Hajime Sawada

Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells.

Bone marrow stromal cells (MSCs) are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. We now demonstrate that MSCs can be induced to differentiate into cells with Schwann cell characteristics, capable of eliciting peripheral nervous system regeneration in adult rats. MSCs treated with beta‐mercaptoethanol followed by retinoic acid and cultured in the presence of forskolin, basic‐FGF, PDGF and heregulin, changed morphologically into cells resembling primary cultured Schwann cells and expressing p75, S‐100, GFAP and O4. The MSCs were genetically engineered by transduction with retrovirus encoding green fluorescent protein (GFP), and then differentiated by treatment with factors described above. They were transplanted into the cut ends of sciatic nerves, which then responded with vigorous nerve fibre regeneration within 3 weeks of the operation. Myelination of regenerated fibers by GFP‐expressing MSCs was recognized using confocal and immunoelectron microscopy. The results suggest that MSCs are able to differentiate into myelinating cells, capable of supporting nerve fibre re‐growth, and they can therefore be applied to induce nerve regeneration.

Treatment of retinal detachment resulting from myopic macular hole with internal limiting membrane removal

PURPOSE To examine the efficacy of vitrectomy with internal limiting membrane removal for retinal detachment resulting from a macular hole in highly myopic eyes. METHODS Eleven consecutive highly myopic eyes (11 patients) with retinal detachment resulting from a macular hole were treated by vitrectomy with removal of the internal limiting membrane, which was stained with indocyanine green and sulfur hexafluoride gas injection. Postoperatively, the patients were instructed to remain prone for 2 weeks. The excised specimens were evaluated with transmission electron microscopy. RESULTS The mean postoperative follow-up was 9.2 +/- 2.3 months (range, 7 to 13 months). In 10 of the 11 eyes (91%) the retina was reattached during the initial surgery. Redetachment occurred in one eye, which was successfully treated during the second surgery. Best-corrected visual acuity improved in all eyes and ranged from 20/400 to 20/50. Pathologic examination showed that the internal limiting membrane and epiretinal tissues were present in all specimens. CONCLUSIONS The use of indocyanine green staining can facilitate removal of a macular internal limiting membrane and overlying epiretinal membrane, resulting in complete relief of the macular traction. Primary removal of the internal limiting membrane may contribute to a high initial success rate for retinal reattachment and be an important adjuvant to the treatment of retinal detachment resulting from a macular hole in highly myopic eyes.

Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach

We developed a new in vivo electroporation method to deliver genes into retinal ganglion cells (RGCs). Efficiency and degree of tissue damage were evaluated using green fluorescent protein (GFP) gene and TUNEL. Soon after the intravitreous injection of the GFP gene, electroporation (five electric pulses of 99 ms duration each and 12V/cm delivered twice 5 min apart) was carried out on the adult rat eyeball with the aid of tweezer-type disc electrodes attached to corneal (cathode) and scleral (anode) surfaces. GFP expression, exhibiting a maximum on day 7, was detectable for up to 21 days. DiI retrograde labeling of RGCs showed that 41.5% of the total ganglion cells in the electroinjected area were GFP-positive. Therefore, this new method may be a useful tool for the delivery of genes into RGCs.

Quantitative Comparison of Anti-Fading Mounting Media for Confocal Laser Scanning Microscopy:

Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)–phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading; fluorescent intensity at the first scan (EM 1); and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EM n = EM 1 • A (n–1). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.

Familial frontotemporal dementia and parkinsonism with a novel N296H mutation in exon 10 of the tau gene and a widespread tau accumulation in the glial cells

Abstract. We report a 62-year-old Japanese man with familial frontotemporal dementia and a novel missense mutation (N296H) in exon 10 of the tau gene. The patient presented with frontal signs followed by temporal signs and parkinsonism. The brain showed localized frontotemporal lobe atrophy including the precentral gyrus and discoloration of the substantia nigra, and revealed severe neuronal loss with proliferation of tau-positive protoplasmic astroglia in the affected cerebral cortex, tau-positive coiled bodies and threads in the subcortical white matter, and tau-positive pretangle neurons in the subcortical and brain stem nuclei. There were no tau-positive neurofibrillary tangles, Pick bodies, tuft-shaped astrocytes or astrocytic plaques in the cerebral cortex. Immunoelectron microscopically, phosphorylated tau accumulated in both neurons and glial cells in different modalities, such as glial filaments in protoplasmic astroglia, straight tubules in coiled bodies, and free ribosomes in pretangle neurons. These findings suggest that tau proteins are not always assembled in abnormal filaments such as twisted ribbons, paired helical filaments and straight tubules in neurons and glial cells, which have been shown in previous cases with frontotemporal dementia and parkinsonism linked to chromosome 17. Immunoblotting of sarkosyl-insoluble tau exhibited accumulation of four-repeat tau isoforms in the brain. The N296H mutation may interfere with the ability of mutated tau to bind with microtubules and lead to tau aggregation. Further study is necessary to determine whether this mutation can account for the characteristic tau pathology of this case.

