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Stem cell therapy is used to restore neurological function in stroke patients. We have previously reported that ischemia-induced multipotent stem cells (iSCs), which are likely derived from brain pericytes, develop in poststroke human and mouse brains. Although we have demonstrated that iSCs can differentiate into neural lineage cells, the factors responsible for inducing this differentiation remain unclear. In this study, we found that LDN193189, a bone morphogenetic protein (BMP) inhibitor, caused irreversible changes in the shape of iSCs. In addition, compared with iSCs incubated without LDN193189, the iSCs incubated with LDN193189 (LDN-iSCs) showed upregulated expression of neural lineage-related genes and proteins, including those expressed in neural stem/progenitor cells (NSPCs), and downregulated expression of mesenchymal and pericytic-related genes and proteins. Moreover, microarray analysis revealed that LDN-iSCs and NSPCs had similar gene expression profiles. Furthermore, LDN-iSCs differentiated into electrophysiologically functional neurons. These results indicate that LDN193189 induces NSPC-like cells from iSCs, suggesting that bioactive molecules regulating BMP signaling are potential targets for promoting neurogenesis from iSCs in the pathological brain, such as during ischemic stroke. We believe that our findings will bring us one step closer to the clinical application of iSCs.

Nerves in the renal parenchyma comprise sympathetic nerves that act on renal arteries and tubules to decrease blood flow and increase primary urine reabsorption, respectively. Synaptic vesicles release neurotransmitters that activate their effector tissues. However, the mechanisms by which neurotransmitters exert individual responses to renal effector cells remain unknown. Here, we investigated the spatial and molecular compositional associations of renal Schwann cells (SC) supporting the nerve terminals in male rats. The nerve terminals of vascular smooth muscle cells (SMCs) enclosed by renal SC processes were exposed through windows facing the effectors with presynaptic specializations. We found that the adrenergic receptors (ARs) α2A, α2C, and β2 were localized in the SMC and the basal side of the tubules, where the nerve terminals were attached, whereas the other subtypes of ARs were distributed in the glomerular and luminal side, where the norepinephrine released from nerve endings may have indirect access to ARs. In addition, integrins α4 and β1 were coexpressed in the nerve terminals. Thus, renal nerve terminals could contact their effectors via integrins and may have a structure, covered by SC processes, suitable for intensive and directional release of neurotransmitters into the blood, rather than specialized structures in the postsynaptic region.

Vibration is detected by mechanoreceptors, including Pacinian corpuscles (PCs), which are widely distributed in the human body including the adventitia of large blood vessels. Although the distribution of PCs around large limb vessels has been previously reported, there remains no consensus on their distribution in the adventitia of the human deep blood vessels in the upper arm. In addition, the physiological functions of PCs located around the deep limb blood vessels remain largely unknown. This study aimed to elucidate detailed anatomical features and physiological function of lamellar sensory corpuscles structurally identified as PCs using the immunohistochemical methods around the deep vessels in the upper arm. We identified PCs in the connective tissue adjacent to the deep vessels in the upper arm using histological analysis and confirmed that PCs are located in the vascular sheath of the artery and its accompanying vein as well as in the connective tissue surrounding the vascular sheath and nerves. PCs were densely distributed on the distal side of deep vessels near the elbow. We also examined the relationship between vascular sound and pulsating sensation to evaluate the PCs functions around deep arteries and veins and found that the vascular sound made by pressing the brachial arteries in the upper arm was associated with the pulsating sensation of the examinee. Our results suggest that PCs, around deep vessels, function as bathyesthesia sensors by detecting vibration from blood vessels.

Interleukin-18 (IL-18) is an inflammatory cytokine linked to major depressive disorder (MDD). MDD is closely related to metabolic disorders, such as diabetes mellitus (DM) and obesity. Moreover, DM is associated with cognitive impairment and promotes apoptosis of hippocampal cells by activating pro-apoptotic and inhibiting anti-apoptotic factors. IL-18-deficient (Il18-/-) mice are obese and have DM. Therefore, we hypothesized a close relationship between IL-18 and death of hippocampal cells, affecting neurogenesis related to behavioral changes such as MDD. Il18-/- male mice were generated on the C57Bl/6 background and Il18+/+ mice were used as controls. Behavioral, histopathological, and molecular responses, as well as responses to intracerebral recombinant IL-18 administration, were examined. Compared with Il18+/+ mice, Il18-/- mice had impaired learning and memory and exhibited lower motivation. In the Il18-/- mice, degenerated mitochondria were detected in synaptic terminals in the molecular layer, the polymorphic layer, and in mossy fibers in the dentate gyrus, suggesting mitochondrial abnormalities. Because of the degeneration of mitochondria in the dentate gyrus, in which pro-apoptotic molecules were upregulated and anti-apoptotic factors were decreased, apoptosis inducers were not cleaved, indicating inhibition of apoptosis. In addition, neurogenesis in the dentate gyrus and the maturity of neuronal cells were decreased in the Il18-/- mice, while intracerebral administration of recombinant IL-18 promoted significant recovery of neurogenesis. Our findings suggested that IL-18 was indispensable for mitochondrial homeostasis, sustaining clearance of degenerative neural cells, and supporting neurogenesis, normal neuronal maturation and hippocampal function.

Transcription of the platelet-derived growth factor receptor α (PDGFRA/Pdgfra) gene is considered to be precisely regulated. We have previously reported that the PDGFRA/Pdgfra gene is regulated by a dual promoter system in human and mouse, in which a novel PDGFRA/Pdgfra transcript has a first exon (exon 1β) different from that of the canonical PDGFRA/Pdgfra transcript (exon 1α). To elucidate the function of each transcript, we first investigated the contribution of different PDGFRA transcripts to final protein levels. Notably, knockdown experiments suggested the existence of other PDGFRA transcripts, and we identified five additional first exons (exons 1γ, 1δ, 1ε, 1ζ, and 1η) in intron 1 in both the human and mouse genes. The first exons of the mouse Pdgfra gene showed unique expression patterns: exon 1α was broadly expressed; exon 1β was highly expressed in embryos; exon 1γ was observed at relatively high levels in the adult central nervous system (CNS); and exon 1δ was expressed at relatively high levels in the developing CNS. Furthermore, in silico analysis of common putative transcription factor binding sites in the upstream regions of the first exons of both human and mouse PDGFRA/Pdgfra genes predicted common (such as Sry, Mzf1, and Cdx) and unique (such as Sox5, Lmo2, and GATA) transcription factors. Our findings show the diversity of the transcriptional regulation of the PDGFRA/Pdgfra gene.

