High concentration of extracellular nucleotides suppresses cell growth via delayed cell cycle progression in cancer and noncancer cell lines.

Tumor necrosis frequently occurs in malignant tumors, showing rapid growth and invasion. This phenomenon is generally regarded as simple ischemic necrosis due to insufficient tumor vessels and blood supply. However, the necrotic tissue contains high amount of nuclear substances, DNA, and nucleoproteins that may affect the surrounding tumor cells by promoting or suppressing the tumor cell growth in vivo. This study focused on the effects of an externally administered water-soluble nuclear crude extract (SNE) containing nuclear protein and oligonucleotides on several human cancer and noncancer cell lines. The results demonstrated that the SNE suppressed cell growth in cancer and noncancer cells in vitro. Through the flow cytometry analysis of the nuclear DNA content, it was observed that the SNE increased and decreased cell proportion in the S and G2/M phases, respectively, thereby suggesting that the cell growth inhibition was due to cell cycle delay, and not due to apoptosis. These studies suggest that the high-concentration of extracellular nucleotides generated as a result of tumor necrosis and/or released from infiltrated neutrophils could suppress the growth of surrounding cancer and intrinsic cells, which provides us some insights into an alternative anticancer strategy for patients with highly malignant necrotic tumor.

PACAP suppresses dry eye signs by stimulating tear secretion.

Dry eye syndrome is caused by a reduction in the volume or quality of tears. Here, we show that pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dry eye-like symptoms such as corneal keratinization and tear reduction. PACAP immunoreactivity is co-localized with a neuronal marker, and PACAP receptor (PAC1-R) immunoreactivity is observed in mouse infraorbital lacrimal gland acinar cells. PACAP eye drops stimulate tear secretion and increase cAMP and phosphorylated (p)-protein kinase A levels in the infraorbital lacrimal glands that could be inhibited by pre-treatment with a PAC1-R antagonist or an adenylate cyclase inhibitor. Moreover, these eye drops suppress corneal keratinization in PACAP-null mice. PACAP eye drops increase aquaporin 5 (AQP5) levels in the membrane and pAQP5 levels in the infraorbital lacrimal glands. AQP5 siRNA treatment of the infraorbital lacrimal gland attenuates PACAP-induced tear secretion. Based on these results, PACAP might be clinically useful to treat dry eye disorder.

Accumulation of autofluorescent storage material in brain is accelerated by ischemia in chloride channel 3 gene-deficient mice.

Autofluorescent storage material (ASM) is an aging pigment that accumulates during the normal course of senescence. Although the role of ASM has yet to be fully elucidated, ASM has been implicated in age-related neurodegeneration. In this study, we determined the level of ASM in chloride channel 3 (ClC-3) gene-deficient (KO) mice both in response to aging and following mild global ischemia. To understand the mechanism of action of the ASM, mice subjected to ischemia were treated with the cyclooxygenase (COX) inhibitor indomethacin or with the noncompetitive glutamate receptor antagonist MK-801. ClC-3 KO mice displayed age-related neurodegeneration of the neocortex as well as the hippocampus. The cortical layers in particular granular layers became thinner with aging. ASM accumulated in the brains of ClC-3 KO mice was increased seven- to 50-fold over that observed in the corresponding regions of their wild-type littermates. Young wild-type mice survived longer than age-matched ClC-3 KO mice after permanent global ischemia. However, in the case of older animals, the survival curves were similar. The ASM also increased four- to fivefold 10 days after mild global ischemia, an effect that was suppressed by treatment with indomethacin and MK-801. These results suggest that temporary ischemia might trigger a process similar to aging in the brain, mimicking the effect of age-related neurodegenerative diseases.

