Tamotsu Seki

Distribution and ultrastructural localization of a receptor for pituitary adenylate cyclase activating polypeptide and its mRNA in the rat retina.

Localization and gene expression of pituitary adenylate cyclase activating polypeptide receptor (PACAPR) in the rat retina were studied by immunocytochemistry and in situ hybridization, respectively. Antisera were raised against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR. Strong PACAPR mRNA expression and PACAPR-like immunoreactivity (PACAPR-LI) were detected in ganglion cells, amacrine cells, and in the inner plexiform layer. PACAPR-LI appeared to be concentrated predominantly in the neuronal perikarya and processes. At the ultrastructural level, strong immunostaining for PACAPR was visible in the plasma membranes, rough endoplasmic reticulum and cytoplasmic matrix in neurons. This study provides the basis for a better understanding of the functions of PACAP in the rat retina.

Neuroprotective Effect of PACAP against Kainic Acid-Induced Neurotoxicity in Rat Retina

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) is well known to protect delayed neuronal cell death in the brain of rodents. In order to investigate the neuroprotective action of PACAP in the retina, we examined the effects of PACAP on kainic acid (KA)‐induced neurotoxicity in the rat retina. Many ganglion cells in the retina died after KA injection in the control group and PACAP treatment significantly promoted cell survival. These findings strongly suggest that PACAP plays very important roles in preventing cell death in the retina.

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina☆☆

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.

Gene Expression for PACAP Receptor mRNA in the Rat Retina by in Situ Hybridization and in Situ RT‐PCR

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor (PAC 1 -R) are localized in the rat retina by in situ hybridization and in situ RT-PCR. PAC 1 -R is distributed in the rat brain and eye. 1,2 PAC 1 -R exists in at least eight splice variants that are differentially coupled to signal transduction pathways, 3 but the distribution and localization of these splice variants in the retina remain to be determined. There are at least eight isoforms of the PAC 1 -R, and the signal transduction system is different for each subtype. We studied gene expression of PAC 1 -R mRNA in the rat retina by in situ hybridization. In addition, we tried to identify PAC 1 -R subtypes in the rat retina by in situ RT-PCR.

Pituitary adenylate cyclase-activating peptide (PACAP) stimulates production of interleukin-6 in rat Müller cells

Pituitary adenylate cyclase-activating peptide (PACAP) is known to regulate not only neurons but also astrocytes. Here, we investigated, both in vitro and in vivo, the effects of PACAP38 on rat Müller cells, which are the predominant glial element in the retina. Müller cells isolated from juvenile Wistar rats were treated with PACAP38 or PACAP6-38, a PACAP selective antagonist. Cell proliferation was determined by measuring the incorporation of bromodeoxyuridine with ELISA. Interleukin-6 (IL-6) levels in the culture medium were determined by a bioassay using B9 cells, IL-6 dependent hybridoma. In adult Wistar rats, the expression of IL-6 in the retina after intravitreal injection of PACAP38 (10 pmol) was assessed by immunohistochemistry. PACAP38 stimulated IL-6 production in Müller cells at a concentration as low as 10(-12) M, which did not induce cell proliferation. This elevation of IL-6 production was inhibited by PACAP6-38. Radial IL-6 expression was observed throughout the retina at 2 and 3 days after PACAP38 injection. These data demonstrate that Müller cells are one of the target cells for PACAP. IL-6, which is released from Müller cells with stimulation by PACAP, may play a significant role in the retina.

PACAP Attenuates NMDA-Induced Retinal Damage in Association with Modulation of the Microglia/Macrophage Status into an Acquired Deactivation Subtype

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been known as a neuroprotectant agent in several retinal injury models. However, a detailed mechanism of this effect is still not well understood. In this study, we examined the retinoprotective effects and associated underlying mechanisms of action of PACAP in the mouse N-methyl-d-aspartic acid (NMDA)-induced retinal injury model, focusing on the relationship between PACAP and retinal microglia/macrophage (MG/MΦ) status. Adult male C57BL/6 mice received an intravitreal injection of NMDA to induce retinal injury. Three days after NMDA injection, the number of MG/MΦ increased significantly in the retinas. The concomitant intravitreal injection of PACAP suppressed NMDA-induced cell loss in the ganglion cell layer (GCL) and significantly increased the number of MG/MΦ. These outcomes associated with PACAP were attenuated by cotreatment with PACAP6-38, while the beneficial effects of PACAP were not seen in interleukin-10 (IL-10) knockout mice. PACAP significantly elevated the messenger RNA levels of anti-inflammatory cytokines such as transforming growth factor beta 1 and IL-10 in the injured retina, with the immunoreactivities seen to overlap with markers of MG/MΦ. These results suggest that PACAP enhances the proliferation and/or infiltration of retinal MG/MΦ and modulates their status into an acquired deactivation subtype to favor conditions for neuroprotection.

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.

Midbrain hemorrhage presenting with oculomotor nerve palsy: case report

BACKGROUND We report a case of isolated oculomotor nerve palsy caused by a midbrain hemorrhage. CASE DESCRIPTION A 75-year-old man visited our hospital complaining of double vision and left eye ptosis without headache. Neuro-ophthalmic examination showed that his left and right pupils were 3.5 mm and 3 mm in diameter, respectively, that left downward eye movement was limited, and that convergence of the right eye was limited. Magnetic resonance imaging (MRI) demonstrated that there was a hematoma located in the anterior tegmentum of the left midbrain. Two weeks after admission and treatment, including conservative therapy, his double vision gradually disappeared. CONCLUSION To date, 73 cases have been reported in the literature. Most cases of isolated oculomotor nerve palsy have been caused by diabetes mellitus, aneurysm, or infarction. However, focal midbrain hemorrhage incidentally produces third nerve palsy. MRI is extremely helpful in diagnosing a small hemorrhage of the midbrain in such cases.

PACAP Stimulates the Release of Interleukin-6 in Cultured Rat Müller Cells

Abstract:  We have investigated the in vivo effect of PACAP on rat Müller cells that are the predominant glial element in the retina. Müller cells were treated with PACAP38, either alone or in the presence of the PACAP‐selective antagonist, PACAP6–38. Cellular proliferation was determined by measuring the incorporation of bromodeoxyuridine, while interleukin‐6 (IL‐6) levels in the culture medium were examined using a B9 cell bioassay. In cultured rat Müller cells, the expression of PACAP receptor (PAC1‐R) was assessed with immunohistochemistry using a PAC1‐R‐specific antiserum. PACAP stimulated IL‐6 production in Müller cells at a concentration as low as 10−12 M, which was not sufficient to induce cell proliferation. This elevation of IL‐6 production was significantly inhibited by PACAP6–38. These data suggest that Müller cells are one of the target cells for PACAP, stimulating the release of IL‐6, and providing a mechanism whereby PACAP exerts a significant neuroprotective effect in the retina.

Distribution and protective function of pituitary adenylate cyclase-activating polypeptide in the retina

Pituitary adenylate cyclase-activating polypeptide (PACAP), which is found in 27- or 38-amino acid forms, belongs to the VIP/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert a protective effect against several types of neural damage. The retina is considered to be part of the central nervous system, and retinopathy is a common cause of profound and intractable loss of vision. This review will examine the expression and morphological distribution of PACAP and its receptors in the retina, and will summarize the current state of knowledge regarding the protective effect of PACAP against different kinds of retinal damage, such as that identified in association with diabetes, ultraviolet light, hypoxia, optic nerve transection, and toxins. This article will also address PACAP-mediated protective pathways involving retinal glial cells.