Katsumi Miyahara

Correlations between both the expression levels of inflammatory mediators and growth factor in medial perimeniscal synovial tissue and the severity of medial knee osteoarthritis

An enhanced expression of the inflammatory mediators in the perimeniscal synovium in knee osteoarthritis (OA) has been suggested to contribute to progressive cartilage degeneration. However, whether the expression levels of these molecules correlated with the severity of OA still remained unclear. Medial perimeniscal synovial samples were obtained from 23 patients with Kellgren-Lawrence (K/L) grades 2 to 4 of medial knee OA. Immunohistochemical analysis of the synovium revealed that the MMP-1, COX-2 and IL-1β expression of the patients with K/L 4 to be significantly reduced in comparison to those with either K/L 2 or 3, while the TGF-β expression showed the opposite. The synovial expression of MMP-1 and IL-1β showed a significant negative correlation with the severity of OA, while that of TGF-β again showed the opposite. In conclusion, although synovial inflammation remained active, the MMP-1, COX-2 and IL-1β expression in synovium decreased depending upon the severity of OA, while the TGF-β expression increased.

Visualization of enteric neural crest cell migration in SOX10 transgenic mouse gut using time-lapse fluorescence imaging.

BACKGROUND Enteric neural crest cells (ENCCs) were labeled with VENUS, an enhanced green fluoroscein protein, to record their migration in genetically engineered transgenic (SOX10-VENUS) mice. MATERIALS AND METHODS Pregnant SOX10-VENUS mice were killed on day 12.5 of gestation. The colorectum was excised from each embryo (n = 20) and placed in tissue culture medium. Time-lapse images captured using fluorescence microscopy at 10-minute intervals for 3000 minutes were compiled into a video to display ENCC migration. RESULTS At 0 minutes, VENUS(+) ENCC were observed to be clustered in the cecum and proximal colon (vagal ENCC), and similar cells were also seen in the rectum/sacrum (sacral ENCC). After 500 minutes, vagal VENUS(+) ENCC had migrated caudally from the proximal colon to the midcolon, reaching the distal colon after 800 minutes. Sacral VENUS(+) ENCC had migrated rostrally and transversely by 1250 minutes and had integrated with vagal ENCC by 2500 minutes. CONCLUSION We recorded the actual rostral-to-caudal migration of vagal ENCC, caudal-to-rostral migration of sacral ENCC, and their integration in the developing mouse hindgut. Such direct evidence of ENCC migration may further elucidate understanding of ENCC development, thus providing insight into the histopathology of bowel dysmotility disorders.

Neuronal immaturity in normoganglionic colon from cases of Hirschsprung disease, anorectal malformation, and idiopathic constipation

AIM Immaturity of neurons in normoganglionic colon in Hirschsprung disease (HD), anorectal malformation (AM), idiopathic constipation (IC), and normal controls (C) was assessed using polysialyated neural cell adhesion molecule. METHODS Polysialyated neural cell adhesion molecule immunoreactivity in 3 sections of normoganglionic colon from HD (n = 48), AM (n = 25), IC (n = 36), and C (n = 18) were scored semiquantitatively according to age; 1 day to 11 months (G1), 1 to 4 years (G2), and 5 years and older (G3). RESULTS Neurons in all specimens appeared mature irrespective of age on hematoxylin-eosin stain. Polysialyated neural cell adhesion molecule was positive (immaturity) in all specimens during G1 (1.34 in HD, 1.60 in AM, 0.89 in IC, and 1.59 in C) and decreased significantly with age in C (0.34* for G2, 0.25* for G3; *P < .01), decreased after 4 years old in IC (0.93 for G2, 0.10(#) for G3; (#)P < .05), decreased gradually in AM (1.10 for G2, 0.75( section sign) for G3; ( section sign)P < .05), but remained strongly positive in HD (1.34 for G1, 1.26 for G2, and 1.21 for G3; P = not significant), which after 4 years was significantly higher than C (P < .05). CONCLUSION Postoperative colonic dysmotility may be because of persistence of immature neurons in HD and impaired maturation of neurons in AM and IC.

Abnormal enteric innervation identified without histopathologic staining in aganglionic colorectum from a mouse model of Hirschsprung's disease