Interleukin-18 (IL-18) is an inflammatory cytokine that has been linked to energy homeostasis and psychiatric symptoms such as depression and cognitive impairment. We previously revealed that deficiency in IL-18 led to hippocampal abnormalities and resulted in depression-like symptoms. However, the impact of IL-18 deficiency on other brain regions remains to be clarified. In this study, we first sought to confirm that IL-18 expression in neural cells can be found in human brain tissue. Subsequently, we examined the expression of genes in the prefrontal cortex of Il18−/− mice and compared it with gene expression in mice subjected to a chronic mild stress model of depression. Extracted genes were further analyzed using Ingenuity® Pathway Analysis, in which 18 genes common to both the chronic mild stressed model and Il18−/− mice were identified. Of those, 16 were significantly differentially expressed between Il18+/+ and Il18−/− mice. We additionally measured protein expression of α-2-HS-glycoprotein (AHSG) and transthyretin (TTR) in serum and the brain. In the prefrontal cortex of Il18−/− mice, TTR but not AHSG was significantly decreased. Conversely, in the serum of Il18−/− mice, AHSG was significantly increased but not TTR. Therefore, our results suggest that in IL-18-deficit conditions, TTR in the brain is one of the mediators causally related to depression, and AHSG in peripheral organs is one of the regulators inducing energy imbalance. Moreover, this study suggests a possible “signpost” to clarify the molecular mechanisms commonly underlying the immune system, energy metabolism, neural function, and depressive disorders.

Background The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis. Methods Il18−/− male mice were generated on the C57Bl/6 background, and littermate C57Bl/6 Il18+/+ male mice were used as controls. To reveal the direct effect of IL-18, primary cell cultures derived from both mice were established. Moreover, for molecular analysis, microarray, quantitative reverse transcription PCR and western blotting were performed using 6 and 12 weeks old mice. To evaluate the short- and long-term effects of IL-18 on BAT, recombinant IL-18 was administered for 2 and 12 weeks, respectively. Results Compared with Il18+/+ mice, BAT of Il18−/− mice showed earlier differentiation and lipid accumulation. To examine the direct effect of IL-18 on BAT, BA cell cultures were established. Myogenic factor 5-expressing adipose precursor cells were extracted from Il18+/+ and Il18−/− mice. PR domain containing 16 (PRDM16), a differentiation inducer, was strongly expressed in Il18−/− BAs, and uncoupling protein 1, a thermogenic and differentiation marker, was upregulated, resulting in the promotion of BA differentiation. Moreover, PRDM16-dependent and independent molecules related to BAT function, such as fibroblast growth factor 21, were activated. These findings were confirmed by comparing Il18+/+ and Il18−/− mice at 6 and 12 weeks of age. Additional analyses of the molecular mechanisms influencing the ‘Quantity of adipocytes’ identified three associated genes, apolipoprotein C3 (Apoc3), insulin-induced gene 1 (Insig1) and vitamin D (1,25-dihydroxyvitamin D3) receptor (Vdr). Intravenous administration of IL-18 not only significantly improved the expression of some of these genes, but it also significantly decreased the adipocytes’ size. Conclusions This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance. Electronic supplementary material The online version of this article (10.1186/s12967-018-1684-3) contains supplementary material, which is available to authorized users.

Pacinian corpuscles are vibration-sensing mechanoreceptors that are densely distributed in the dermis of the human hand. Although they are also known to occur in various other regions/structures throughout the human body, including the adventitia of large vessels, their precise distribution and function in arteries remain unclear. In the present study, we identified Pacini-like lamellar corpuscles (LCs) adjacent to the femoral artery, and investigated their distribution with respect to that structure via a histological analysis. We identified nine LCs that were localized in the connective tissue surrounding the femoral artery and vein. We showed that although their distribution was heterogeneous, they were predominantly concentrated on the dorsal side of the femoral artery. Immunohistochemical analyses revealed that the identified femoral artery LCs exhibited features characteristic of typical LCs located in the dermis of the index finger. Thus, the results of the present study contribute to an improved understanding of the function of femoral artery LCs. Anat Rec, 301:1809-1814, 2018. © 2018 Wiley Periodicals, Inc.

A network of myelinated nerve fibers in the peritoneum covers the abdominal wall. We studied the topographic distribution of this network, explored the fibers' destination in the central nervous system, and examined the markers in these fibers in order to identify the nature of the sensation conveyed by the network of nerve fibers in rats. We used Sihler's method, which stains myelinated fibers in whole mount materials, and observed a dense nerve network and endings toward the peritoneal cavity in the peritoneum that covers the abdomen's lateral bulge. We studied the axonal transport of cholera toxin subunit B to investigate the central projections of this network in order to identify its function. After applying the tracer in the peritoneum, we observed many labeled terminals in the medial part of laminae 3-5 of the spinal cord. A small number of labeled terminals was observed in the dorsal nucleus of Clarke and gracile nucleus. Labeled somata were observed in the dorsal root ganglia (DRG). Most (96%) were larger than 35 μm. We performed immunohistochemistry of the abdominal wall, using antiserum against the 200-kD neurofilament (a marker for mechanosensory neurons). We observed many positive nerve fibers in the peritoneum. Because cell bodies in the DRG were large, their nerve terminals ended in the base of the dorsal horn, which is known to transmit proprioceptive information, and the network possesses the marker for mechanosensitive fibers; therefore, it appears that the myelinated nerve network conveys information about distension and/or contraction of the abdominal wall. Anat Rec, 300:1662-1669, 2017. © 2017 Wiley Periodicals, Inc.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine and an important mediator of peripheral inflammation and host immune response. IL-18 functions through its binding with the IL-18 receptor (IL-18R), which consists of two chains, an IL-18-binding α chain (IL-18Rα) and a signaling β chain. IL-18 and IL-18R are expressed in the brain; however, limited information is available on IL-18R expression and the role of IL-18 in neurosecretory cells. In the present study, we used immunohistochemical techniques to investigate the distribution of IL-18Rα and IL-18 in the hypothalamus of male mice and rats. IL-18Rα-positive and IL-18-positive perikarya and fibers were found scattered throughout the medial septal nucleus, the nuclei of the vertical and horizontal limbs of the diagonal band, the organum vasculosum of the laminae terminalis, the preoptic area, and the anterior hypothalamic area. It is well known that gonadotropin-releasing hormone (GnRH) neuronal somata and/or fibers are found in these regions. Therefore, we performed double-label immunofluorescence for IL-18Rα/IL-18 and GnRH. IL-18Rα was expressed in approximately 60% of GnRH-immunopositive perikarya, and IL-18 was distributed in all GnRH-immunopositive perikarya. These observations suggest that IL-18 exerts direct effects upon the GnRH neuron via IL-18Rα and acts on GnRH neurons through an autocrine or paracrine pathway.

