUSIONS: Ras contributes to cerebral vasospasm, and inhibitors of Ras FTase may have potential in the management of cerebral vasospasm.

Gene transfer of extracellular superoxide dismutase failed to prevent cerebral vasospasm after experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: We examined the therapeutic effect of human extracellular superoxide dismutase (ECSOD) gene transfer in the prevention of delayed cerebral vasospasm after experimental subarachnoid hemorrhage (SAH) because it was reported ECSOD relieved early-stage vasospasm. METHODS: Twenty mongrel dogs were divided randomly into 4 groups to serve as control, SAH, SAH+adenovirus ECSOD (AdECSOD), and SAH+no transgene (AdBglII) groups, respectively. An established canine double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on day 0 and day 2. Angiography was performed at day 0 and day 7. Clinical behavior, cerebrospinal fluid (CSF) ECSOD activity, CSF leukocyte count, morphology, and human ECSOD expression (RT-PCR) in the basilar arteries were evaluated. RESULTS: Severe vasospasm was obtained in SAH, SAH+AdECSOD, and SAH+AdBglII gene-transferred dogs, and the residual diameters of the basilar artery were 41+/-1%, 39+/-4%, and 49+/-4%, respectively. Increased CSF activity of ECSOD was obtained in SAH+AdECSOD (162+/-23 U/mL) when compared with SAH (26+/-2) and SAH+AdBglII (25+/-3) dogs. RT-PCR confirmed successful gene transfer in the basilar arteries from SAH+AdECSOD dogs. Increased leukocyte counts were observed in the CSF and in the subarachnoid space, especially in SAH+AdECSOD and SAH+AdBglII dogs. CONCLUSIONS: Gene transfer of human ECSOD failed to prevent delayed cerebral vasospasm.

Caspase inhibitors prevent endothelial apoptosis and cerebral vasospasm in dog model of experimental subarachnoid hemorrhage.

Apoptosis in the endothelium of major cerebral arteries may play a role in the initiation and maintenance of cerebral vasospasm after subarachnoid hemorrhage (SAH). We tested the therapeutic effect of caspase inhibitors on endothelial apoptosis and on cerebral vasospasm in an established dog double-hemorrhage model. Thirty-one mongrel dogs were divided into five groups: control; SAH; SAH treated with vehicle [DMSO]; SAH treated with Ac-DEVD-CHO [a specific caspase-3 inhibitor]; and SAH treated with Z-VAD-FMK [a broad caspase inhibitor]. The inhibitors (100 microM) were injected into the cisterna magna daily from Day 0 through Day 3. Angiography was performed on Day 0 and Day 7. Histology, TUNEL staining, and immunohistochemistry were conducted on basilar arteries collected on Day 7 after SAH. Positive staining of TUNEL, poly(ADP)-ribose polymerase (PARP), caspase-3, and caspase-8 was observed in the endothelial cells of the spastic arteries. Double fluorescence labeling demonstrated co-localization of TUNEL with caspase-3 and TNFalpha receptor-1 (TNFR1). Ac-DEVD-CHO and Z-VAD-FMK prevented endothelial apoptosis and reduced angiographic vasospasm. The mechanism of apoptosis in endothelial cells involves TNFR1 and the caspase-8 and caspase-3 pathways. Caspase inhibitors may have potential in the treatment of cerebral vasospasm.

Interlaminar fixation using the atlantoaxial posterior fixation system (3XS system) for atlantoaxial instability: surgical results and biomechanical evaluation.