PURPOSE The piebald lethal mouse with a deletion of endothelin-B receptor gene (EDNRB) is a model for Hirschsprungs disease (HD), whereas the SOX10 gene is vital for the development of intestinal neural crest-derived cells. Recently, we created a SOX10 transgenic mouse with intestinal neural crest-derived cells visible with enhanced green fluorescent protein (VENUS), that is, SOX10-VENUS(+)/EDNRB(sl/sl) to investigate intestinal innervation in HD. METHODS SOX10-VENUS(+)/EDNRB(sl/sl) (n = 30) were compared with wild-type littermates as controls (EDNRB(s/s), n = 30). Mice were killed on days 3, 7, or 12 of age. The entire colorectum was excised, fixed with 4% paraformaldehyde, and examined using fluorescence microscopy alone without staining. RESULTS In normoganglionic colorectum from controls, a grid network of nerve fibers/glial cells was visualized that connected smoothly with extrinsic nerve fibers running along the colorectal wall. In aganglionic colorectum from SOX10-VENUS(+)/EDNRB(sl/sl) mice, there was no grid network and more extrinsic nerve fibers than controls that invaded the colon wall becoming elongated with branching fibers. Normoganglionic colon from controls and SOX10-VENUS(+)/EDNRB(sl/sl) mice appeared the same. Innervation patterns did not change over time. CONCLUSION This is the first time for abnormal enteric innervation in aganglionic colon in a model for HD to be visualized without staining.

Transplantation of infantile bladder in rats: an alternative procedure for bladder augmentation.

Background. Our purpose was to evaluate whether bladder transplantation (BTx) can be used for bladder augmentation (BA). Methods. Bladders from infantile Brown-Norway rats (less than 21 days old) were excised and each transplanted into a pouch created in the distal omentum of a 6-week-old Lewis rat (fully allogeneic BTx). No immunosuppressant was used in group I (n=12). Intramuscular FK506 was used daily from the day of BTx in group II (n=16; 0.2 mg/kg), group III (n=22; 0.6 mg/kg), and group IV (n=16; 1.2 mg/kg) until harvesting 3, 4, 5, or 6 weeks after BTx. FK506 was used for only 2 weeks in group V (n=12; 0.6 mg/kg/day) and group VI (n=12; 1.2 mg/kg/day). Syngeneic bladder transplants acted as controls (n=16). Hematoxylin and eosin staining was used to examine all grafts. In six rats from group III, BA was performed by anastomosing the graft to the recipient bladder 10 days after BTx. Results. Each successfully transplanted graft appeared macroscopically as a thin-walled cyst. Rejection was seen in all grafts from groups I, II, V, and VI, and was minimal or absent in groups III and IV. On medium to long-term follow-up the only side effect of FK506 observed was reduced weight gain. Graft survival in the control group was 100%. BA was successful in all six cases, and the mucosa was normal throughout each augmented bladder. Conclusions. This is the first report of the successful transplantation of infantile tissue without vascular anastomosis. Because of the efficient, safe immunosuppression possible with FK506, our BTx technique could find clinical application for creating viable vesical tissue that could be used for BA.

BLADDER TRANSPLANTATION IN RATS USING FK-506

PURPOSE We created viable bladder tissue by transplantation with immunosuppression. MATERIALS AND METHODS For bladder transplantation the bladder of newborn Brown-Norway rats was excised and each was transplanted into a pouch created in the distal omentum of a 5-week-old Lewis rat. In 15 group 1 rats no immunosuppressive agent was used. In 20 group 2 rats 0.6 mg./kg. FK-506 daily were given intramuscularly until a predetermined day of harvest. Recipient rats were sacrificed on day 3, 5, 7 or 14 after bladder transplantation, and the bladder grafts were harvested and formalin fixed. Hematoxylin and eosin staining was done to examine bladder graft survival and the degree of rejection, and immunohistochemical testing was performed for assessing the vesical nervous system. In 5 rats in the control group bladder augmentation was performed by anastomosing the bladder graft to the native bladder. Each augmented bladder was harvested 21 days later for histopathological assessment. RESULTS Overall bladder graft survival was 96.4%. Each successfully transplanted bladder graft appeared macroscopically as a thin walled cyst. In group 1 all bladder grafts showed rejection with cellular infiltration. In group 2 there was mild rejection in 5 rats and no evidence of rejection in the remaining 15. All group 2 bladder grafts had intact nerve distribution. Bladder augmentation was successful in all 5 cases and the mucosa was normal throughout each augmented bladder. CONCLUSION Because FK-506 successfully prevents rejection, our technique would appear to have the potential for creating viable bladder tissue that may be used for bladder augmentation in cases of vesical exstrophy or neurogenic bladder.

Recipient non-hematopoietic bone marrow cells in the intestinal graft after fetal small intestinal transplantation.

We examined whether non-hematopoietic BM cells can migrate into the intestinal graft after fetal small intestinal transplantation (FSITx). Fetal small intestine from donor C57BL/6 mice was transplanted into the rectus abdominis of recipient C57BL/6 mice with only green fluorescent protein (GFP) BM cells (syngeneic FSITx). Intestinal grafts were harvested on days 5, 10, and 30 after FSITx and stained immunohistochemically using anti-CD45 antibody (a marker for hematopoietic BM cells). Although there were no GFP-positive cells identified in the epithelium of the graft intestinal villi, there were a few cells positive for both GFP and CD45 in the lamina propria on day 5 after FSITx, and many present on days 10 and 30. In some grafts there were only cells that were GFP positive/CD45 negative (i.e., non-hematopoietic BM cells) found in the lamina propria on days 10 and 30. These data indicate that non-hematopoietic BM cells as well as hematopoietic BM cells can migrate from the recipient’s bone marrow, suggesting that recipient mesenchymal stem cells may be strongly implicated in graft regeneration and development after FSITx.

