Yoshinao Z. Hosaka

Topography of ganglion cells and photoreceptors in the sheep retina

Retinal topographies of some cell types and distribution of the tapetum lucidum in the sheeps eye were investigated in this study. The tapetum was observed macroscopically in the fundus. The topographical distributions of retinal ganglion cells (RGCs), cones, and rods were simultaneously analyzed in retinal whole mounts stained with cresyl violet. Short‐wavelength‐sensitive (S) cones were immunocytochemically identified in retinal whole mounts. The tapetum was located dorsal to the optic disc, with the nasal part elongated horizontally and the temporal part expanded dorsally. RGCs were distributed densely in the area centralis, horizontal visual streak, and anakatabatic area. The highest density in the area centralis was approximately 18,000 RGCs/mm2. Cones showed high density in the horizontal area crossing the optic disc and dorsotemporal area, whereas rods showed high density in the horizontal area, which was greater in height than the horizontal area of high cone density. S cones showed high density in the dorsotemporal retina. The rod/cone ratios were high horizontally in the dorsal retina to the optic disc, with a mean value of 11:1. The cone/RGC and rod/RGC ratios were lower in the horizontal and dorsotemporal retina, and the rod/cone/RGC ratio was lowest in the area centralis (9:1:1). The retinal topographies and distribution of the tapetum were specialized in the horizontal and dorsotemporal fundus. This suggests that sheep have better visual acuity in horizontal and anteroinferior visual fields and that this specialization is related to the visual ecology of sheep. J. Comp. Neurol. 518:2305–2315, 2010.

Graded Arrangement of Collagen Fibrils in the Equine Superficial Digital Flexor Tendon

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.

Full-length transient receptor potential vanilloid 1 channels mediate calcium signals and possibly contribute to osmoreception in vasopressin neurones in the rat supraoptic nucleus

Neurones in the supraoptic nucleus (SON) of the hypothalamus possess intrinsic osmosensing mechanisms, which are lost in transient receptor potential vanilloid 1 (Trpv1)-knock-out mice. The molecular nature of the osmosensory mechanism in SON neurones is believed to be associated with the N-terminal splice variant of Trpv1, although their entire molecular structures have not been hitherto identified. In this study, we sought for TRPV1-related molecules and their function in the rat SON. We performed RT-PCR and immunohistochemistry to detect TRPV1-related molecules in the SON, and patch-clamp and imaging of the cytosolic Ca(2+) concentration ([Ca(2+)]i) to measure responses to osmolality changes and TRPV-related drugs in acutely dissociated SON neurones of rats. RT-PCR analysis revealed full-length Trpv1 and a new N-terminal splice variant, Trpv1_SON (LC008303) in the SON. Positive immunostaining was observed using an antibody against the N-terminal portion of TRPV1 in arginine vasopressin (AVP)-immunoreactive neurones, but not in oxytocin (OT)-immunoreactive neurones. Approximately 20% of SON neurones responded to mannitol (50 mM) with increased action potential firing, inward currents, and [Ca(2+)]i mobilization. Mannitol-induced responses were observed in AVP neurones isolated from AVP-eGFP transgenic rats and identified by GFP fluorescence, but not in OT neurones isolated from OT-mRFP transgenic rats and identified by RFP fluorescence. The mannitol-induced [Ca(2+)]i responses were reversibly blocked by the non-selective TRPV antagonist, ruthenium red (10 μM) and the TRPV1 antagonists, capsazepine (10 μM) and BCTC (10 μM). Although the TRPV1 agonist, capsaicin (100 nM) evoked no response at room temperature, it triggered cationic currents and [Ca(2+)]i elevation when the temperature was increased to 36°C. These results suggest that AVP neurones in the rat SON possess functional full-length TRPV1. Moreover, differences between the responses to capsaicin or hyperosmolality obtained in rat SON neurones and those obtained from dorsal root ganglion neurones or TRPV1-expressing cells indicate that the osmoreceptor expressed in the SON may be a heteromultimer in which TRPV1 is co-assembled with some other, yet unidentified, molecules.

Comparative study of the characteristics and properties of tendinocytes derived from three tendons in the equine forelimb.

The aim of this study was to determine the characteristic differences in tendinocytes derived from tendons in the equine forelimb, superficial digital flexor tendon (SDFT), deep digital flexor tendon (DDFT) and common digital extensor tendon (CDET), in morphology, proliferation, collagen production ability and ability for synthesis of matrix metalloproteinases (MMPs). Significant differences were observed in cell number in vivo. The cellular number was largest in the SDFT and smallest in the CDET. The values of in vitro proliferation ratios and ability for synthesis of collagen and MMPs were largest in the SDFT and smallest in the CDET. Addition of TNFalpha to culture of all three types of tendinocytes increased the synthesis of both proMMP-9 (except CDET) and collagen and decreased proMMP-13 synthesis and had no effect on proMMP-2 synthesis. Flexor tendons in forelimbs (SDFT and DDFT) restore energy during locomotion and are more easily injured than are extensor tendons. This structural property would cause active ECM and MMPs synthesis. And CDET have very low turnover potential; in the small number of cells, low cellular proliferation, lower ability for synthesis of collagen and MMPs. The isolated tendinocytes provided much information on the characteristics and properties of tendons for the ECM turnover system and responsiveness of tendinocytes to complex inflammatory responses in tendinopathy.

Downregulation of Decorin and Transforming Growth Factor-β1 by Decorin Gene Suppression in Tendinocytes

Scars formed after tendonitis result in altered tissue mechanical properties after injury. The interaction of collagen molecules with decorin affects collagen fibrogenesis, and scar tissue is fragile as a consequence of a large amount of decorin in the scar. We hypothesized that scar formation could be prevented by controlling decorin expression in tendinocytes. As a preliminary experiment, we treated tendinocytes with decorin antisense oligodeoxynucleotides (ODNs). Tendinocytes were isolated from Achilles tendons of New Zealand white rabbits and treated with ODN. When tendinocytes were transfected with decorin sense ODN, there was no alteration, whereas decorin antisense ODN-treated tendinocytes showed suppression of transforming growth factor (TGF)-β1 production. Decorin and TGF-β1-production of tendinocytes is regulated by decorin gene suppression. The results showed that the antisense approach is an attractive therapeutic strategy not only for preventing decorin deposition in scar tissue, which decreases collagen fibril diameter, but also for controlling TGF-β1 production, which leads to organ fibrosis.

Comparative study of muscle regeneration following cardiotoxin and glycerol injury.

In the present study, we examined muscle regeneration following two types of chemical injuries, cardiotoxin (CTX) and glycerol, in order to compare their effect on the morphological characteristics during muscle regeneration, in addition we studied the structural changes of the intramuscular connective tissue (IMCT) during the regeneration process, by scanning electron microscopy (SEM) after digestion of the cellular elements of the muscle with sodium hydroxide. Tibialis anterior (TA) muscles of adult male mice were injected either with CTX or glycerol. Muscle degeneration was greater in the CTX-injured model than in the glycerol-injured model at day 4 post injection. Muscle regeneration started at day 7 in both the CTX and glycerol models. However, the CTX-injured model showed a higher myotube density and larger myotube diameter than the glycerol-injured model at days 10 and 14 post injection. On other hand, adipocyte infiltration was detected in the glycerol-injured model. In contrast, no adipocytes could be detected in the CTX-injured model. Furthermore, ultrastructural analysis showed a significant difference in myofiber damage and regeneration between the two models. SEM of the IMCT showed a transient increase in endomysial collagen deposition at early stages of regeneration in the CTX-injured model. In contrast, glycerol-injured model showed slight endomysial collagen deposition. Our results suggest that changes in IMCT affect the efficiency of muscle regeneration. Studying the three dimensional structure of IMCT may help clinical therapies to reduce skeletal muscle fibrosis. To our knowledge this is the first time the changes in IMCT following CTX and glycerol injury using SEM-cell maceration technique have been compared.

Concerted and adaptive alignment of decorin dermatan sulfate filaments in the graded organization of collagen fibrils in the equine superficial digital flexor tendon

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small‐diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large‐diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small‐ and large‐diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface‐to‐surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center‐to‐center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT.

Tumor suppressor candidate TUSC3 expression during rat testis maturation.

Analysis of microarray data obtained by comparing gene expression between 2-week-old infant and 7-week-old mature SD rat testes revealed novel targets involved in tumor suppression. Reverse-transcription polymerase chain reaction and Northern blotting indicated that Tusc3 gene expression was upregulated in the normal maturing testis and prostate and other organs such as the cerebrum and ovary. Tumor suppressor candidate 3 protein expression was detected in these same organs at a size of about 40 kDa, in accord with the predicted molecular size. In situ hybridization and immunohistochemistry showed that mRNA and protein localization were prevalent in the testis spermatocytes and interstitial cells such as the Leydig cells, as well as prostate epithelial cells. These data suggest that TUSC3 is deeply involved in spermatogenesis in the testis, inducing sperm differentiation and maturation, and plays a role in normal prostate development and tumor suppression.

Efficacy of self-assembled hydrogels composed of positively or negatively charged peptides as scaffolds for cell culture.

KASEA16(+) and KASEA16(−) peptides, the net charges of which are positive and negative, respectively, under a neutral condition could undergo self-assembly into nanofibers and form transparent hydrogels without peptide aggregation upon rapid pH neutralization. The numbers of NIH3T3 cells attached to the KASEA16(+) hydrogel and KASEA16(−) hydrogel were similar, and cells proliferated with time on both hydrogels. Cells on the KASEA16(+) hydrogel had spindle-like morphology, while cells on the KASEA16(−) hydrogel formed clusters without extending cytoplasmic processes. Comparison of differently charged peptides under a neutral condition suggested that the charges of the scaffolds should be taken into consideration for the best design and selection of scaffolds for cell culture. Since the KASEA16(+) peptide could form a stable hydrogel under a neutral condition and the hydrogel served as a scaffold for cell proliferation, the KASEA16(+) hydrogel will be a useful scaffold for cell culture.

Histological analysis of glucagon-like peptide-1 receptor expression in chicken pancreas

Glucagon-like peptide-1 (GLP-1) released from intestinal L cells in response to nutrient ingestion inhibits both gastrointestinal emptying and gastric acid secretion and promotes satiety. The main biological effect of GLP-1 is the stimulation of insulin secretion (thereby fulfilling the criterion for an incretin hormone) in order to reduce blood glucose levels in mammalian species. Chicken GLP-1 receptor (cGLP-1R) has also been identified in various tissues by gene expression analysis. Although certain effects of GLP-1 in mammals and birds are consistent, e.g., inhibition of food intake, whether GLP-1 has the same insulinotropic activity in chickens as in mammals is debated. Moreover, the expression of cGLP-1R in chicken pancreatic B cells has not been reported. The localization of cGLP-1R and its mRNA in pancreatic islets is studied by triple-immunofluorescence microscopy and in situ hybridization. Triple-immunofluorescence microscopy with antisera against cGLP-1R, somatostatin and insulin or glucagon revealed that cGLP-1R protein was exclusively localized in D cells producing somatostatin in chicken pancreatic islets. The D cells were localized in peripheral areas of the pancreatic islets and cGLP-1R mRNA was detected in the same areas, indicating that cGLP-1R mRNA was also expressed in D cells. This is the first report to demonstrate that cGLP-1R is expressed by D cells, not B cells as in mammals. Our study suggests that chicken GLP-1 performs its insulinotropic activity by a different mode of action from that of the mammalian hormone.