This study evaluated the surgical results for patients with atlantoaxial instability due to various lesions treated using the atlantoaxial posterior fixation system (3XS system; Kisco DIR, Paris, France), together with a biomechanical study of this system. The strength of the 3XS system during torsion was examined using a biomechanical simulation model. The 3XS system consists of a transverse unit, hooks, and rods. The lower part of the biomechanical simulation machine was rigidly fixed and the upper part was movable, allowing torsion to be applied until the point of failure. The test was started at 1.5 newton-meters, thought to be the maximum load on the upper cervical spine. The 3XS system tolerated torsion of up to 20 newton-meters, but became deformed. The instrument was fractured at 30 newton-meters. Fifteen patients, four with atlantoaxial instability, seven with os odontoideum, and four with odontoid fractures, underwent surgery using the 3XS system and an iliac bone fragment inserted between the C-1 and C-2 laminae. Postoperative rigid fixation of the lesion and optimal cervical alignment was obtained in all patients, and the patients were discharged within 2 weeks after surgery. Follow-up radiography showed bony fusion between C-1 and C-2 in all patients. Posterior fixations between C-1 and C-2 using the 3XS system were easy to perform and no surgical complications were encountered. The biomechanical study showed the 3XS system can tolerate torsions unlikely to occur during rotation movements in the atlantoaxial region in humans. The surgical use of the 3XS system for the treatment of atlantoaxial instability has several advantages.

Differences in diffusion-weighted and T2-weighted magnetic resonance imaging findings in the acute and chronic stages of ischemic cerebrovascular disease--two case reports.

A 71-year-old man presented with sudden onset of vertigo and a 77-year-old man suffered consciousness disturbance. Diffusion-weighted magnetic resonance (MR) imaging on admission showed hyperintense areas in the left cerebellar hemisphere in the first patient and in the brainstem in the second patient. Both patients were treated with argatroban and edaravone, and the neurological deficits markedly improved one month after admission. T2-weighted MR imaging one month after the onset showed much smaller hyperintense areas compared with the findings on admission in both patients. These results indicate that findings of hyperintense areas by diffusion-weighted MR imaging in the acute stage of ischemic cerebrovascular disease indicate not only the ischemic core but also parts of the reversible incomplete ischemic lesion and suggest that intensive treatment in the acute stage might reverse ischemic brain damage in some patients.

Matrix metalloproteinase inhibition attenuates brain edema in an in vivo model of surgically-induced brain injury.

OBJECTIVE: Neurosurgical procedures can result in brain injury by various means, including direct trauma, hemorrhage, retractor stretch, and electrocautery. This surgically-induced brain injury (SBI) can cause postoperative complications such as brain edema after blood-brain barrier (BBB) disruption. The present study seeks to test a matrix metalloproteinase (MMP) inhibitor for preventing postoperative brain edema and BBB disruption in an in vivo model of surgically-induced brain injury. METHODS: A rodent model of SBI was used which involves resection of a part of the right frontal lobe. A total of 89 Sprague-Dawley male rats (weight, 300-350 g) were randomly divided into four groups: 1) SBI with vehicle treatment (0.1% dimethyl sulfoxide), 2) SBI with single treatment of MMP inhibitor-1 (an inhibitor of MMP-9 and MMP-2), 3) SBI treated daily (total 3 times) with MMP inhibitor-1, and 4) sham surgical group. Postoperative assessment at different time periods included evaluation of BBB permeability, brain water content (brain edema), neurological scoring, histology, immunohistochemistry, and zymography for MMP enzymatic activity. Temporal magnetic resonance imaging studies were also performed to assess postoperative edema. RESULTS: The results indicate that SBI caused increased brain water content (ipsilateral frontal lobe) and BBB permeability compared with sham animals. Treatment with MMP inhibitor-1 attenuated MMP-9 and MMP-2 activity and decreased brain water content with preservation of the BBB. CONCLUSION: Inhibition of MMP-9 and MMP-2 attenuates brain edema and BBB disruption after SBI. The study suggests a potential role for MMP inhibition as preoperative therapy before neurosurgical procedures.

Encephalocele in the ethmoid sinus presenting as a massive intracerebral hemorrhage after a "polypectomy": a case report.

The discovery of a congenital transethmoidal encephalocele in an adult patient is very rare and is sometimes misdiagnosed as a polyp of the ethmoid sinus. It is important to note that a congenital transethmoidal encephalocele presenting as a massive intracerebral hemorrhage in an adult patient has never been reported. This study's patient underwent endoscopic polypectomy and suffered from massive intracerebral hemorrhage because the encephalocele contained a frontobasal artery. Transcranial surgery was successfully performed to repair the frontal base. We report on the patient's condition and discuss the clinical implications of the present case.