Effect of intracameral anesthesia on the corneal endothelium

Purpose: To evaluate the effects of intracameral anesthesia on the corneal endothelium. Setting: Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama, Japan. Methods: This study comprised 24 eyes of 12 white rabbits. One eye of 3 rabbits each was injected with preservative‐free lidocaine at concentrations of 0.02, 0.2, or 2% and the fellow eye injected with balanced salt solution (BSS®) as a control. The anesthetic agent was injected into the anterior chamber using a bimanual technique. Immediately after enucleation, the cornea was examined by scanning electron microscopy. Results: Scanning electron microscopy revealed no abnormal findings in the eyes injected with lidocaine 0.02 or 0.2% when compared with eyes in the control group. Scanning electron microscopy of the eyes injected with lidocaine 2% showed irregular hexagonal endothelial cells and a significant loss of microvilli. Conclusion: Intracameral anesthesia with high concentrations of lidocaine risks corneal endothelial damage but at the low concentration usually used in cataract surgery did not appear to have an adverse effect. J Cataract Refract Surg 1998; 24:1377‐1381

Immunocytochemistry of extracellular matrix components in the rat seminiferous tubule: Electron microscopic localization with improved methodology

Cell‐cell interactions between Sertoli, myoid, Leydig, and germ cells are thought to be essential for spermatogenesis. These cells interact with each other through the extracellular matrices (ECMs) of the testicular lamina propria, which thus may have an important function in spermatogenesis. For an understanding of the role of ECMs in spermatogenesis, it is important to investigate the molecular constitution of the testicular ECM. We examined the distribution of type IV, V, and VI collagens (Cols), laminin (LM), fibronectin (FN), and heparan sulfate proteoglycan (HSPG) in the rat testicular lamina propria.

Coexistence of nitric oxide synthase and neuropeptides in the mouse vomeronasal organ demonstrated by a combination of double immunofluorescence labeling and a multiple dye filter

Nitric oxide synthase (NOS)-immunofluorescence techniques were applied to the mouse vomeronasal organ. Immunoreactivity for NOS was found in the nerve fibers distributed in the receptor-free epithelium, and around the blood vessels and glands in the cavernous tissue. No NOS fibers were seen in the receptor area. A combination of double immunofluorescence labeling and multiple dye filter revealed that a part of the substance P (SP)-immunoreactive nerve fibers in the cavernous tissue contained NOS and that all the vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers around the blood vessels and glands in the cavernous tissue contained NOS. A few SP-immunoreactive cell bodies in the trigeminal ganglion showed coexistence with NOS, and almost all VIP-immunoreactive cell bodies in the sphenopalatine ganglion showed coexistence with NOS. Immunoreactivity for NOS without VIP in the cell bodies in the sphenopalatine ganglion was also found. These results suggest that NOS-immunoreactive nerve fibers in the mouse vomeronasal organ originate from the trigeminal and the sphenopalatine ganglia, and may modulate the vascular tone and the glandular secretion. In addition, these functions may be controlled in part by the interaction of nitric oxide and neuropeptides.

Expression of matrix metalloproteinase-7 in choroidal neovascular membranes in age-related macular degeneration

PURPOSE Matrix metalloproteinases are a family of extracellular matrix-degrading enzymes associated with neovascularization. We evaluated the expression and localization of matrix metalloproteinase-7 in choroidal neovascular membranes in age-related macular degeneration. METHODS Immunofluorescence and transmission electron microscopic examinations were performed on subfoveal neovascular membranes that had been surgically removed from seven eyes of seven patients with age-related macular degeneration. RESULTS Matrix metalloproteinase-7 was expressed in all specimens and distinctly expressed in the thickened layer of Bruch membrane and basement membrane-like structure around retinal pigment epithelial cells. CONCLUSIONS Matrix metalloproteinase-7 was expressed in Bruch membrane of choroidal neovascular membranes in age-related macular degeneration. Matrix metalloproteinase-7 may be an important factor for the development of the submacular neovascular membrane in age-related macular degeneration.

Ultrastructure of the capillary pericytes and the expression of smooth muscle α‐actin and desmin in the snake infrared sensory organs

The infrared sensory membranes of pit organs of pit vipers have an extremely rich capillary vasculature that forms many vascular loops, each serving a small number of infrared nerve terminals. We clarified the ultrastructure of capillary pericytes in the pit membranes by scanning and transmission electron microscopy, and examined the immunoreactivity in their cytoplasm to two contractile proteins: smooth muscle α‐actin (SM α‐actin) and desmin. The capillary pericytes had two major cytoplasmic processes: thickened primary processes that radiate to embrace the endothelial tube and flattened secondary processes that are distributed widely on the endothelium. Coexpression of SM α‐actin and desmin was observed in the pericytes of entire capillary segments, and SM α‐actin was characterized by prominent filament bundles directed mainly at right angles to the capillary long axis. This expression pattern was different from that of capillary pericytes of the scales, where SM α‐actin was expressed diffusely in the cytoplasm. In a series of electron microscopic sections, we often observed the pericyte processes depressing the endothelial wall. We also observed a close relationship of the pericytes with inter‐endothelial cell junctions, and pericyte processes connected with the endothelial cells via gap junctions.