Pacinian corpuscles are vibration-sensing mechanoreceptors that are densely distributed in the dermis of the human hand. Although they are also known to occur in various other regions/structures throughout the human body, including the adventitia of large vessels, their precise distribution and function in arteries remain unclear. In the present study, we identified Pacini-like lamellar corpuscles (LCs) adjacent to the femoral artery, and investigated their distribution with respect to that structure via a histological analysis. We identified nine LCs that were localized in the connective tissue surrounding the femoral artery and vein. We showed that although their distribution was heterogeneous, they were predominantly concentrated on the dorsal side of the femoral artery. Immunohistochemical analyses revealed that the identified femoral artery LCs exhibited features characteristic of typical LCs located in the dermis of the index finger. Thus, the results of the present study contribute to an improved understanding of the function of femoral artery LCs. Anat Rec, 301:1809-1814, 2018. © 2018 Wiley Periodicals, Inc.

Neurons influence renal function and help to regulate fluid homeostasis, blood pressure and ion excretion. Intercalated cells (ICCs) are distributed throughout the renal collecting ducts and help regulate acid/base equilibration. Because ICCs are located among principal cells, it has been difficult to determine the effects that efferent nerve fibers have on this cell population. In this study, we examined the expression of neurotransmitter receptors on the murine renal epithelial M-1 cell line. We found that M-1 cells express a2 and b2 adrenergic receptor mRNA and the b2 receptor protein. Further, b2 receptor-positive cells in the murine cortical collecting ducts also express AQP6, indicating that these cells are ICCs. M-1 cells were found to express m1, m4 and m5 muscarinic receptor mRNAs and the m1 receptor protein. Cells in the collecting ducts also express the m1 receptor protein, and some m1-positive cells express AQP6. Acetylcholinesterase was detected in cortical collecting duct cells. Interestingly, acetylcholinesterase-positive cells neighbored AQP6-positive cells, suggesting that principal cells may regulate the availability of acetylcholine. In conclusion, our data suggest that ICCs in murine renal collecting ducts may be regulated by the adrenergic and cholinergic systems.

We have examined whether calcitonin gene-related peptide-immunoreactive (CGRP-ir) neurons in the vagal and glossopharyngeal ganglia innervate the larynx. Many CGRP-ir neurons were located mostly in the superior glossopharyngeal–jugular ganglion complex that was fused the superior glossopharyngeal ganglion and the jugular ganglion in the cranial cavity. When Fluorogold was applied to the cut end of the superior laryngeal nerve (SLN) or the recurrent laryngeal nerve (RLN), many Fluorogold-labeled neurons were found in the superior glossopharyngeal–jugular ganglion complex and the nodose ganglion. Double-labeling for CGRP and Fluorogold showed that about 80% of Fluorogold-labeled neurons in the superior glossopharyngeal–jugular ganglion complex expressed CGRP-like immunoreactivity in the case of application to the SLN, and about 50% of Fluorogold-labeled neurons expressed CGRP-like immunoreactivity in the case of the RLN. Only a few double-labeled neurons were found in the nodose ganglion. The number of the Fluorogold-labeled neurons and double-labeled neurons in the superior glossopharyngeal–jugular ganglion complex in the case of the SLN was larger than that in the case of the RLN. These results indicate that sensory information from the larynx might be conveyed by many CGRP-ir neurons located in the superior glossopharyngeal–jugular ganglion complex by way of the SLN and the RLN.

A network of myelinated nerve fibers in the peritoneum covers the abdominal wall. We studied the topographic distribution of this network, explored the fibers' destination in the central nervous system, and examined the markers in these fibers in order to identify the nature of the sensation conveyed by the network of nerve fibers in rats. We used Sihler's method, which stains myelinated fibers in whole mount materials, and observed a dense nerve network and endings toward the peritoneal cavity in the peritoneum that covers the abdomen's lateral bulge. We studied the axonal transport of cholera toxin subunit B to investigate the central projections of this network in order to identify its function. After applying the tracer in the peritoneum, we observed many labeled terminals in the medial part of laminae 3-5 of the spinal cord. A small number of labeled terminals was observed in the dorsal nucleus of Clarke and gracile nucleus. Labeled somata were observed in the dorsal root ganglia (DRG). Most (96%) were larger than 35 μm. We performed immunohistochemistry of the abdominal wall, using antiserum against the 200-kD neurofilament (a marker for mechanosensory neurons). We observed many positive nerve fibers in the peritoneum. Because cell bodies in the DRG were large, their nerve terminals ended in the base of the dorsal horn, which is known to transmit proprioceptive information, and the network possesses the marker for mechanosensitive fibers; therefore, it appears that the myelinated nerve network conveys information about distension and/or contraction of the abdominal wall. Anat Rec, 300:1662-1669, 2017. © 2017 Wiley Periodicals, Inc.

We have determined the localization of calcitonin gene-related peptide-immunoreactive (CGRP-ir) and calretinin-ir neurons in the vagal ganglia that innervate the cervical or subdiaphragmatic esophagus. Many CGRP-ir neurons were found exclusively in the jugular ganglion located in the cranial cavity. Calretinin-ir neurons were distributed throughout the vagal ganglia. Injection of Fluorogold into the cervical esophagus resulted in many Fluorogold-labeled neurons in the jugular and nodose ganglia. Injection of Fluorogold into the subdiaphragmatic esophagus resulted in many Fluorogold-labeled neurons, with most in the nodose ganglion. In the case of Fluorogold injection into the cervical esophagus, double-labeling combining immunohistochemistry and retrograde tracing showed that about 40% of the Fluorogold-labeled neurons in the jugular ganglion express CGRP-like immunoreactivity, and about 20% of the Fluorogold-labeled neurons in both the jugular and nodose ganglia express calretinin-like immunoreactivity. In the case of injection into the subdiaphragmatic esophagus, only a few Fluorogold-labeled neurons express CGRP-like immunoreactivity or calretinin-like immunoreactivity in the vagal ganglia. These results indicate that the cervical esophagus receives projections from many CGRP-ir neurons in the jugular ganglion and from calretinin-ir neurons in the jugular and nodose ganglia, while the subdiaphragmatic esophagus receives projections from only a few CGRP-ir and calretinin-ir neurons in the vagal ganglia.

Aims Acetylcholine (ACh) has been shown to increase ion and water excretion in the kidneys, resulting in hypotension. However, no evidence of renal parasympathetic innervation has been shown, and the source of ACh acting on nephrons is still unknown. The aim of the present study was to identify ACh-producing cells in the rat kidney, by examining the expression of cholinergic agents and localization of an ACh-synthesizing enzyme, choline acetyltransferase (ChAT), in the kidney. Main methods Adult mail Sprague–Dawley rats were used in this study. Expression of mRNA of cholinergic agents, ChAT, vesicular ACh transporter (VAChT), and high-affinity choline transporter (CHT-1), in the kidney was examined by RT-PCR. Localization of ChAT mRNA and protein was examined by in situ hybridization and tyramide-enhanced immunohistochemistry, respectively. Key findings RT-PCR showed the expression of ChAT, VAChT, and CHT-1. In situ hybridization demonstrated that ChAT mRNA is localized to the renal cortical collecting ducts (CCD). Immunohistochemistry showed that the ChAT-positive cells were principal cells, and that they were unevenly distributed in the tubules, and constituted approximately 15.2% of CCD in the cortex, and 3.6% and 1.5% in the outer and inner medulla, respectively. ChAT-positive immunoreactivity was localized to the apical side of principal cells, suggesting that ACh synthesis may occur in the apical compartment of these cells. Significance These results suggest that the cholinergic effects in the nephron may be mediated at least in part by ACh originating from CCD principal cells and its expression may be locally regulated in the rat kidney.

