Masato Nagahama

Tissue Distribution and Immunocytochemical Localization of Neurotrophin‐3 in the Brain and Peripheral Tissues of Rats

Abstract: The tissue distribution of neurotrophin‐3 (NT‐3) was investigated in rats at 1 month of age using a newly established, sensitive two‐site enzyme immunoassay system for NT‐3, as well as the immunocytochemical localization of this protein. The immunoassay for NT‐3 enabled us to quantify NT‐3 at levels > 3 pg per assay. In the rat brain, NT‐3 was detectable only in the olfactory bulb (0.54 ng/g wet weight), cerebellum (0.71 ng/g), septum (0.91 ng/g), and hippocampus (6.3 ng/g). By contrast, NT‐3 was widely distributed in peripheral tissues. Appreciable levels of NT‐3 were also found in the thymus (31 ng/g), heart (38 ng/g), diaphragm (21 ng/g), liver (45 ng/g), pancreas (892 ng/g), spleen (133 ng/g), kidney (40 ng/g), and adrenal gland (46 ng/g). An antibody specific for NT‐3 bound to pyramidal cells in the CA2‐CA4 regions of the hippocampus, to A cells in the islets of Langerhans in the pancreas, to unidentified cells in the red pulp of the spleen, to liver cells, and to muscle fibers in the diaphragm from rats at 1 month of age. Molecular masses of NT‐3‐immunoreactive proteins in the hippocampus and pancreas were 14 and 12 kDa, respectively. Thus, in rats, NT‐3 was detected in restricted regions of the brain and in the visceral targets of the nodose ganglia at high concentrations. Our present results suggest that NT‐3 not only functions as a classical target‐derived neurotrophic factor but also can play other roles.

Inducible nitric oxide synthase-dependent DNA damage in mouse model of inflammatory bowel disease

Increased cancer risk occurs in inflammatory bowel disease (IBD) undergoing long‐term chronic inflammation. To evaluate whether inducible nitric oxide synthase (iNOS)‐dependent DNA damage plays a role in the carcinogenic process triggered by IBD, we prepared a mouse model of IBD induced by transfer of CD45RBhighCD4+ T cells lacking regulatory T cells to female severe combined immunodeficiency (SCID) mice. CD45RBhighCD4+ T cells were isolated from mouse spleen after staining with fluorescein isothiocyanate (FITC)‐conjugated anti‐CD45RB monoclonal antibody, followed by anti‐FITC‐conjugated microbeads. This IBD mouse model showed that the bodyweight increased with aging to a lesser extent than non‐treated controls, and that the intestine was shortened. Pathological findings of this mouse model, which showed severe inflammation in colon tissues, were similar to IBD patients. Double immunofluorescence technique revealed that both 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) were formed mainly in epithelial cells of the IBD mouse model. 8‐Nitroguanine was formed in most of 8‐oxodG‐immunoreactive nuclei of epithelial cells. iNOS, proliferating cell nuclear antigen and p53 protein were also expressed in the colon epithelium. These results indicate that nitrative DNA damage, as well as oxidative DNA damage, is induced in colon epithelial cells of the IBD mouse model followed by proliferation of these cells, which may contribute to colon carcinogenesis. (Cancer Sci 2005; 96: 157–163)

Localization of Various Forms of the γ Subunit of G Protein in Neural and Nonneural Tissues

Abstract: For a study of the localization of various forms of the γ subunit of G proteins, antibodies were raised in rabbits against peptides that corresponded to partial amino acid sequences of bovine γ2, γ3, γ5, and γ7. Affinity‐purified antibodies against γ2, γ3, and γ5 reacted specifically with γ2, γ3, and γ5, respectively, but the antibody against γ7 reacted with γ2, γ3, and a novel γ subunit, designated γs1, as well as with γ7. Because these antibodies reacted with the respective forms of the γ subunit from rat brain, we investigated the localization of γ subunits in the rat. γ2 and γ3 were abundant in all regions in the brain, whereas the concentration of γ5 and γ7 was relatively low with the single exception being a high concentration of γ7 in the striatum. The concentration of γ2 was consistently high during ontogenic development in the rat brain, whereas γ3 appeared about a week after birth and their concentrations then increased until a month after birth. In tissues other than the brain, γ3 was observed only in the pituitary gland, whereas γ2, γ5, and γ7 were found in a variety of tissues. In addition, most tissues contained relatively high concentrations of some other γ subunit, which was detected with an antibody against a γ7‐derived peptide and appeared to be γs1. Among cloned cells tested, γ3 was detected only in PC12 pheochromocytoma cells. Taken together, the results indicated that γ3 was expressed specifically in neuronal cells, and γs1 was the major γ subunit in most nonneural cells. γ2, γ5, and γ7 were distributed in a variety of tissues, but γ2 was dominant in the brain.

L-Arginine Immunoreactive Enteric Glial Cells in the Enteric Nervous System of Rat Ileum

L-Arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.

