Matsuo, Toshihiko

To test transdifferentiation of retinal pigment epithelial (RPE) cells in suspension culture, chick embryonic RPE sheets and dissociated RPE cells were cultured for two months in a non-adherent dish for suspension culture. RPE cells, isolated as a sheet, aggregated immediately and remained the same size with their differentiated characteristics for two months. The presence of basic fibroblast growth factor (bFGF) at concentrations of 10 ng/ml or higher induced the formation of a spherical lentoid structure which was positive for crystallin and bFGF receptor. In contrast, dissociated RPE cells did reaggregate but did not develop the lentoid structure even in the presence of bFGF. The transdifferentiation of RPE cells to the lentoid structure in this study was in contrast to their transdifferentiation to the retina, as reported in a previous study.

To study the expression of the amiloride-sensitive sodium channel, a putative mechano-receptor in the rat eye, reverse transcriptase-polymerase chain reaction and in situ hybridization were done. The gene for the alpha subunit of the amiloride-sensitive sodium channel was shown by polymerase chain reaction to be expressed in mRNA isolated from the whole eye tissue. In situ hybridization demonstrated that the gene was expressed in basal layers of the corneal and conjunctival epithelium, ciliary epithelial cells, lens epithelial cells at the equator, retinal and iris pigment epithelial cells, ganglion cells and cells in the inner and outer nuclear layers of the retina. The results suggest that the amiloride-sensitive sodium channel plays a role in maintaining sodium balance as well as in possible mechanosensation in these ocular tissues.

We analyzed nucleotide changes in 3 genes, ARIX, PHOX2B, and KIF21A, in 6 patients of 3 families with congenital superior oblique muscle palsy. Three exons of ARIX, 3 exons of PHOX2B, and exons 8, 20, and 21 of KIF21A were amplified by polymerase chain reaction from genomic DNA isolated from the peripheral blood. The DNA fragments were directly sequenced in both directions. In 2 different families, a heterozygous nucleotide change, ARIX 153G>A, in the 5’-untranslated region was found in common between a father and daughter with muscle palsy and between a mother and daughter with muscle palsy (Family No. 1 and No. 3). In the other family (Family No. 2), a heterozygous 15-nucleotide deletion, PHOX2B 1124del15, resulting in loss of 5 alanine residues in the alanine repeat of the protein, was found in the daughter with muscle palsy and her father with normal traits, but was not found in the mother with muscle palsy. No KIF21A nucleotide change was found in any patients. The ARIX 153G>A polymorphism might be a genetic risk factor for the development of congenital superior oblique muscle palsy.

Retinal cells from chick embryos aged 7.5 days of gestation were cultured for two months in a non-adherent suspension culture dish to study the effects of growth factors and co-culture with retinal pigment epithelial cells on their differentiation. Dissociated retinal cells became cellular aggregates (multicellular spheroids) within a day, and rosettes were formed in the spheroids after 2 days. Ultrastructurally, neurons of the rosettes developed connecting cilia, ellipsoids (accumulation of mitochondria), and external limiting membrane, indicative of their differentiation into photoreceptor cells. Epidermal growth factor enhanced the expression of rhodopsin by rosette-forming neurons, while basic fibroblast growth factor induced the growth of Mueller cells at 4 weeks, and their transdifferentiation into lens-epithelial-like cells at 8 weeks. Co-culture of retinal cells with retinal pigment epithelial cells enhanced the formation of rosettes in spheroids. Multicellular spheroids formed in a dish for suspension culture would provide a convenient in vitro system to examine differentiation and transdifferentiation of the retina.

To understand the development of the trabecular meshwork of the eye, floating cellular aggregates (multicellular spheroids) were formed from human trabecular cells in a non-adherent environment of culture and incubated for up to one month. Dissociated trabecular cells formed multicellular spheroids within one day in the non-adherent environment, and apoptosis continued to occur in the spheroids which had been initially filled with cells. The final structure after one month appeared as a meshwork of cells with large extracellular spaces. Epidermal and basic fibroblast growth factor (EGF and bFGF) protected trabecular cells in the spheroids from apoptosis and, as a result, kept the spheroids filled with cells even after one month. In the absence of excess EGF or bFGF, the multicellular spheroids grown in vitro from human trabecular cells mimicked the mesh-like structure of normal trabecular tissue. In constrast, under an excess of these growth factors, spheroids of high cellularity, resembling the abnormal trabecular tissues of patients with congenital glaucoma, were formed.

The purposes of this study were to examine whether body sway is altered immediately after strabismus surgery in children and to find preoperative clinical factors associated with body sway. In a prospective study, body sway was measured on 1-3 days before surgery and on the third day after surgery; for the measurements, computerized static stabilometry was carried out on 28 consecutive patients with strabismus (age range: 3 to 12 years old; mean: 7.4) who underwent strabismus surgery under general anesthesia. The linear length of the sway path (cm), the linear length of the sway path in a particular unit of time (cm/second), and the area of the sway path (cm2), indicative of the extent of body sway, all increased significantly among a total of 28 patients in both conditions of the patient's eyes open and closed, as well as among those in a subgroup of 16 patients with exotropia, after they had undergone strabismus surgery (p < 0.05, Wilcoxon signed ranks test). The center of pressure along the Y axis of orientation from the toe to the heel was found to deviate significantly toward the heel postoperatively, as compared with the preoperative center in the subgroup of 16 patients with exotropia (p < 0.05). Before surgery, 15 patients with no stereoacuity exhibited a greater amount of body sway when their eyes were open than did 13 patients with measurable stereoacuity (p < 0.05, Mann-Whitney U-test). In the subgroup of 16 patients with exotropia when their eyes open, 3 patients with abnormal head posture exhibited more extensive body sway than did 13 patients without abnormal head posture (p < 0.05). Body sway was found to significantly increase immediately after strabismus surgery in children with strabismus. Stereoacuity and abnormal head posture are 2 clinical factors associated with preoperative postural instability.

We report herein the disappearance of macular hard exudates after the introduction of hemodialysis in diabetic patients. A 62-year-old woman and a 52-year-old man with diabetes mellitus showed hard exudates in the macula of the left eyes. Both patients had previously undergone panretinal photocoagulation in both eyes. During the follow-up, hemodialysis was introduced for deteriorating chronic renal failure caused by diabetic nephropathy. Half a year later, macular hard exudates in the left eyes disappeared dramatically in both patients, but the visual acuity remained the same. No additional laser treatment was done during the observation period. Hemodialysis is considered to have accelerated the resolution of macular hard exudates in both patients. The deposition of macular hard exudates in diabetic patients is due in part to concurrent poor renal function.