We have studied the connections of calcitonin gene-related peptide immunoreactive (CGRP-ir) sensory neurons in the ganglia of the vagus nerve. Many CGRP-ir neurons were identified in the jugular ganglion located in the cranial cavity, while fewer CGRP-ir neurons were found in the nodose ganglion located at the level of the jugular foramen. Application of Fluorogold to the cut end of the cervical vagus nerve resulted in many Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Application of Fluorogold to the cut end of the subdiaphragmatic vagus nerve resulted in Fluorogold-labeled neurons mostly in the nodose ganglion with only a few labeled neurons in the jugular ganglion. Injection of Fluorogold into the heart resulted in Fluorogold-labeled neurons in both the jugular and the nodose ganglia. Double labeling combining CGRP immunohistochemistry and Fluorogold retrograde tracing showed that in cases of both the application of Fluorogold to the cut end of the cervical vagus nerve and the injection of Fluorogold into the heart, about 40% of the Fluorogold-labeled neurons in the jugular ganglion expressed CGRP-like immunoreactivity. These results indicate that many CGRP-ir neurons in the jugular ganglion innervate the cervical and thoracic visceral organs, including the heart, but only a few CGRP-ir neurons project to the abdominal visceral organs.

To clarify the origin of efferent nerves containing renal plexus, the retrograde neuronal tracing was utilized with a new exact closed injection system with microcapsules. The microcapsule was positioned in the rat left renal plexus, and the capsule was filled with fluoro-gold. Retrograde labeled cells were observed in the ipsilateral sympathetic trunk, especially T12 and T13, and the ipsilateral suprarenal ganglia (SrG). There were no labeled cells in the parasympathetic nuclei in medulla oblongata and sacral cords. These results indicated that the origins of efferent nerves in the rat renal plexus are almost all sympathetic ganglia, such as sympathetic trunk and SrG, and cells in other ganglia may be secondary or accessory innervations.

We have examined whether calcitonin gene-related peptide immunoreactive (CGRP-ir) neurons in the glossopharyngeal ganglia innervate the soft palate, the root of tongue, and the pharynx of the rat. Immunohistochemical observations revealed that numerous CGRP-ir neurons are located in the superior glossopharyngeal ganglion located ventrolateral to the medulla oblongata in the cranial cavity, and that CGRP-ir neurons are also located in the inferior glossopharyngeal ganglion at the jugular foramen. When Fluorogold was injected into the soft palate, the root of tongue, or the pharyngeal constrictor muscles, many retrogradely Fluorogold-labeled neurons were found in the superior glossopharyngeal ganglion and the nodose ganglion, and several Fluorogold-labeled neurons were found in the inferior glossopharyngeal ganglion. Double labeling with immunohistochemistry for CGRP and Fluorogold showed that in every case of injections of Fluorogold into the soft palate, the root of tongue, or the pharynx, about 30% of the Fluorogold-labeled neurons in the superior glossopharyngeal ganglion expressed CGRP-like immunoreactivity, while no double-labeled neurons were found in the inferior glossopharyngeal ganglion or the nodose ganglion. These results indicate that nociceptive sensory information from the soft palate, the root of tongue, and the pharynx might be conveyed by the neurons in the superior glossopharyngeal ganglion to the nucleus tractus solitarii.

Aquaporin-4 (AQP4) is an isoform of membrane water channel aquaporins. It is most abundant in the brain, and is believed to be expressed by astrocytes and ependymal cells. We have reported previously that rat pituitary glands express some aquaporin subtypes, including AQP4, but the precise distribution of AQP4 in the pituitary gland is not known. The present work investigated whether AQP4 immunoreactivity exists in various pituitary cell types in adult rats, using double immunofluorescent staining and confocal microscopy. In the adenohypophysis, co-labeling for S-100 protein indicated that folliculo-stellate (FS) cells and marginal layer cells in Rathke's residual pouch have extensive labeling for AQP4. Some AQP4-expressing cells also contained glial fibrillary acidic protein (GFAP) in parenchymal tissue of the anterior lobe, and cytokeratin in marginal layer cells. AQP4 was not coexpressed in any hormone-producing pituitary cells, however. In the neurohypophysis, AQP4 was expressed in some pituicytes, which have been identified as GFAP-positive. These results suggest that AQP4, which is expressed in FS cells, in marginal layer cells and in pituicytes, but not in hormone-producing cells, is important in water transfer within the pituitary gland; astrocytes and ependymal cells do the same in the brain.

The aim of this study was to investigate the antitumor efficacy of treatment, identify prognostic factors, and construct a prognostic index in patients with hepatocellular carcinoma treated by transcatheter arterial infusion chemotherapy (TAI) using cisplatin suspended in lipiodol.We analyzed the outcomes in a total of 94 consecutive patients with previously untreated hepatocellular carcinoma who were treated by TAI using cisplatin suspended in lipiodol.Twenty-seven patients (29%) showed complete response and 21 patients (22%) showed partial response, with an overall response rate of 51% (95% confidence interval, 41-61%). The median survival time was 2.5 years and the proportions of survivors at 1, 2, and 5 years were 81.6, 65.2, and 18.3%, respectively. The results of multivariate analysis indicated a significant association of serum albuminor =3.0 g/dL, maximum tumor sizeor =3.0 cm, absence of ascites, and unilateral distribution of the tumors with a favorable survival. For clinical application, we also propose a prognostic index based on a combination of these prognostic factors. Based on this index, the patients were classified into three groups: those with good, intermediate, and poor prognosis. The median survival times in these three groups were 4.3, 2.7, and 1.1 years, respectively (p0.01).TAI with cisplatin suspended in lipiodol exhibited favorable tumor efficacy and survival in patients with hepatocellular carcinoma. The prognostic factors identified and the index proposed based on these factors may be useful for predicting life expectancy, determining treatment strategies, and designing future clinical trials.