IL-6 and PACAP receptor expression and localization after global brain ischemia in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a neuroprotective action against ischemic damage. This action is mediated by the interleukin-6 (IL-6) pathway. However, as the expression patterns of PACAP receptors and IL-6 following ischemia are not understood, we evaluated them in the mouse hippocampus in response to ischemia induced by bilateral common carotid artery occlusion. Real-time PCR determination of PAC1R mRNA expression in the hippocampus was significantly elevated on day 7 after ischemia. VPAC1R mRNA expression was significantly decreased 3 days after the ischemic episode, while VPAC2R mRNA expression showed a nonsignificant tendency to increase on day 7. IL-6 mRNA expression was significantly increased on day 3 and peaked on day 7 after ischemia. The mRNA expression of activity-dependent neuroprotective protein, which is a neuroprotective factor stimulated by PACAP, remained virtually unchanged in response to ischemia. IL-6 immunoreactivity was detected in the CA1 pyramidal cell layer and colocalized with the neuronal marker NeuN on day 1 after ischemia. On day 3, irregularly shaped IL-6-immunopositive cells colocalized with the astrocytic marker glial fibrillary acidic protein but not with the microglial marker Iba1. PAC1R immunoreactivity co-labeled with IL-6 immunoreactivity. These results suggest that PACAP could stimulate IL-6 secretion by neurons during the acute phase after an ischemic episode and thereafter by astrocytes during the subacute phase.

Expression and localization of the orexin-1 receptor (OX1R) after traumatic brain injury in mice.

Orexins are neuropeptides that have a wide range of physiological effects, and recent studies have suggested that the orexin system may be involved in traumatic brain injury. However, the expression and localization of orexin receptors have not been examined yet under brain injury conditions. In the present study, we used immunohistochemical techniques to investigate the expression of orexin-1 receptor (OX1R) and its time-dependent changes in the mouse brain after controlled cortical impact (CCI) injury. OX1R-like immunoreactivity was first detected 6 h after injury in the surrounding penumbra of the injury. The intensity of this immunoreactivity was increased at 12 h, peaked at day 1, and then decreased from day 2 to day 7. To identify the cellular localization of OX1R, we also performed double-immunohistochemical staining with OX1R and several cell marker antibodies. OX1R-like immunopositive cells were clearly co-localized with immunoreactivity for the neuronal marker NeuN at day 7. It was also expressed on the periphery of cells immunopositive for CD11b, a microglial cell marker, at days 1 and 7. These results suggest that orexin and its receptor may play roles in traumatic brain injury, and that OX1R is induced in neurons and microglial cells after traumatic brain injury.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates proliferation of reactive astrocytes in vitro.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalamus. Recently, we have shown that the PACAP receptor (PAC1-R) is expressed in reactive astrocytes following an in vivo stub wound brain injury. However, the functional role of PACAP has not yet been clarified. In order to investigate the effect of PACAP on the proliferation of reactive astrocytes, a scratch wound paradigm was applied to astrocytic monolayers. Following injury, there was an increase in PAC1-R and glial fibrillary acidic protein (GFAP) immunoreactivity in the astrocytes surrounding the scratch line. PACAP at concentrations of 10(-15) to 10(-7) M was applied immediately after scratching, and the proliferating astrocytes were visualized by multiple immunofluorescence labeling. The percentage of cells that colabeled for Ki67 (a marker of proliferating cells) and GFAP increased in the 10(-11)- and 10(-13)-M PACAP-treated groups. The proliferating astrocytes induced by PACAP treatment mainly occurred in the proximal wound area where many reactive astrocytes were observed. Pretreatment with the PACAP receptor antagonist PACAP6-38 significantly suppressed the PACAP-induced effects. These results strongly suggest that PACAP plays an important role in the proliferation of reactive astrocytes following nerve injury.

Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury.

BACKGROUND: We hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation. METHODS: Unilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNgamma or IL-4. RESULTS: Gp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNgamma, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox. CONCLUSIONS: Classical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

Cardioprotective effect of endogenous pituitary adenylate cyclase-activating polypeptide on Doxorubicin-induced cardiomyopathy in mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a cytoprotective polypeptide. PACAP and its receptors are expressed in the heart, but it is unclear whether PACAP exerts its protective effect on the myocardium in vivo. The aim of the present study was to investigate whether endogenous PACAP has a cardioprotective effect on Doxorubicin (Dox)-induced cardiomyopathy. METHODS AND RESULTS: Dox was intraperitoneally injected to induce cardiomyopathy in wild type (WT) and PACAP knockout (ie, PACAP+/- and PACAP-/-) mice. The survival rates up to 15 days of PACAP+/- mice and PACAP-/- mice were significantly less than that of WT mice. Cardiac function, measured by echocardiography, was significantly lower in PACAP+/- mice than in WT mice at day 10. Morphological examination of sections of myocardium showed degenerative change and fibrosis in PACAP+/- mice at day 10. Serum reactive oxygen metabolites (a marker of oxidative stress), the number of 8-hydroxy-deoxyguanosine-positive nuclei and TdT-mediated dUTP nick end-labeling (TUNEL) positive nuclei in the myocardium were higher in PACAP+/- mice than WT mice. However, continuous subcutaneous administration of PACAP38 was able to prevent the myocardial damage typically caused by Dox injection in PACAP+/-. CONCLUSIONS: These results suggest that endogenous PACAP might attenuate Dox-induced myocardial damage and that its mechanism of action is likely to be associated with the reduction of oxidative stress and mediated via anti-apoptotic effects.