A study of the myenteric plexus of the congenital aganglionosis rat (spotting lethal)

SummaryThe entire bowel of a mutant strain of rats, the congenital aganglionosis rat (spotting lethal), was investigated using the acetylcholinesterase reaction and immunohistochemical staining for tyrosine hydroxylase and substance P in whole-mount preparations. The histology of the bowel of mutant rats was also studied by light- and electron microscopy.In all examined mutant rats, a constricted region of intestine followed a dilated region of the bowel. In 29 cases constricted segments extended from rectum to distal ileum; in 3 cases from rectum to middle colon.In controls the myenteric plexus appeared as a mesh-work consisting of ganglion strands and internodal strands, showing a rather regular ladder-like pattern from duodenum to rectum. The myenteric plexus of mutants was very different from that of controls, showing conspicuous regional differences. Even in the duodenum, where there was no macroscopical disorder, the plexus showed an irregular pattern, the meshes varying greatly in size and shape. Ganglion strands were shorter than those in controls. The plexus in the dilated segment gradually decreased in density, finally disappearing above the proximal terminal of the constricted segment. In some areas, below this transition, i.e., the anal portion of the constricted segment in 29 cases (long constricted segment type only), there were neither ganglion cells nor nerve fibers except for scarcely distributed tyrosine hydroxylase-immunoreactive nerve fibers. In the distal part of the upper colon some fine nerve bundles, gradually increased in number and mixed with thicker nerve bundles in the lower portion of the colon. Finally, at the level of the rectum, nerve bundles of various sizes interlaced irregularly with one another to form a network. However, this network was free from ganglion cells.

iNOS-dependent DNA damage in patients with malignant fibrous histiocytoma in relation to prognosis

Malignant fibrous histiocytoma (MFH) is one of the most common soft tissue sarcomas. MFH has been proposed to be a lesion accompanied with inflammatory responses. During chronic inflammation, reactive nitrogen and oxygen species generated from inflammatory cells are considered to participate in carcinogenesis by causing DNA damage. 8‐nitroguanine is a mutagenic nitrative DNA lesion formed during chronic inflammation. We examined whether nitrative DNA damage is related to the prognosis of MFH patients. We performed immunohistochemical analyses to examine the distribution of DNA damage and the expression of inflammation‐related molecules including inducible nitric oxide synthase (iNOS), nuclear factor‐κB (NF‐κB), and cyclooxygenase‐2 (COX‐2) in clinical specimens from 25 patients with MFH. We also analyzed the correlation of DNA damage or the expression of these genes with the prognosis of MFH patients. Immunohistochemical staining revealed that the formation of 8‐nitroguanine and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG), an oxidative DNA lesion, occurred to a much greater extent in MFH tissue specimens from deceased patients than in live patients. iNOS, NF‐κB and COX‐2 were colocalized with 8‐nitroguanine in MFH tissues. It is noteworthy that the statistical analysis using the Kaplan‐Meier method demonstrated strong 8‐nitroguanine staining to be associated with a poor prognosis. In conclusion, 8‐nitroguanine appears to participate in not only the initiation and promotion of MFH, but also in the progression of MFH, and could therefore be used as a promising biomarker to evaluate the prognosis of cancer patients. (Cancer Sci 2007; 98: 163–168)

Localization of d-serine and serine racemase in neurons and neuroglias in mouse brain

Abstractd-Serine is a novel candidate for an intrinsic ligand for the glycine site of N-methyl-d-aspartate (NMDA) receptors in mammalian brain. d-Serine and serine racemase, which produces d-serine from l-serine, have long been presumed to be localized in astrocytes. However, we have reported that d-serine immunoreactivity was observed in neurons in rats. In the present study, the distributions of d-serine and serine racemase were investigated in combination with marker proteins for neurons, astrocytes and oligodendrocytes in mice. Immunoreactivities for d-serine and serine racemase were found in neurons and oligodendrocytes. These results suggest that d-serine can be produced in neurons as well as glias and used as a neurotransmitter, which control the synaptic function of NMDA receptors.

Aquaporin 1 immunoreactive enteric neurons in the rat ileum

Most neurons in the central nervous system and peripheral nervous system do not express water transporting protein, aquaporin (AQP). In the present study, we have demonstrated the presence of AQP1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ENS) of the rat ileum. AQP1-immunoreactive (IR) neurons simultaneously expressed a neuronal marker HuC/D. Moderate numbers of AQP1-IR neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. AQP1-IR neurons can be classified as Dogiel type I cells, which have several short processes and a single long process. Many AQP1-IR fibers were found both in the myenteric and submucosal plexi. Many AQP1-IR varicose fibers were closely associated with neuronal somata in the ganglia, whereas other AQP1-IR fibers penetrated into the muscle layers. These results suggest that AQP1-IR neurons probably play a significant role within the ENS to control gut functions.

Cellular Localization of Group IIA Phospholipase A2 in Rats

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA2 isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.

Light and electron microscopic studies of pituitary adenylate cyclase-activating peptide (PACAP) – immunoreactive neurons in the enteric nervous system of rat small and large intestine

Abstract Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1–15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility.