A growing body of clinical cases suggests that a kind of nematode larva, type X larva of the suborder Spirurina that inhabits firefly squids ( Watasenia scintillans , or ‘Hotaru‐ika’ in Japanese), can cause acute ileus in humans. However, the larva itself has rarely been found in the wall of the obstructed intestine. We describe here a case of acute ileus, in which a type X spirurina larva was found histologically. A 60‐year‐old Japanese man suffered from acute abdomen, and an emergency laparotomy revealed a marked stenosis of the ileum. Histological study of the surgically resected ileum showed severe eosinophilic enteritis and a nematode larva. The morphological features of this larva were identical to those of the type X spirurina larva. Interestingly, the larva that was found existed within a small blood vessel, suggesting that the larva migrans of type X spirurina can take place via vasculature. The patient in the present case did not recall ingesting raw squids prior to the onset of his disease. Hence, this indicates that even if the ingestion of raw firefly squids is uncertain, spirurina infection should be included in the differential diagnosis of acute ileus or eosinophilic enteritis.

Proliferation and differentiation of ependymal cells in the injured carp spinal cord were studied by immunohistochemistry using proliferating cell nuclear antigen (PCNA) and H-3-thymidine (H-3-TdR) autoradiography. A surge of proliferation of ependymal cells occurred in the lesion with a peak around 6th day after surgical operation (complete transection) of the spinal cord. The proportion of PCNA-positive ependymal cells at 6th post-operative day (6 POD) was over 14 times that in the normal state. Electron microscopic autoradiography revealed that most of the ependymal cells incorporating H-3-TdR at 5 POD, especially those located in the caudal side of the transection site, contained numerous free ribosomes in their apical portion, but the other organellae, such as rough endoplasmic reticulum and Golgi apparatus, were poorly developed in this portion. In three weeks thereafter, the ependymal cell layer was reconstructed through the lesion, and the apical part of the H-3-TdR-labeled ependymal cells became elongated and morphologically differentiated: that is, it had free ribosomes decreased and glial filaments increased in number. Many bundles of regenerating axons were observed to course within the reconstructed ependymal cell layer. These results may suggest that proliferation, differentiation and reconstruction of the ependymal cell layer following injury of the carp spinal cord are requisite to make the permissive milieu for elongation of the regenerating axons.

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (ICa,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca2+ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5–E11.5) than late developmental stage (LDS, E16.5–E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, ICa,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca2+ influx via LTCCs on AC activity, leading to the inability to inhibit AC.

To identify how the gp130-signaling cytokine oncostatin M (OSM), acting alone or in concert with IL-1β or TNFα, affects synovial fibroblast expression of genes relevant to inflammation and bone erosion in inflammatory arthritis.Synovial fibroblasts (SFs) were isolated from non-arthritic wild type (WT) or OSM receptor deficient (OSMR−/−) mice and stimulated with OSM, IL-1β or TNFα and their combinations. Cytokine gene expression was assessed by quantitative RT–PCR. ELISA, flow cytometry and immunohistochemistry identified protein expression. Gene expression patterns were confirmed in SFs isolated from patients with osteoarthritis (OASFs) and rheumatoid arthritis (RASFs).Expression of OSM and its receptors, gp130, OSMR and LIFR, was increased in synovial tissue from the mouse antigen-induced arthritis model. In isolated WT mouse synovial fibroblasts OSM alone, or in synergy with IL-1β, or together with TNFα, potently induced expression of the pro-inflammatory cytokine IL-6. OSM also induced a sustained increase in mRNA levels of the pro-osteoclastic cytokine RANKL. Combining OSM with IL-1β, but not with TNFα, further increased RANKL expression. Importantly these effects of OSM were all dependent on the expression of OSMR. Furthermore, OSM also increased expression of its own receptors, gp130 and OSMR and the IL-1 receptor, IL1-R1; the latter effects were also observed in both human OASFs and RASFs.Together our data suggests that OSM signaling via OSMR in SFs has the potential to contribute significantly to joint destruction in inflammatory arthritis. It not only induces expression of pro-inflammatory and pro-osteoclastic cytokines but can also augment its own actions and that of IL-1 by inducing expression of OSMR and IL-1R1.

Effect of pituitary adenylate cyclase activating polypeptide (PACAP) on growth hormone (GH) release from GH3 cells was studied in a dynamic superfusion system. PACAP-38 and PACAP-27 stimulated GH release from superfused GH3 cells. The stimulatory effect of PACAP-38 was comparable to those of vasoactive intestinal polypeptide (VIP) and PACAP-27 at a concentration of 1 nM, but the duration of action was more prolonged in PACAP-38 than in the other two peptides. PACAP(6–38), a selective antagonist of PACAP, as well as a VIP antagonist blunted the GH release induced by PACAP-38 and VIP. An antagonist of GH-releasing factor (GRF) at a concentration of 1 μM, however, did not affect the GH release induced by PACAP-38. These findings suggest that PACAP and VIP stimulate GH release from GH3 cells through type II PACAP receptor but not through the GRF receptors.

Glial cells of the cerebellum originate from cells of the ventricular germinative layer, but their lineage has not been fully elucidated. For studying the glial cell lineage in vivo by retrovirus-mediated gene transfer, we introduced a marker retrovirus into the ventricular germinative layer of embryonic day 13 mice. In the resulting adult cerebella, virus-labeled glial cells were grouped in discrete clusters, and statistical analysis showed that these clusters represented clones in high probability. Of 71 of the virus-labeled glial clusters, 33 clusters were composed of astrocytes/Bergmann glia, 10 were composed of only white matter astrocytes, and 24 were composed of only oligodendrocytes. No glial clusters contained virus-labeled neurons. These results suggest that astrocytes/Bergmann glia, white matter astrocytes and oligodendrocytes immediately arise from separate glial precursors: these three glial lineages may diverge in the course of cerebellar development.

Granule cells of the mammalian cerebellar cortex originate from embryonic progenitors present in the ventricular germinal layer. To investigate the allocation fate of these ventricular progenitors in the mouse, we labeled a few of them on embryonic day 13 with a recombinant retrovirus carrying lacZ which encodes E. coli beta-galactosidase (beta-gal), and the labeled cells in the postnatal cerebellar cortex were detected by beta-gal histochemistry. In the postnatal cerebellar cortex, the virally-labeled beta-gal+ granule cells formed discrete clusters. These clusters were not compactly packed with the beta-gal+ cells, and there was intermingling with beta-gal- granule cells. Neither beta-gal+ Purkinje cells nor glia were found to be included in the clusters. Most of the granule cell clusters were incompatible with the functional areas of the cortex. These results suggest: (1) granule cells derived from individual ventricular progenitors are allocated in clusters and are not extensively dispersed, (2) granule cells descended from one progenitor may mix with their neighbors that are descended from another progenitor, (3) the allocation fate of the ventricular progenitors of granule cells is not restricted to the functional areas of the cerebellar cortex.

Astrocyte proliferation in the stab-wounded cerebral cortex of mice was studied using double immunohistochemistry for proliferating cell nuclear antigen (PCNA) and glial fibrillary acidic protein (GFAP). The number of GFAP-positive astrocytes increased markedly from day 0.5 to day 3 after stab wounding. Some GFAP-positive astrocytes in the immediate vicinity of the wound were found to be positive for PCNA. However, the maximum number of these double positive astrocytes was only 5-6% of the number of GFAP-positive astrocytes. This maximum value was observed on days 2.5 and 3. The present study revealed that astrocytes are able to reactively express PCNA, an intrinsic marker of DNA replication. On the other hand, it is suggested that the proliferation of astrocytes in the wounded cerebral cortex is limited, in contrast with their marked reactive up-regulation of GFAP.

Proliferation of microglia in the mouse cerebral cortex around a stab wound was studied, using thiamine pyrophosphatase (TPPase) histochemistry combined simultaneously with 3H-thymidine (3H-TdR) autoradiography. Many cells with cell membrane TPPase activity (TPPase-positive cells) were scattered in the cortical parenchyma apart from the stab wound. Light and electron microscopically, TPPasepositive cells were identified as microglia but not as astrocytes, oligodendrocytes, or neurons. TPPasepositive cells were immunohistochemically negative for astrocytic markers, i. e., S-100 protein and glial fibrillary acidic protein. Autoradiography showed that approximately 92% of all cells labeled with 3H-TdR within 2 days after stab-wounding were TPPase-positive cells. Approximately 60% of all labeled cells 3 and 4 days after stab-wounding were also TPPase-positive cells. These results suggest that microglia, but not astrocyte, begins active proliferation immediately after injury and is the major cell type having high proliferative activity in the injured cerebral cortex.

Reactions of glial cells after stab wounding of mouse cerebral cortex were studied by [3H]thymidine autoradiography combined with immunohistochemistry for S-100 protein. S-100-positive cells in the stabbed cortex had the light and electron microscopic characteristics of astrocytes, and they showed remarkable hypertrophic changes 4 to 5 days after stabbing. There were many cells labeled with [3H]thymidine in the stabbed cortex from 24 h to 8 days after stabbing, and the number of labeled cells was maximum at 48 h. A few of the labeled cells were S-100-positive, and the labeled S-100-positive cells were seen 24 h to 6 days after stabbing, mostly after 72-96 h. By successive injections of [3H]thymidine for 6 days after stabbing, about 90% of labeled cells were S-100-negative, and about 90% of S-100-positive cells were unlabeled with [3H]thymidine. The increase in number of S-100-positive cells by day 6 after stabbing was not statistically significant (P greater than 0.05). These results suggest that reactive proliferation of astrocytes is a minor phenomenon in gliosis of injured cerebral cortex, in contrast with their remarkable reactive hypertrophy.

Cell number and proliferation of reactive astrocytes were studied quantitatively in the stabbed cerebral cortex of adult mice, using immunohistochemistry for glial fibrillary acidic protein (GFAP) and [3H]thymidine autoradiography. GFAP-positive astrocytes increased in cell number gradually from 24 to 96 h after stabbing, and their immunoreactivity became intense. The maximum number of GFAP-positive cells was about 4.5 times normal in the layers II–VI of the cortex, whereas it was only 1.5 times normal in the layer I (molecular layer). In contrast to the gradual increase in cell number, no GFAP-positive astrocytes were labeled with [3H]thymidine prior to 48 h after stabbing, in either the layer I or the layers II–VI. Then 3–5% of them were labeled at 72 and 96 h, but very few again after 6 days. By injecting [3H]thymidine successively for 6 days after stabbing, only 17% of GFAP-positive astrocytes of the layer I or the layers II–VI were labeled. These results reveal that, in the cortical layers II–VI, many GFAP-negative source cells initially express much more GFAP-antigen without proliferation and change into GFAP-positive reactive astrocytes. Proliferation of reactive astrocytes is not the major factor for the marked increase in number of them. The cortical layer I would have few GFAP-negative source cells for reactive astrocytes. These source cells may be protoplasmic astrocytes.

In an attempt to analyse the kinetics of angiogenesis in the brain, we developed a new lectin-histochemical staining technique for identifying the vasculature. Three horseradish-peroxidase-conjugated lectins, i.e., Griffonia simplicifolia agglutinin 1 (GS1), Ricinus communis agglutinin 1 (RCA1) and soybean agglutinin (SBA), selectively stained vascular walls in brain-tissue sections. When these lectins were injected into the circulation of ether-anesthetized animals via the pulsating left ventricle, they bound specifically to the inner surface of endothelial cells and revealed the three-dimensional architecture of the vascular network within thick tissue preparations. When this technique, referred to a lectin angiography, was combined with 5-bromo-2-deoxyuridine (BudR) immunohistochemistry, proliferating capillary cells could be easily identified in three-dimensional structures of the developing vasculature. Because of its simplicity and wide applicability, lectin angiography should be useful for analysing the kinetics of angiogenesis in developmental, regenerative, and pathological conditions in various tissues and organs.

The ability to record transient cellular events in the DNA or RNA of cells would enable precise, large-scale analysis, selection, and reprogramming of heterogeneous cell populations. Here, we report a molecular technology for stable genetic tagging of cells that exhibit activity-related increases in intracellular calcium concentration (FLiCRE). We used FLiCRE to transcriptionally label activated neural ensembles in the nucleus accumbens of the mouse brain during brief stimulation of aversive inputs. Using single-cell RNA sequencing, we detected FLiCRE transcripts among the endogenous transcriptome, providing simultaneous readout of both cell-type and calcium activation history. We identified a cell type in the nucleus accumbens activated downstream of long-range excitatory projections. Taking advantage of FLiCRE’s modular design, we expressed an optogenetic channel selectively in this cell type and showed that direct recruitment of this otherwise genetically inaccessible population elicits behavioral aversion. The specificity and minute resolution of FLiCRE enables molecularly informed characterization, manipulation, and reprogramming of activated cellular ensembles.

We investigated the expression patterns of (1) intermediate filament proteins in the developing CNS according to the immunohistochemical and immunoblotting techniques and (2) glycosyl residues exposed on the cell membranes in the fetal brain using histochemical techniques.In the early neural tube which was composed of matrix cells (stage I), vimentin was found predominantly in the basal processes of these cells. After the beginning of neuron production (stage II), vimentin was present in “radial fibers”, guideropes for migrating neuroblasts, which could be regarded as elongated basal processes of matrix cells. In this stage of neurogenesis, neurofilament protein was detected in the processes running parallel to radial fibers. These processes seemed to be those of migrating neuroblasts. Glial fibrillary acidic protein was detected for the first time after the cessation of neuron production (stage III). These results indicate the absence of glial cells in the early stages of neurogenesis (stages I and II).The lectin-histochemistry of the fetal brain implied the presence of abundant terminal sialyl and N-acetylglucosaminyl residues on the plasma membranes of various cells and fibers. On the other hand, it was suggested that terminal galactosyl residues on radial fibers are much fewer in the corticogenetic region than in the non-corticogenetic place. This difference in the amount of terminal galactosyl residues could be related to the mechanism of corticogenesis. A possible role of galactosyltransferase in this mechanism was suspected.