Regulation of oxidative stress by pituitary adenylate cyclase-activating polypeptide (PACAP) mediated by PACAP receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional peptide that has been shown to be neuroprotective following a diverse range of cell injuries. Although several mechanisms regulating this effect have been reported, no direct evidence has linked PACAP to the regulation of oxidative stress, despite the fact that oxidative stress is a factor in the injury progression that occurs in most models. In the present study, we investigated the plasma oxidative metabolite and anti-oxidation potential levels of PACAP-deficient mice, as well as those of wild-type animals treated with PACAP38. These were assayed by the determination of Reactive Oxidative Metabolites (d-ROMs) and the Biological Anti-oxidant Potential (BAP) using the Free Radical Electron Evaluator system. We also investigated the direct radical scavenging potency of PACAP38 and the functional role of its receptor in the regulation of oxidative stress by PACAP, by using vasoactive intestinal peptide (VIP) and the PACAP receptor antagonist, PACAP6-38. Although younger PACAP null mice displayed no significant effect, greater d-ROMs and lower BAP values were recorded in older animals than in their wild-type littermates. Intravenous injection of PACAP38 in wild-type mice decreased the plasma d-ROMs and BAP values in a dose-dependent manner. These effects were not reproduced using VIP and were abolished by co-treatment with PACAP38 and the PAC1R antagonist PACAP6-38. Taken together, these results suggest that PACAP plays an important role in the physiological regulation of oxidative stress.

Nucleoprotein Diet Ameliorates Arthritis Symptoms in Mice Transgenic for Human T-Cell Leukemia Virus Type I (HTLV-1).

Because rheumatoid arthritis (RA), an autoimmune disease, the patients often recognize side-effects due to the medication, alternative therapeutic strategies might potentially offer a clinical advantage. We evaluated the effect of nucleoprotein from salmon soft roe on animal model of arthritis. Mice transgenic for human T-cell leukemia virus type I (HTLV-1 Tg) were divided into three experimental groups and supplemented on either nucleoprotein-free (nonNP), or 0.6% or 1.2% nucleoprotein mixed (NP0.6 or NP1.2) diet for 3 months. The mice were evaluated arthritis by morphology, and measured with rheumatoid factor (RF). Moreover, macrophages and oxidative metabolites were assessed in the ankle and/or serum. Anti-oxidative potentials in nucleoprotein were determined with biological anti-oxidative potential (BAP) test, and electron spin resonance (ESR) analysis. NonNP-diet HTLV-1 Tg mice increased an arthritis symptoms and RF. The symptoms were ameliorated in NP-diet groups. Macrophages detected by F4/80 staining, and oxidative metabolites in the serum and/or joints were clearly decreased in 1.2% NP-diet HTLV-1 Tg mice. Nucleoprotein and DNA-nucleotide, but less protamine, had direct anti-oxidative potency with BAP test and/or ESR in vitro. These observations suggest that dietary nucleoprotein ameliorates arthritis symptoms in HTLV-1 Tg mice and offers hope as an alternative treatment for this debilitating medical condition.

Endogenous pituitary adenylate cyclase activating polypeptide is involved in suppression of edema in the ischemic brain.