Altered expression of laminin alpha1 in aganglionic colon of endothelin receptor-B null mouse model of Hirschsprung’s disease

PurposeLaminin, an extracellular matrix molecule, is essential for normal development of the nervous system. The alpha1 subunit of laminin-1 (LAMA1) has been reported to promote neurites and outgrowth and is expressed only during embryogenesis. Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize Enteric neural crest-derived cell (ENCC)s with a green fluorescent protein, Venus. We designed this study to investigate the expression of LAMA1 using Sox10-VENUS mice gut.MethodsWe harvested the gut on days 13.5 (E13.5) and 15.5 (E15.5) of gestation. Sox10-VENUS+/Ednrb−/− mice (n = 8) were compared with Sox10-VENUS+/Ednrb+/+ mice (n = 8) as controls. Gene expression of LAMA1 was analysed by real-time RT-PCR. Fluorescent immunohistochemistry was performed to assess protein distribution.ResultsThe relative mRNA expression levels of LAMA1 were significantly increased in HD in the proximal and distal colon on E15.5 compared to controls (p < 0.05), whereas there were no significant differences on E13.5. LAMA1 was expressed in the serosa, submucosa and basal lamina in the gut, and was markedly increased in the proximal and distal colon of HD on E15.5.ConclusionsAltered LAMA1 expression in the aganglionic region may contribute to impaired ENCC migration, resulting in HD. These data could help in understanding the pathophysiologic interactions between LAMA1 and ENCC migration.

Activating transcription factor 3 is not up-regulated in hypospadias patients in Japan

Background: The aetiology of hypospadias is largely uncharacterized. Some of the researchers have advocated that activating transcription factor 3 (ATF3), an oestrogen-responsive transcription factor, is up-regulated in patients with hypospadias. The purpose is to evaluate the universality of this fact; we studied the expression of ATF3 protein in prepuce tissue obtained from hypospadias and phimosis patients living in metropolitan Tokyo. Materials and Methods: Prepuce tissue was obtained from outer foreskin at the time of surgery, quickly prepared for paraffin-embedded sectioning and stained immunohistochemically for ATF3. Two researchers blindly evaluated immunoreactivity and scored it semi-quantitatively as nil = 0, weak = 1, or strong = 2, to give a final staining intensity score (SIS). Subjects were 18 hypospadias patients and 17 phimosis patients (as controls) who had surgery between January, 2009 and March, 2010. Results: All subjects lived in metropolitan Tokyo, Japan. Mean ages at surgery were 2.9 ± 1.0 and 3.9 ± 2.4 years, respectively (P > 0.05). SIS was not statistically different between hypospadias patients (1.4 ± 0.5) and controls (1.5 ± 0.5), (P > 0.05). Conclusions: Our data suggest that ATF3 is not highly associated with hypospadias in metropolitan Tokyo. Differences in ethnicity might have influenced our results.

Does Abnormal Expression of Acetylcholine Receptors in Hirschsprung's Disease Induce Acetylcholine Esterase Positive Nerve Fibres?

OBJECTIVE Alpha bungarotoxin (alpha-BTX) is a neurotoxin isolated from the venom of Bungarus multicinctus that binds specifically to the beta-subunits of nicotinic acetylcholine receptors (nAChR) on myotube membranes. The purpose of the present study was to investigate the distribution of alpha-BTX-sensitive nAChR in Hirschsprungs disease (HD) to understand the histopathological features of HD, especially the increase in acetylcholine esterase (AChE) positive nerve fibres. METHODS Confocal microscopy was used to study the expression of FITC (fluorescein isothiocyanate)-alpha-BTX, anti-synaptophysin (A-SY) antibody, and anti-neurofilament (A-NF) antibody to determine the distribution of nAChR and ganglion cell and nerve fibres in colon specimens from five cases of HD and three normal controls. RESULTS Quantitative assessment of the immunoreactivity of colonic muscle and colonic mucosal epithelium from an aganglionic segment of HD bowel demonstrated markedly increased nAChR compared with colonic muscle and colonic mucosal epithelium from a ganglionic segment of HD bowel and normal bowel (p < 0.0001, respectively), both of which have only a few positive nAChR. In colonic muscle from aganglionic and transitional segments of HD, there were many nAChR around hypertrophic nerve trunks identified by A-NF and A-SY staining. CONCLUSION We suggest that abnormal expression of nAChR in HD might be implicated in causing gastrointestinal dysmotility because of their localization around hypertrophic nerve trunks.