The stomach receives sympathetic projections from the celiac ganglion. To determine what kinds of neurons in the celiac ganglion project to the cardia or the pylorus of the stomach, we injected the retrograde tracer Fluoro-Gold into the cardia and the retrograde tracer cholera toxin subunit b into the pylorus of the same animal. A few neurons (about 10%) innervating the cardia sent collateral projections to the pylorus. Ultrastructural observations revealed that the celiac ganglion contained oval, medium-sized to large neurons. They had a dark cytoplasm containing numerous free ribosomes, rough endoplasmic reticulum, mitochondria, lysosomes, several Golgi apparatuses, and an oval nucleus. The axon terminals were small and usually contacted thin processes extending from the dendrites or the soma. About half of the terminals contained round vesicles, while the rest contained pleomorphic vesicles. Both types of terminals made asymmetric synaptic contacts. We then retrogradely labeled the neurons projecting to the cardia and the pylorus with wheat germ agglutinin conjugated horseradish peroxidase to examine their ultrastructural characteristics. The neurons projecting to the cardia (33.3x22.4 microm) were similar to the neurons projecting to the pylorus (33.4x24.7 microm) in their size and ultrastructural appearance. The neurons not projecting to the stomach (40.4x28.0 microm) were significantly larger than the neurons projecting to the cardia or the pylorus. Only a few axosomatic terminals were found on the neurons projecting to the cardia (1.6 per somatic profile), the pylorus (1.3) or the neurons not projecting to the stomach (0.9). These results provide morphological bases for the sympathetic motor neurons innervating the stomach.

We investigated the expression of aquaporin (AQP) subtypes (AQP1-AQP9) in the rat pituitary gland by the reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Specific amplification by RT-PCR was observed in AQP1, AQP3, AQP4 and AQP5 but not in the others. AQP1 immunoreactivity was detected in the endothelial cells of blood vessels. No positive reaction to AQP3 was observed by immunohistochemistry, in spite of the detection of mRNA. AQP4 was localized in the supporting cells of the adenohypophysis and neurohypophysis. AQP5 was expressed in parts of the marginal cells in Rathke's residual pouch. These observations suggest that several subtypes of the AQP family are involved in regulation of water homeostasis in the rat pituitary gland.

We investigated potential pathophysiological relationships between interleukin 18 (IL-18) and dyslipidemia, nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH). Compared with Il18(+/+) mice, IL-18 knockout (Il18(-/-)) mice developed hypercholesterolemia and hyper-high-density-lipoprotein-cholesterolemia as well as hypertriglyceridemia as they aged, and these disorders occurred before the manifestation of obesity and might cause secondary NASH. The analyses of molecular mechanisms involved in the onset of dyslipidemia, NAFLD, and NASH in Il18(-/-) mice identified a number of genes associated with these metabolic diseases. In addition, molecules related to circadian rhythm might affect these extracted genes. The intravenous administration of recombinant IL-18 significantly improved dyslipidemia, inhibited the body weight gain of Il18(+/+) mice, and prevented the onset of NASH. The expression of genes related to these dysfunctions was also affected by recombinant IL-18 administration. In conclusion, this study demonstrated the critical function of IL-18 in lipid metabolism and these findings might contribute to the progress of novel treatments for NAFLD or NASH.

The motility of the alimentary canal is regulated not only by neurons that contain acetylcholine or adrenaline, but also by nonadrenergic noncholinergic neurons. There are many neurons containing dopamine in the dorsal motor nucleus of the vagus (DMV). We examined the projections of these dopaminergic neurons to the alimentary canal with double-labeling immunohistochemistry for tyrosine hydroxylase (TH) and the retrograde tracer cholera toxin subunit b following its injection into the subdiaphragmatic esophagus, the cardia, the pylorus, the duodenum, the jejunum, and the ascending colon. Almost all double-labeled neurons were found in the half of the DMV caudal to the area postrema. In the caudal half of the DMV, about 58% of the TH-immunoreactive neurons projected to the cardia, about 36% projected to the pylorus, and about 28% projected to the subdiaphragmatic esophagus. Only a few TH-immunoreactive neurons projected to the duodenum, the jejunum, or the ascending colon. As a whole, less than 10% of the neurons in the DMV that projected to the alimentary canal showed TH-like immunoreactivity. These results suggest that some of the dopaminergic neurons in the DMV might regulate the activities of the stomach and the subdiaphragmatic esophagus.

The intermediate subnucleus of the nucleus tractus solitarii (imNTS) receives somatosensory inputs from the soft palate and pharynx, and projects onto the nucleus ambiguus, thus serving as a relay nucleus for swallowing. The ultrastructure and synaptology of the rat imNTS, and its glossopharyngeal afferent terminals, have been examined with cholera toxin-conjugated horseradish peroxidase (CT-HRP) as an anterograde tracer. The imNTS contained oval or ellipsoid-shaped, small to medium-sized neurons (18.2 x 11.4 microm) with little cytoplasm, few cell organelles and an irregularly shaped nucleus. The cytoplasm often contained one or two nucleolus-like stigmoid bodies. The average number of axosomatic terminals was 1.8 per profile. About 83% of them contained round vesicles and formed asymmetric synaptic contacts (Gray's type I), while about 17% contained pleomorphic vesicles and formed symmetric synaptic contacts (Gray's type II). The neuropil contained small or large axodendritic terminals, and about 92% of them were Gray's type I. When CT-HRP was injected into the nodose ganglion, many labeled terminals were found in the imNTS. All anterogradely labeled terminals contacted dendrites but not somata. The labeled terminals were usually large (2.69+/-0.09 mum) and exclusively of Gray's type I. They often contacted more than two dendrites, were covered with glial processes, and formed synaptic glomeruli. A small unlabeled terminal occasionally made an asymmetric synaptic contact with a large labeled terminal. The large glossopharyngeal afferent terminals and the neurons containing stigmoid bodies characterized the imNTS neurons that received pharyngeal afferents.

The high emetic response (HER) strain and low emetic response (LER) strain of musk shrews (Suncus murinus) markedly differ in the emetic reflex in adults. However, there have been no studies on young musk shrews. We gave a shaking stimulus to young musk shrews aged 10 days or more that were obtained by mating within each strain and observed emetic responses. In the HER strain, no animal aged 10 days vomited, but vomiting was observed in 1 of 5 animals each aged 12 and 14 days, 2 of 5 animals aged 16 days, and all animals aged 18 days or more. In the LER strain, no vomiting was observed until the age of 14 days, but at the age of 16 days or more, 1 or 2 of 5 animals at each age vomited. After stimulation, activated neurons of the dorsal vagal complex and the dorsal reticular formation of the nucleus ambiguus (Amb) were examined by Fos immunohistochemistry. This morphometric study demonstrated that the numbers of Fos-positive neurons in the nucleus of the solitary tract and the dorsal reticular formation of the Amb were significantly larger in the animals that vomited in the HER strain than animals that did not vomit in the LER strain. We suggest that neurons in these regions are involved in emetic responses, as is the case in adult animals.