Pituitary adenylate cyclase activating polypeptide is a pleiotropic neuropeptide. We previously showed that heterozygous PACAP gene knockout (PACAP(+/-)) mice had larger infarct volumes and worse neurological scores after middle cerebral artery occlusion (MCAO). However, the relationship between endogenous PACAP levels and edema in the ischemic brain has not yet been evaluated. In this study, the formation of edema in the ischemic brain as well as cerebral blood flow was compared between PACAP(+/-) and wild-type (PACAP(+/+)) mice. The amount of brain edema was calculated by subtracting the contralateral volume from the ipsilateral volume 24 h after permanent MCAO. PACAP(+/-) mice showed significantly greater brain edema than PACAP(+/+) mice. To investigate the effects of endogenous PACAP on blood flow during ischemia, cerebral blood flow in the ipsilateral and the contralateral cortices was compared between PACAP(+/-) and PACAP(+/+) mice for 25 min after ischemia. With a two-dimensional laser Doppler perfusion imaging system, the blood flow in the ipsilateral and contralateral cortices was shown to be similar in PACAP(+/-) and PACAP(+/+) mice during ischemia. These results suggest that endogenous PACAP suppresses the formation of edema in the ischemic brain.

Novel free radical monitoring in patients with neurological emergency diseases.

Recent experimental studies have demonstrated that oxidative stress has important roles in various neuronal conditions. Stroke and traumatic brain injury are also related to oxidative stress. However few studies prove the existence of free radicals in humans because they are difficult to measure. We recently developed a technique for free radical and oxidative stress monitoring using the ex vivo electron spin resonance (ESR) spin trapping method in patients with neuroemergency. Blood samples were collected by catheterization of the internal jugular bulb. The alkoxyl radical level was measured by ex vivo ESR spectrometry using 5,5-dimethyl-1-pyrroline-1-oxide (Dojin Chemical, Tokyo, Japan) as a spin trap. Electron spin response detection of the spin adduct was performed at room temperature using a JESREIX X-band spectrometer (JEOL, Tokyo, Japan). As a marker of reactive oxygen species, we also used the diacron-reactive oxygen metabolites test (d-ROM). This method is not invasive for patients, and it is technically easy to execute.Oxidative stress monitoring is useful and may prove valuable for clarifying the pathophysiology of neuroemergency diseases, which has long been hampered by technical difficulties in measuring and monitoring oxidative stress.

Pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) co-localizes with activity-dependent neuroprotective protein (ADNP) in the mouse brains.

Activity-dependent neurotrophic protein (ADNP) was discovered as a novel response gene for vasoactive intestinal polypeptide. We have reported that PACAP strongly stimulated ADNP mRNA expression in a mouse neuron/glial cell culture; however, the distribution of ADNP in the brain and its possible co-expression with the PACAP receptor (PAC1R) are unknown. In this study, the specificity of the ADNP antibody used in subsequent immunohistochemistry experiments was first characterized. Mouse brain lysates were analysed by Western blot, with an ADNP-immunopositive signal corresponding to the expected molecular weight of ADNP detected as a 124 kDa band. Immunohistochemical staining to identify ADNP and PAC1R immunoreactivity in mouse brain was then performed. ADNP immunoreactive cells were observed in the cerebral cortex, cerebellum, hippocampus, and medial septum of brain slices. ADNP-immunoreactive cells in the cerebral cortex were multi-polar-shaped and co-immunostained with the astrocyte marker, glial fibrillary acidic protein (GFAP). ADNP-immunoreactive cells in the cerebellum were found to surround Purkinje cells and showed GFAP immunoreactivity. On the other hand, ADNP-immunoreactive cells in the hippocampus and septum were round in shape and co-immunostained with the neuron marker, neuron-specific enorase. All of the ADNP-immunopositive cells co-localized with PAC1R immunoreactivity. These observations suggest that ADNP is expressed in both astrocytes and neurons, and that ADNP expression may be regulated by PACAP.

Distribution and localization of pituitary adenylate cyclase-activating polypeptide-specific receptor (PAC1R) in the rostral migratory stream of the infant mouse brain.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to participate in the regulation of neuronal proliferation and differentiation. While these processes are considered to be mediated via PACAP's actions on the PACAP-specific receptor, PAC1R, the precise distribution of PAC1R during neurodevelopment has not yet to be elucidated in detail. The purpose of this study is to examine the distribution of PAC1R in the neurogenic region of the rostral migratory stream (RMS) from the apical subventricular zone (SVZa) to the olfactory bulb (OB) in infant mice using immunostaining. Co-immunostaining for PAC1R in a variety types of cell were carried out using different markers. These included the neural stem cell markers, nestin and glial fibrillary acidic protein (GFAP), a marker for migrating neuroblasts (doublecortin, DCX), a marker for immature neurons betaIII-tubulin, (Tuj1), and a marker for mature neurons, neuronal nuclei (NeuN). PAC1R-like immunoreactivity (LI) was observed in the RMS. However, the intensity of PAC1R- LI was different depending on the regions which were investigated. PAC1R-LI was strong in nestin- and GFAP-positive cells in the SVZa and was also observed in NeuN-positive cells in the OB. However, the intensities of PAC1R-LI in DCX- and Tuj1-positive cells were weaker than the other markers. These results suggest that PACAP may participate in the neurodevelopment with the stage-specific expression of PAC1R and that PACAP plays important roles in neurons as well as in glial cells.

