Masahiko Nakamura

Differential distribution of 68 Kd and 200 Kd neurofilament proteins in the gerbil hippocampus and their early distributional changes following transient forebrain ischemia

SummaryThe distribution of neurofilament (NF) proteins was studied immunohistochemically in the gerbil hippocampus with antibodies against NF68 (68 Kd molecular weight) and NF200 proteins, and changes in the distribution of NF proteins after transient ischemia were observed in order to investigate the temporal correlation between NF and delayed neuronal death. In the normal hippocampus, NF68-like immunoreactivity (NF68-LI) was densely distributed in nerve processes in CA2, CA3 and the hilus of the dentate gyrus but was less intense in CA1. In contrast, processes with NF200-LI appeared to be evenly distributed in CA1, CA2, CA3 and the dentate gyrus. Mongolian gerbils were subjected to transient ischemia for 5 min by bilateral carotid occlusion and subjected to immunohistochemistry 1, 2, 3 and 4 days after ischemia. Following transient ischemia, prior to neuronal cell death, the intensity of both NF68-LI and NF200-LI decreased in the whole hippocampal formation. This decrease was more obvious in the case of NF68-LI than NF200-LI. Four days after ischemia, when neuronal death of CA1 pyramidal cells had occurred, processes in CA1, particularly 68 Kd components, showed marked decreases in number and staining intensity, although processes in most layers of CA2, CA3 and the dentate gyrus appeared to be stained similarly to normal brain. Since NF68 protein is considered to be the major component of NF proteins and NF200 is an associated accessory protein, the current observations suggest that the poor distribution of NF68 in CA1 and the early loss of NF proteins may be closely related to selective vulnerability of CA1 pyramidal cells and delayed neuronal death.

Muscarinic acetylcholine receptors in rat gastric mucosa

SummaryThe muscarinic cholinergic innervation of the rat gastric mucosa was investigated by localizing the muscarinic receptors using a tritiated muscarinic antagonist, pirenzepine. Radioautography was performed by freeze drying stomach tissue, which was then embedded in Epon and wet sectioned with ethylene glycol, and dry mounting on emulsion film by the wire-loop method to prevent loss of the labelled substance during fixation and the radioautographic procedure. Light and electron microscopy showed that the specific pirenzepine-binding sites were localized predominantly on parietal cells, chief cells and perivascular plexuses. Analysis of the grain distribution on parietal cells revealed that the silver grains corresponding to the pirenzepine-binding sites were mainly on the basolateral plasma membrane. On the other hand, the surface mucous or mucous neck cells had few pirenzepine-binding sites.

Autoradiographic demonstration of gastrin-releasing peptide-binding sites in the rat gastric mucosa

The location of [125I]iodotyrosyl gastrin-releasing peptide-binding sites in the rat fundic mucosa was studied. Peptide specificity was demonstrated by competitive binding studies using the addition of a large amount of cold gastrin-releasing peptide or substance P. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by the wire-loop method to prevent loss of the labeled substance. Specific binding sites of gastrin-releasing peptide were found on D cells, surface mucus cells, and parietal cells, whereas few binding sites were seen on the chief or mucus neck cells.

Effect of basic fibroblast growth factor on reinnervation of gastric microvessels. Possible relevance to ulcer recurrence.

Basic fibroblast growth factor (bFGF) has well-established angiogenic and ulcer healing actions. bFGF has also been found to induce neural regeneration in the central nervous system. Thus, the present study was undertaken to clarify the effect of basic fibroblast growth factor on the regeneration of autonomic nerves in the granulation tissues following the induction of experimental gastric ulcer induced by acetic acid in rats. Rats were divided into control, acetic acid alone, and acetic acid plus acid-stable human recombinant basic fibroblast growth factor (CS23, 1 μg/100 g body wt., every 12 hr for three days, or one or two weeks, through oral gastric intubation) groups. As a result, few autonomic nerves were recognized surrounding the newly formed arterioles and venules in the acetic acid alone group. In the CS23-treated group, the cholinergic, calcitonin gene-related peptide and vasoactive intestinal peptide-immunoreactive nerves were clearly recognized near the microvessels, but few adrenergic nerves were seen even after CS23 treatment. From these observations, basic fibroblast growth factor was suggested to promote the reinnervation of the newly formed microvessels.

Autoradiographic demonstration of gastrin binding sites in rat gastric mucosa.

The location of 125I-iodotyrosyl gastrin I binding sites in rat gastric mucosa was studied. Peptide specificity was demonstrated by competitive binding studies through the addition of a large dose of cold human gastrin I or cholecystokinin-octapeptide. Autoradiography of the stomach tissue was carried out by freeze-drying, embedding in Epon, wet-sectioning with ethylene glycol, and dry-mounting the emulsion film by means of the wire-loop method to prevent loss of the labeled substance. Specific binding sites for gastrin were found on parietal and chief cells, whereas few binding sites were seen on the surface mucous or mucous neck cells. Binding sites on the parietal cells were dispersed in the cytoplasm, while those on the chief cells were found near the basal plasma membrane.