Objective: This study compared the antitumor effect, adverse effects and survival between transcatheter arterial embolization (TAE) and transcatheter arterial infusion chemotherapy (TAI) in patients with hepatocellular carcinoma (HCC). Methods: The study population consisted of 168 consecutive patients with advanced HCC treated with transcatheter arterial treatments using cisplatin suspended in lipiodol. Among these, 74 patients were treated with TAE, and the remaining 94 patients were treated with TAI. Results: There were no significant differences in any baseline characteristics except hemoglobin, platelets, albumin, and glutamic pyruvic transaminase. Complete or partial tumor response was achieved in 54 patients (73%) in the TAE group and in 48 patients (51%) in the TAI group (p < 0.01). There were two treatment-related deaths caused by acute hepatic failure and acute renal failure in the TAE group. Nausea and deterioration of serum transaminase after TAE were significantly more severe than after TAI. Median survival time and survival rates at 5 years were 3.1 years and 25% in the TAE group, and 2.5 years and 18% in the TAI group (p = 0.37). Conclusion: TAE has a higher antitumor effect than TAI, but does not significantly improve the survival of patients with HCC.

The central subnucleus of the nucleus tractus solitarii (ceNTS) receives afferent projections from the esophageal wall and projects to the nucleus ambiguus, thus serving as a relay nucleus for peristalsis of the esophagus. Here we examine the synaptic organization of the ceNTS, and its esophageal afferents by using transganglionic anterograde transport of cholera toxin-conjugated horseradish peroxidase (CT-HRP). When CT-HRP was injected into the subdiaphragmatic esophagus, many anterogradely labeled terminals were found only in the ceNTS. The ceNTS was composed of round or oval-shaped, small neurons (14.7x8.7 micro m) containing sparse organelles and an irregularly shaped nucleus. The average number of axosomatic terminals was only 1.3 per section cut through the nucleolus. Most of them (92%) contained round vesicles and formed asymmetric synaptic contacts (Gray's type I), and a few (8%) contained pleomorphic vesicles and formed symmetric synaptic contacts (Gray's type II). All anterogradely labeled terminals contacted dendrites but not the neuronal somata. The labeled terminals were large (2.55+/-0.07 micro m) and exclusively Gray's type I. More than half of them (60%) contacted small dendrites (less than 1 micro m in diameter), and contained dense-cored vesicles. More than 40% of the labeled terminals contacted two to four dendrites, thus forming a synaptic glomerulus. Sometimes a labeled terminal that contacted an unlabeled terminal by an adherent junction was found within the glomerulus. The large terminals and these complex synaptic relations appeared to characterize the esophageal afferent projections in the ceNTS.

The upper cervical esophagus is exerted on swallowing and peristalsis by somatic and visceral motoneurons, whereas the lower esophagus is exerted on only peristalsis by visceral motoneurons. We examined the origin of the esophageal motoneurons and whether there were any differences between the distributions of the upper and the lower esophageal motoneurons in the medulla and the spinal cord using cholera toxin subunit b (CTb) as the retrograde tracer. Following injection of CTb into the cervical esophagus resulted in heavy labeling of the neurons in the nucleus ambiguus including the compact (AmC), semicompact (AmS) and loose (AmL) formations, and the medial column of lamina IX at the C1-C5 levels of the cervical spinal cord corresponding to the spinal accessory nucleus. A few labeled neurons were found in the inferior salivatory nucleus, the rostral division of the dorsal motor nucleus of the vagus (DMX), the accessory facial nucleus and the lateral column of lamina IX at the C2 and C3 levels. All these labeled neurons showed ChAT immunoreactivity. When CTb was injected into the cut end of the unilateral recurrent laryngeal nerve, many labeled neurons were found in the ipsilateral AmC, the AmL, and the bilateral medial column at the C1 and C2 levels. Following injection of CTb into the subdiaphragmatic esophagus resulted in heavy labeling of the neurons only in the AmC and the DMX. When CTb was injected into the sternomastoid muscle, many labeled neurons were found in the medullary reticular formation, the facial nucleus, the medial column at the C1-C3, C5 and C6 levels, and the lateral column at the C2, C3, C5 and C6 levels. Injections of a Fluoro-Gold into the cervical esophagus and a CTb into the sternomastoid muscle or the subdiaphragmatic esophagus in the same animal showed many double labeled neurons in the medial column of the accessory nucleus at the C1 and C2 levels, but no double labeled neurons in the AmC. These results indicated that the upper cervical esophagus is innervated by the visceral medullary vagal motoneurons as well as the somatic spinal accessory motoneurons. The lower esophagus is innervated only by the visceral medullary vagal motoneurons.

We subcutaneously injected 0.5 mg/kg veratrine into the musk shrew (Suncus murinus), observed the presence or absence, latency, and the incidence of vomiting in each animal for 90 min, and selected animals that frequently vomited (FV group) and those that did not vomit (NV group). Subsequently, animal brains were removed, and the induction of c-fos protein (Fos) was immunohistochemically examined to evaluate neuronal activity in the medulla oblongata. The distribution of Fos-positive neurons in the medulla oblongata was similar between FV and NV groups, with numerous neurons along the entire length of the nucleus of the solitary tract and in the ventrolateral reticular formation. Both veratrine-injected groups showed higher numbers of positive neurons than the saline administered group. However, while the FV group showed a high concentration of positive neurons in the dorsal-dorsomedial reticular formation of the nucleus ambiguus in the rostral medulla, the NV group showed few positive neurons in this area. Fos activity in neurons in this area appeared to be higher in animals with a higher incidence of vomiting.

We previously reported three kinds of nitric oxide synthase-immunoreactive (NOS-ir) axons in the guinea pig heart: the sparse fiber network covering the right atrium, the basket-like endings around intracardiac neurons, and the axons in the septal region. The sparse NOS-ir nerve fiber network in the right atrium remained after vagotomy and has been suggested to be originated from intrinsic cardiac ganglia. Using Chorera toxin B as a retrograde tracer, we determined a part of them were derived from cardiac ganglionic neurons located in the area near the vena cavae.

The cricothyroid (CT) and the posterior cricoarytenoid (PCA) muscles in the larynx are activated by the laryngeal motoneurons located within the nucleus ambiguus; these motoneurons receive the laryngeal sensory information from the nucleus tractus solitarii (NTS) during respiration and swallowing. We investigated whether the neurons in the NTS projected directly to the laryngeal motoneurons, and what is the synaptic organization of their nerve terminals on the laryngeal motoneurons using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) was injected into the CT muscle or the PCA muscle, the anterogradely WGA-HRP-labeled terminals from the NTS were found to directly contact the retrogradely CT-HRP-labeled dendrites and soma of both the CT and the PCA motoneurons. The labeled NTS terminals comprised about 4% of the axosomatic terminals in a section through the CT motoneurons, and about 9% on both the small (PCA-A) and the large (PCA-B) PCA motoneurons. The number of labeled axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray's type I) was almost equal to that of the labeled terminals containing pleomorphic vesicles and making symmetric synaptic contacts (Gray's type II) on the CT motoneurons. The labeled axosomatic terminals were mostly Gray's type II on the PCA-A motoneurons, while the majority of them were Gray's type I on the PCA-B motoneurons. These results indicate that the laryngeal CT and PCA motoneurons receive a few direct excitatory and inhibitory inputs from the neurons in the NTS.