Increased mitochondrial DNA oxidative damage after transient middle cerebral artery occlusion in mice.

Oxidative stress and DNA oxidation play important roles in the induction of ischemic neuronal cell death. However, the subcellular source of oxidized DNA detected by 8-hydroxy-2'-deoxyguanosine (8-OHdG) after ischemia has not been clarified although it is known to increase in the brain after ischemia. One-hour transient ischemia of the middle cerebral artery was induced in mice utilizing an intraluminal filament. The occurrence of superoxide anion as an ethidium (Et) signal, 8-OHdG, cytochrome c release and neuronal cell death were examined using immunohistological and biochemical techniques in sham-operated control (0h) and 1, 3, 6, 24, or 96h after reperfusion. Et signals were prominent in the cortical neurons of ipsilateral hemisphere 3h after reperfusion. Strong 8-OHdG immunoreactivity was observed 3-6h after reperfusion. Immunoassays after cell fractionation revealed a significant increase of 8-OHdG in mitochondria 6h after reperfusion. Immunohistochemistry revealed that the 8-OHdG immunoreactivity colocalized with a neuronal marker, microfilament 200 and a mitochondrial marker, cytochrome oxidase subunit I. Cytochrome c rose in cytoplasm at 6h and TUNEL-positive neurons noted 6-24h after ischemia. The present results suggest the possibility that the mitochondrial damage including mitochondrial DNA oxidation might be responsible for the induction of ischemic neuronal cell death.

Does edaravone (MCI- 186) act as an antioxidant and a neuroprotector in experimental traumatic brain injury?

Edaravone (MCI-186) is a novel synthetic free radical scavenger intended to have neuroprotective effect against ischemic insult. It is currently used on patients with cerebral infarction. Here, we note beneficial pharmaceutical effects of edaravone in rat experimental traumatic brain injury. Under specific experimental conditions, edaravone minimized traumatic brain injury by functioning as a synthetic antioxidant. Clinical trials testing the efficacy of edaravone are warranted.

Neuronal damage in rat brain and spinal cord after cardiac arrest and massive hemorrhagic shock.

OBJECTIVE: Severe global ischemia often results in severe damage to the central nervous system of survivors. Hind-limb paralysis is a common deficit caused by global ischemia. Until recently, most studies of global ischemia of the central nervous system have examined either the brain or spinal cord, but not both. Spinal cord damage specifically after global ischemia has not been studied in detail. Because the exact nature of the neuronal damage to the spinal cord and the differences in neuronal damage between the brain and spinal cord after global ischemia are poorly understood, we developed a new global ischemia model in the rat and specifically studied spinal cord damage after global ischemia. Further, we compared the different forms of neuronal damage between the brain and spinal cord after global ischemia. DESIGN: Randomized, controlled study using three different global ischemia models in the rat. SETTING: University research laboratory. SUBJECTS: Male, adult Sprague-Dawley rats (300 g). INTERVENTIONS: Animals were divided into three experimental groups, group A (n = 6, survived for 7 days), 12 mins of hemorrhagic shock; group B (n = 6, survived for 7 days), 5 mins of cardiac arrest; or group C (n = 6, each for 6 hrs, 12 hrs, 1 day, 3 days, and 7 days), 7 mins of hemorrhagic shock and 5 mins of cardiac arrest. Motor deficit of the hind limbs was studied 6 hrs to 7 days after resuscitation. Also, nonoperated animals (n = 6) were used as the control. Histologic analysis (hematoxylin and eosin, Fluoro-Jade B, terminal deoxynucleotidyl transferase- mediated dUTP end-labeling [TUNEL], Klüver-Barrera) and ultrastructural analysis using electron microscopy were performed on samples from the CA1 region of the hippocampus and lumbar spinal cord. Demyelination of the white matter of the lumbar spinal cord was analyzed semiquantitatively using Scion Image software. MAIN RESULTS: No paraplegic animals were observed in either group A or B. All group C animals showed severe hind-limb paralysis. Severe neuronal damage was found in the CA1 region of the hippocampus in all groups, and the state of delayed neuronal cell death was similar among the three groups. Neuronal damage in the lumbar spinal cord was detected only in group C animals, mainly in the dorsal horn and intermediate gray matter. Demyelination was prominent in the ventral and ventrolateral white matter in group C. A significant difference was observed between control and group C rats with Scion Image software. Ultrastructural analysis revealed extensive necrotic cell death in the intermediate gray matter in the lumbar spinal cord in group C rats. CONCLUSION: The combination in the global ischemia model (i.e., hemorrhagic shock followed by cardiac arrest) caused severe neuronal damage in the central nervous system. Thereby, hind-limb paralysis after global ischemia might result from spinal cord damage. These results suggest that therapeutic strategies for preventing spinal cord injury are necessary when treating patients with severe global ischemia.

