Sachiko Yofu

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

BackgroundWe 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.MethodsUnilateral 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 IFNγ or IL-4.ResultsGp91phox 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 IFNγ, 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.ConclusionsClassical 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.

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.

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.

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.

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.

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.

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 PACAPs 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.

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.

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.

Controlled normothermia during ischemia is important for the induction of neuronal cell death after global ischemia in mouse

A stable model of neuronal damage after ischemia is needed in mice to enable progression of transgenic strategies. We performed transient global ischemia induced by common carotid artery occlusions with and without maintaining normal rectal temperature (Trec) in order to determine the importance of body temperature control during ischemia. We measured brain temperature (Tb) during ischemia/reperfusion. Mice with normothermia (Trec within +/- 1 degrees C) had increased mortality and neuronal cell death in the CA1 region of hippocampus, which did not occur in hypothermic animals. If the Trec was kept within +/- 1 degrees C, the Tb decreased during ischemia. After reperfusion, Tb in the normothermia group developed hyperthermia, which reached > 40 degrees C and was > 2 degrees C higher than Trec. We suggest that tightly controlled normothermia and prevention of hypothermia (Trec) during ischemia are important factors in the development of a stable neuronal damage model in mice.