内視鏡的経鼻胆管ドレナージ (endoscopic naso-biliary drainage: ENBD) による胆管クーリング下経皮的マイクロ波凝固療法 (PMCT) を試行した. 症例は60歳の女性. 後区域グリソン鞘に近接した20×17mm の高分化型肝細胞癌を認めた. C型肝硬変を合併しており, 臨床病期はII期であった.ENBD チューブから冷却した生理食塩水の注入と吸引を繰り返すことにより胆管をクーリングした.1.6mmの深部針で60W, 30秒凝固の条件で5回のPMCT を試行した. 術後に一過性の高アミラーゼ血症を認めたが, 保存的に治癒した. 治療後の造影CTでは血管損傷を認めることなく, 腫瘍は周囲肝組織を含めて直径30mmの造影効果のない低吸収域となった. 胆管についてはMRCPで経過観察を行っているが, 狭窄や拡張を認めていない. 術後半年の現在, 無再発生存中である.本法は肝内グリソン鞘に近接した肝細胞癌治療における新しい選択肢となりうる.

A small ganglion, named the peri-trigeminal ganglion (PTG), was found in the ventromedial border of the rostral half of the trigeminal ganglion (TG) in the musk shrew (Suncus murinus). In frontal sections, the PTG was semicircular or elliptical in shape. Most of the neurons constituting this ganglion were round in shape and much smaller than those of the TG. The retrograde fluorescent tracer fluoro-gold was injected into various regions of the face in order to investigate innervation by the PTG neurons. When the tracer was injected subcutaneously around the external acoustic meatus and around the circumference of the orbit, a number of labeled neurons were seen not only in the TG but also in the PTG. After applying the tracer to the lacrimal gland (LG) and the harderian gland (HG), numerous labeled neurons were detected only in the PTG. A few labeled neurons were found in the PTG after injection into the palatoglossal arch. Immunohistochemically, most of the neurons constituting the PTG were positive for vasoactive intestinal polypeptide (VIP) antiserum. And a moderate number of somatostatin (SOM)-immunoreactive neurons and a small number of leucine-enkephalin (L-ENK)-immunoreactive neurons were detected. Numerous substance P-immunoreactive nerve fibers and varicosities were found in the PTG, and fewer L-ENK-, SOM- and VIP-immunoreactive fibers were observed. The present results suggest that the PTG is an autonomic ganglion that resembles in part the pterygopalatine ganglion in other species, and mainly innervates the HG and LG.

We measured adult mandibles of Ryukyu wild pigs from Amami-Oshima, Kakeroma, Okinawa, Ishigaki and Iriomote Islands. The size cline was not statistically recognized among the populations of Nansei Islands. The Ishigaki population was significantly larger than Iriomote one in mandible length. Some measurement ratios indicated that the ramus is enlarged laterally and that the body of mandible is dorso-ventrally developed in Ishigaki and Iriomote populations. The Amami-Oshima mandibles were relatively smaller than those of Okinawa and Iriomote populations in length item of body of mandible. These results will contribute to the zoo-archaeology on the historical change of size and shape in the island populations, and to the evolutionary biology on the morphological adaptation of this animal.

ニワトリ胚の始原生殖細胞(PGCs)を含む血液をウズラ胚血流への導入し,ニワトリPGCsを特異的に染色するモノクローナル抗体2C9およびウズラPGCsを特異的に染色するモノクローナル抗体QCR1を用いて,二重免疫組織染色により,それぞれのPGCsを識別した.Hamburger and Hamiltonのステージ14-16のウズラ胚からは0.5-2μlの血液を抜き取ることができたが,ステージ29における生殖腺中のウズラのPGCsを有意に減少させるには至らなかった.抜血後,ステージ13-14のニワトリPGCsを0.5-3μl導入したウズラ胚はステージ29において,生殖腺にニワトリPGCsが定住し,全体の5.6%を占めた.

Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatozoa. In the present study, to determine the expression pattern of MCS mRNA in the hamster testis, northern blot and in situ hybridization analyses using digoxigenin-labeled riboprobes for the hamster MCS were performed in the testes of 10-week-old golden hamsters. According to the northern blot analysis, hamster MCS was detected as a single transcript of about 1 kb in the testis. During spermatogenesis, the hamster MCS mRNA first appeared in step 6 spermatids, gradually increased in round spermatids during spermiogenesis, reached a peak in step 8 spermatids, and persisted a high level until step 13 spermatids. After step 14, the signal began to show a progressive decline in the spermatids and was weakly detected in the tails of step 17 spermatids. However, the signal was not observed in spermatogonia, spermatocytes, Sertoli cells, peritubular myoid cells, and interstitial cells. These findings indicate that hamster MCS is mainly related to the spermiogenesis during spermatogenesis.

We obtained three kinds of novel cytoplasmic dynein light-intermediate chain (LIC) isoforms from rat testis and brain by reverse transcription polymerase chain reaction (RT-PCR). The primers for RT-PCR were designed according to LIC-2 (LIC 53/55) (15). In one novel isoform, the 42 bp specific sequence named TDL was inserted between 1,106 and 1,107 nucleotides (nts) of LIC-2, whereas the 57 bp sequence corresponding to LIC-2 1,339-1,395 nts (BDL) was absent. The TDL and BDL regions were specifically digested with restriction enzymatic treatment and followed by subcloning of non-digested cDNA band in testis and brain, producing an isoform without TDL and BDL regions. BDL specific RT-PCR of testis cDNA followed by sequencing produced an isoform with two specific regions. By Northern blot hybridization using TDL and BDL specific antisense oligo DNA probe, 4.4, 3.5, and 2.0 kb of signals were detected. With both TDL and BDL probes, the 2.0 kb signal was intensely detected in testis, while the 4.4 kb was defected in brain. This indicates that TDL and BDL are derived from the same size of mRNAs. In situ hybridization method using these probes showed that all seminiferous epithelial cells, especially late pachytene spermatocytes, were positive, indicating that LIC 53/55 isoforms were coexpressed in these cells. These findings indicate that LIC 53/55 isoforms provide a variety of dynein subunits, and thus may regulate the dynein-dependent intracellular transport system.

