Masahiko Takano

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.

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.

Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.

We developed an in vivo electroporation method to introduce foreign genes into retinal ganglion cells (RGCs). After the intravitreous injection of the plasmid gene (20 μg), five electric pulses (6 V/cm, 100 ms duration) were each delivered twice with 5 min interval to the rat eye using a contact lens-type electrode (cathodal) attached to the cornea and a needle electrode (anodal) inserted to the middle of the forehead. The efficiency of the genetic introduction into RGCs and tissue damage to the eyeball was evaluated using a green fluorescent protein (GFP) gene, TUNEL and histological observation. DiI retrograde labeling revealed that 24.4±4.7% of all RGCs were electrointroduced with the GFP gene. TUNEL and histological analysis showed a few tissue damages in the cornea, lens and retina. To confirm whether this method can actually rescue damaged RGCs, glial cell line-derived neurotrophic factor (GDNF) was electrointroduced into RGCs after optic nerve transection. After the electrointroduction, a significant increase in the number of surviving RGCs was observed 2 and 4 weeks after the optic nerve transection, and the decrease of caspase 3 and 9 was detected by RT-PCR. These results suggest that this method may be useful for the delivery of genes into RGCs with simplicity and minimal tissue damage.

Diabetes alters neurite regeneration from mouse retinal explants in culture

We examined the effect of experimental diabetes on neurite regeneration from adult mouse retinal explants cultured in the presence of different concentrations of glucose. The numbers of regenerating neurites at 3, 6 and 10 days in culture at normal glucose concentration (7 mM) were significantly smaller in streptozotocin-induced diabetic C57BL/6 mice than in normal control mice. In contrast, treatment of retinal explants with high glucose concentration (57 mM) significantly diminished the number of regenerating neurites in the control mice, but not in the diabetic mice. These results suggest that retina in diabetic mice has impaired capability of neurite regeneration in a normal glucose environment, but is adaptable to a high glucose environment in vitro.

Critical period for degradation of adult rat retinal ganglion cells and their regeneration capability after axotomy.

The optic nerve (ON) in adult rats was transected intraorbitally followed by retrograde labelling of retinal ganglion cells (RGCs) with fluorescence dye (DiI). A two phase reduction of axotomized RGCs was represented in the retina until 15 days after ON transection. The critical period of RGC loss is around 7 days after axotomy. In this period, the capability of neurite regeneration from RGCs in culture was strongly intensified by ON transection. These findings indicate that the 7th day after axotomy is important for both RGC survival and regeneration.

Scleral Buckling and Pars Plana Vitrectomy for Rhegmatogenous Retinal Detachment: An Analysis of 542 Eyes

Abstract Purpose: To investigate the anatomical success rates of scleral buckling (SB) and pars plana vitrectomy (PPV) performed for rhegmatogenous retinal detachment (RRD) in a large case series and to identify prognostic factors for the primary anatomical success rates of surgical techniques. Methods: We reviewed 542 consecutive eyes for primary RRD in this retrospective study. Follow-ups were performed for at least six months. In each of the two groups, 271 eyes were examined. The main outcome measure was the primary anatomical success rate. Multivariate analysis was also performed to determine whether independent risk factors of the preoperative parameters for redetachment exist. Results: The primary anatomical success rates were 93.7% and 96.3% in the SB and PPV groups; and the final anatomical success rates were 100% in both groups (each with 271 eyes). In the SB group, eyes with macula-off had significantly lower primary anatomical success rates than those with macula-on (p = 0.002). Preoperative break location or lens status had no significant effect on primary anatomical success rates in either group. Multivariate logistic regression analysis using four variables, namely, sex, posterior vitreous detachment, macular status and preoperative visual acuity, showed that the macular status was an independent risk factor for redetachment in the SB group (p = 0.039, odds ratio 3.7). The six-month follow-up visual acuity was significantly better than the preoperative visual acuity in both groups (p ≤ 0.001). Conclusions: Both SB and PPV gave excellent primary and final anatomical success rates. The macula-off status was associated with a lower success rate in the SB group, although break location and lens status had no significant effect on success rates in either group.

Involvement of retinal neurons and pigment epithelial cells in a murine model of sandhoff disease.

Background/Aims: To investigate the effects of deficient degradation of glycolipids on the morphological appearance of all retinal cells in a murine model of GM2 gangliosidosis (Sandhoff disease). Methods: The morphological appearance of the retina in Sandhoff mice at symptomatic stages (3 and 4 months of age) was examined by immunohistochemistry, lectin histochemistry and electron microscopy. Results: Under a light microscope, intense immunoreactivity for GM2 ganglioside was observed in the ganglion cell, inner plexiform, and inner nuclear layers in the Sandhoff mice. The ganglion cell layers and retinal pigment epithelium in the Sandhoff mice were stained intensely withconcanavalin A agglutinin and succinylated wheat germ agglutinin. Ultrastructural studies revealed numerous inclusions in the cytoplasm of retinal ganglion cells and other neuronal cells (particularly amacrine cells), whereas we failed to detect apparent involvement of photoreceptor cells. In addition to the cytoplasmic inclusions in the retinal neurons, vacuolation was evident in the retinal pigment epithelium. Conclusion: These findings suggest that neuronal cells and pigment epithelial cells are more vulnerable to the deficient ganglioside degradation than other retinal cells in Sandhoff mice.

Fixation Point After Successful Macular Hole Surgery with Internal Limiting Membrane Peeling

BACKGROUND AND OBJECTIVE This study evaluated the fixation points of macular holes using scanning laser ophthalmoscope microperimetry before and after surgery with internal limiting membrane peeling. PATIENTS AND METHODS This prospective non-comparative study examined 21 eyes (21 patients) with macular holes. Hole size and distance between preoperative and postoperative fixation points were measured. RESULTS The fixation points were located on the superior edge of the macular hole in 18 (86%) cases preoperatively and shifted centrally in 15 (71.4%) cases postoperatively. Macular hole size correlated well with the distance moved by the fixation points (r = .83, P < .0001). CONCLUSION Scanning laser ophthalmoscope microperimetry facilitated pinpointing of fixation points before and after surgery. The correlation between macular hole size and fixation point shift suggests that the retina around the hole moves centripetally after surgery.

Diabetes-induced reduction of neuronal survival in hypotonic environments in culture

Dorsal root ganglion (DRG) neurons from streptozotocin (STZ)-induced diabetic and normal C57BL mice were exposed to three different hypotonic environments (1/2, 1/4, and 1/8 osmolar solutions). After rapid applications of these hypotonic solutions to the neurons, the cell volume autoregulatory mechanism operated in 1/2 osmolar solution but was disrupted in superhypotonic solutions below 1/4 osmolar in both kinds of mice. None of the neurons could survive 12 h after treatment with superhypotonic solutions. On the other hand, a gradual reduction of osmolarity of the culture medium enabled neurons in the normal mice to survive in 1/2 and 1/4 osmolar solutions as well as in an isotonic solution. However, this reduction of osmolarity increased neuronal cell death in the diabetic mice. These results suggest that the ability of DRG neurons to survive in hypotonic environments in culture may be lost in diabetes.

Regional differences in neurite regeneration of postnatal mouse retinal explants

Retinal explants from mice at postnatal days 2-15 were three-dimensionally cultured in collagen gel. The regeneration of neurites from the explants was investigated at each stage after birth. The explants from the central region in the retina could not regenerate neurites after postnatal day 9, but those from peripheral regions expressed such ability until postnatal day 12. This regional difference in the disappearance stages of retinal regenerative capability coincides with that of retinal maturation.