Pituitary adenylate cyclase-activating polypeptide (PACAP) decreases ischemic neuronal cell death in association with IL-6.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to decrease ischemic neuronal damage and increase IL-6 secretion in rats. However, the mechanisms underlying neuroprotection are still to be fully elucidated. The present study was designed to investigate the role played by PACAP and IL-6 in mediating neuroprotection after ischemia in a null mouse. Infarct volume, neurological deficits, and cytochrome c in cytoplasm were higher in PACAP(+/-) and PACAP(-/-) mice than in PACAP(+/+) animals after focal ischemia, although the severity of response was ameliorated by the injection of PACAP38. A decrease in mitochondrial bcl-2 was also accentuated in PACAP(+/-) and PACAP(-/-) mice, but the decrease could be prevented by PACAP38 injection. PACAP receptor 1 (PAC1R) immunoreactivity was colocalized with IL-6 immunoreactivity in neurons, although the intensity of IL-6 immunoreactivity in PACAP(+/-) mice was less than that in PACAP(+/+) animals. IL-6 levels increased in response to PACAP38 injection, an effect that was canceled by cotreatment with the PAC1R antagonist. However, unlike in wild-type controls, PACAP38 treatment did not reduce the infarction in IL-6 null mice. To clarify the signaling pathway associated with the activity of PACAP and IL-6, phosphorylated STAT (signal transducer and activator of transcription) 3, ERK (extracellular signal-regulated kinase), and AKT levels were examined in PACAP(+/-) and IL-6 null mice after ischemia. Lower levels of pSTAT3 and pERK were observed in the PACAP(+/-) mice, whereas a reduction in pSTAT3 was recorded in the IL-6 null mice. These results suggest that PACAP prevents neuronal cell death after ischemia via a signaling mechanism involving IL-6.

Effect of pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) on tissue oxygen content--treatment in central nervous system of mice.

It has been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in preventing neuronal cell death and is also a potent vasodilator. Cerebral hypotension and hypoperfusion during cerebral ischemia and neurodegenerative diseases are well known as some of the negative factors which aggravate neuronal cell death. Nevertheless, the effect of PACAP on the cerebral circulation was not understood well. Therefore, in the present study, we determined the mean arterial blood pressure (MBP), regional cerebral blood flow (rCBF) and cerebral oxygen content (pO2) in mice, and estimated the therapeutically useful doses of PACAP. Under barbiturate anesthesia, polyethylene tubes were inserted into mice to monitor MBP and to administer PACAP (5 x 10(-13)-5 x 10(-8) mol/kg) or vasoactive intestinal peptide (VIP; 5 x 10(-12) and 5 x 10(-9) mol/kg). Then, MBP, rCBF and cerebral pO2 were simultaneously measured in the mice. PACAP (5 x 10(-10)-5 x 10(-9) mol/kg) injections transiently decreased MBP, and cerebral pO2. PACAP (5 x 10(-8) mol/kg) injections produced a long-lasting potent decline of MBP, rCBF and cerebral pO2. Therefore, PACAP should be applied at low doses which do not influence the MBP and cerebral circulation to determine the therapeutically useful doses of PACAP for neuroprotection.