ウズラ初期胚血流循環中の始原生殖細胞(PGCs)をニワトリ胚血流に導入した.ウズラとニワトリのPGCsを識別するために,3種のモノクローナル抗体,QCR1,QB2,2C9について,ニワトリおよびウズラ生殖細胞における免疫組織化学的染色性を調べた.QCR1,QB2はウズラ特異的であり,2C9はニワトリ特異的であった.さらに,3種とも発生段階特異的染色性を示した.ウズラPGCsを導入したニワトリ胚をHamburger and Hamiltonのステージ29で調べたところ,生殖腺にウズラPGCsが定住しており,全体の14.2%を占めた.ウズラPGCsを導入したニワトリ雄ヒナ精巣に外来のウズラおよび内在のニワトリの精原細胞の2つの集団が観察された.

The monoclonal antibody (MAb), named TSd-1, specific to spermatogenic cells of the common tree shrew (Tupaia glis) was established and characterized using immunohistochemistry and immunoblotting. MAb TSd-1 reacted with elongating and matured spermatids in a stage-dependent manner. TSd-1 recognized a 94 kilodalton (kDa) peptide in the plasma membrane and cytosol. Additionally, an extremely weak 107 kDa band was detected only in the cytosol. The reactions were not detected in round spermatids. In elongating Stage VI spermatids, the plasma membrane and the granular structure within the cytoplasm were intensely positive, and most intense after the appearance of new round spermatids in the lower layer (Stage I). The reactions were observed neither in the other organs of the common tree shrew nor in the testes of other animals, indicating that TSd-1 antigen is specific to the spermatogenic cells of the common tree shrew, and may act on elongating or matured spermatids.

The vagal motor neurons project to the gastrointestinal tract by way of the gastric, celiac and hepatic branches of the vagus trunk. We have examined whether single neurons in the dorsal motor nucleus of the vagus nerve (DMV) have collateral projections to the stomach, the duodenum and the intestines using a double-labeling tracing method. Following application of Fluorogold to the cut end of the accessory celiac branch and injection of cholera toxin subunit b (CTb) into the body of stomach, many Fluorogold- and CTb-labeled neurons were found throughout the DMV. Most CTb-labeled neurons (about 90%) were also labeled with Fluorogold. When Fluorogold was applied to the cut end of the accessory celiac or the gastric branch and CTb was injected into the duodenum, many Fluorogold-labeled neurons and CTb-labeled neurons were found in the DMV. About 20% of CTb-labeled neurons were also labeled with Fluorogold. These results indicate that many neurons in the DMV send collateral projections to both the stomach and the intestines innervated by way of the celiac branch. However, many neurons in the DMV projecting to the duodenum do not project to the stomach or the intestines caudal to the duodenum.

We investigated the effects of lipopolysaccharide (LPS)-induced endotoxemia on the expression of aquaporin-4 (AQP4) in the rat anterior pituitary gland, using the real-time polymerase chain reaction and immunohistochemistry. After intraperitoneal injection of LPS, the level of AQP4 mRNA doubled at 2, 4 and 8 hr. Immunohistochemical analysis showed an increase with time in AQP4 immunostaining in folliculo-stellate cells following LPS injection; the intensity of immunoreactivity peaked at 8 hr. At the same time, some cyst-like structures, formed by AQP4-positive cells, were observed. These findings indicate that LPS induces the expression of AQP4 in the anterior pituitary gland. The present results should provide an important key to elucidate the pathogenesis of the anterior pituitary gland during endotoxemia.

The classification of the cycle of the seminiferous epithelium was carried out in the common tree shrew (Tupaia glis). The tree shrew captured in Thailand were fixed with Bouin's fixative, embedded in paraffin wax, and stained with PAS-hematoxylin. The cycle was classified into twelve stages on the basis of the acrosomal changes of spermatids. Relative frequencies of stages form I to XII were 11.9, 7.2, 8.9, 22.5, 12.9, 9.7, 8.0, 5.9, 4.0, 3.2, 2.9, and 3.6%, respectively. Different stages did no appear in a cross-sectioned tubule as did in primates. The head of matured spermatid was discoidal in shape and different from that of primates and rodents. Spermatogenesis of the common tree shrew is different from that of primates and rodents according to its morphological features.

We established a mouse antiserum by immunizing BALB/c mice with sexually undifferentiated 6-day chick embryonic gonads. The antiserum had a higher affinity to the plasma membrane and cytoplasm of primordial germ cells (PGCs) than to the somatic cells in 6-day chick gonads. PGCs at this stage appear to have a higher specificity with more antigenic capacity than gonadal somatic cells; thus, the antiserum should be useful as a cell marker of chick PGCs. Subsequently, an immunohistochemical examination of PGCs settling in the genital ridges of chick embryos was carried out by the use of the antiserum. The reaction was clearly detected in the plasma membrane and cytoplasm of 5-day PGCs. After sexual differentiation, the reaction changed in distribution in females. At 8 days, it was limited to the perinuclear region of PGCs. In males, although the reaction persisted for longer period than in females, only a weak reaction was detectable at 9 days. These results suggest that the occurrence of periodic molecular changes may be different between male and female PGCs.

Cardiac myocytes in the left ventricle and atrium of the greater bandicoot rat (Bandicota indica) were examined by transmission electron microscopy. The fine structure showed typical features of mammalian cardiac myocytes. In atrial myocytes, however, the areas occupied by mitochondria were much smaller than that filled with myofibrils. The decrease in mitochondria and the abundance of myofibrils are thought to be an ultrastructural adaptation to the large body size of this species. Many conducting myocytes were observed in both atrium and ventricle. The atrial conducting myocytes were ultrastructurally different from the Purkinje fibers of the ventricle. We suggest that the abundance and the ultrastructural variation of conducting myocytes are related to the physiological aspects, such as body size, of this animal.

The distribution and the ultrastructure of afferent fibers innervating the parietal peritoneum in the rat was studied with immunohistochemistry using an antiserum against the neuronal marker protein gene product 9.5. The immunoreactive fibers were distributed throughout the peritoneum. They generally ran straight and parallel to the intercostal nerves running in the abdominal muscles underlying the peritoneum. They sometimes branched and terminated by forming club-like endings. The number of nerve endings on the peritoneal surface was 3.25 ± 1.66 mm(-2) . Electron microscopic observations revealed both unmyelinated and myelinated nerve fibers. The unmyelinated fibers were thin and about 1 μm in diameter. Their endings formed slight swellings located just inside the peritoneal cell layer. The myelinated fibers often formed a bundle that was composed of two or three nerve fibers. Each myelinated fiber kept in contact with a Shwann cell and projected toward the peritoneal cavity. Finally, they penetrated the peritoneal cell layer to reach the peritoneal cavity. These fibers then made contacts with the peritoneal cells and became free from the myelin sheath. The ending had a club-like shape covered with collagen fibers, and contained many neurofilaments, a few mitochondria, but no synaptic vesicles. These results suggest that since the sensory endings are exposed at the peritoneal cavity, the sensory fibers are highly sensitive to somatic or nociceptive stimuli.