Plasticity of myofibroblasts appearing in granulation tissues after acetic acid treatment effect of bFGF

To clarify the origin of the myofibroblasts appearing in the healing process of the acetic acid-induced ulcer and effect of basic fibroblast growth factor (bFGF) on these myofibroblasts, we conducted an immunohistochemical study using antibody to intermediate filaments, desmin and vimentin. The binding sites of bFGF on the regenerative tissues were also studied by the radioautographic study of soluble compounds. As a result, the binding sites of bFGF were accumulated on the fibroblasts and myofibroblasts as well as on endothelial cells. The effect of CS23, acid-stable human recombinant bFGF was shown on distribution of myofibroblasts and regeneration of the microvascular system in the mucosal and submucosal layer.

Alteration of gastric microcirculation in ulcer healing and recurrence: Significance of autonomic nervous regeneration and mesenchymal cell

After the introduction of potent antisecretagogues to the peptic ulcer treatment, such as H2 receptor antagonists and proton pump inhibitors, more than 90% of peptic ulcers have been found to be cured within 8 weeks after these treatments, except intractable or resistant ulcers. Remission after the cessation of these treatments is also common. The factors that could exert influence on ulcer healing and recurrence were reevaluated. Microcirculatory disturbance in the regenerative tissue after the gastric ulcer formation has been thought to be closely related to the ulcer healing and recurrence. The present study was undertaken to study: (1) the microvascular pattern of the regenerative tissues from the acetic acid-induced ulcer, and (2) the distribution of the mesenchymal cells and autonomic nerves in the regenerative tissue and the effect of basic fibroblast growth factor (b-FGF), one of the potent angiogenic factors, on ulcer healing.

Fluorescent Histochemical Study on the Localization of Myofibroblasts in the Healing of Acetic Acid-Induced Gastric Ulcers in the Rat

The changes in the localization of FITC-phalloidin-positive smooth muscle cells and interstitial cells were studied in control and acetic acid-treated rat fundic mucosae. In the control rats, the FITC-phalloidin-positive cells mostly corresponded to smooth muscle cells of the muscularis mucosae, and arteriolar and venular smooth muscle cells. On the other hand, the fluorescence of the smooth muscle cells of the muscularis mucosae disappeared and a large number of fluorescent interstitial cells appeared in the regenerated mucosal layer in rats one week after the acetic acid-treatment, while three weeks after the application, severely thickened fluorescent muscularis mucosae were formed.

Wound healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin, with special reference to prolylhydroxylase and collagenase enzyme activity

The healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin were investigated with reference to the enzyme activity of both prolylhydroxylase and collagenase as related to histological findings. The rats were observed by endoscopy on the 3rd day after the subserosal injection of acetic acid; rats with ulcers were divided into three groups: non-treated, and cimetidine- and calcitonin-treated. The latter two groups were treated for 7 days. Prolylhydroxylase activity in active ulcers in the non-treated group was slightly higher on the 3rd day and significantly higher on the 10th day than the activity in control rats that had received subserosal injections of physiological saline solution on the respective days. In non-treated rats, the healed ulcer on the 10th day showed lower prolylhydroxylase activity than that in the active ulcer on the same day. Cimetidine did not affect prolylhydroxylase activity, but, with calcitonin, there was higher prolylhydroxylase activity in the healed than in the active ulcer, although the difference was not significant. Interstitial collagenase showed the highest activity on the 3rd day and decreased on the 10th day in non-treated rats. Collagenase activity was higher in the cimetidine-treated group, than that in the non-treated group, and numerous peroxidase-positive granulocytes were seen in the mucosa and submucosa. Calcitonin did not affect collagenase activity. The participation of both enzymes is indispensable in the healing process and the effects of anti-ulcer agents on these enzymes must be considered.

A novel immunocytochemical staining method that preserves cell membranes: Application for demonstrating Ca2+ pump ATPase in the liver

We have devised a method for immunogold staining of unosmicated, plastic-embedded cells that gives high levels of specific staining without sacrificing cell ultrastucture. Important conditions include PLP (periodate-lysine-paraformaldehyde) fixation, postfixation with uranyl acetate to preserve membrane phospholipids, dehydration with acetone, low-temperature embedding in LR gold resin, and use of osmium tetroxide to stain thin sections after immunogold labeling. We developed this method to localize plasma membrane calcium pump ATPase in rat hepatocytes and hepatic sinusoidal endothelial cells. Most gold particles of Ca2+ pump ATPase were easily assigned to bile canalicular membranes in rat hepatocytes, and the gold particles of Ca2− pump ATPase were located on the labyrinthlike structures of the endothelial sinusoidal fenestrae in rat hepatic sinusoidal endothelial cells. In these studies, it was useful to preserve the cell membrane for postembedding methods.