F. Germain

Functional and structural modifications during retinal degeneration in the rd10 mouse

Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degeneration in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm(2). In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with age-matched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities.

Age-related functional and structural retinal modifications in the Igf1−/− null mouse

BACKGROUND Mutations in the gene encoding human insulin-like growth factor-I (IGF-I) cause syndromic neurosensorial deafness. To understand the precise role of IGF-I in retinal physiology, we have studied the morphology and electrophysiology of the retina of the Igf1(-/-) mice in comparison with that of the Igf1(+/-) and Igf1(+/+) animals during aging. METHODS Serological concentrations of IGF-I, glycemia and body weight were determined in Igf1(+/+), Igf1(+/-) and Igf1(-/-) mice at different times up to 360days of age. We have analyzed hearing by recording the auditory brainstem responses (ABR), the retinal function by electroretinographic (ERG) responses and the retinal morphology by immunohistochemical labeling on retinal preparations at different ages. RESULTS IGF-I levels are gradually reduced with aging in the mouse. Deaf Igf1(-/-) mice had an almost flat scotopic ERG response and a photopic ERG response of very small amplitude at postnatal age 360days (P360). At the same age, Igf1(+/-) mice still showed both scotopic and photopic ERG responses, but a significant decrease in the ERG wave amplitudes was observed when compared with those of Igf1(+/+) mice. Immunohistochemical analysis showed that P360 Igf1(-/-) mice suffered important structural modifications in the first synapse of the retinal pathway, that affected mainly the postsynaptic processes from horizontal and bipolar cells. A decrease in bassoon and synaptophysin staining in both rod and cone synaptic terminals suggested a reduced photoreceptor output to the inner retina. Retinal morphology of the P360 Igf1(+/-) mice showed only small alterations in the horizontal and bipolar cell processes, when compared with Igf1(+/+) mice of matched age. CONCLUSIONS In the mouse, IGF-I deficit causes an age-related visual loss, besides a congenital deafness. The present results support the use of the Igf1(-/-) mouse as a new model for the study of human syndromic deaf-blindness.

Morphological signs of apoptosis in axotomized ganglion cells of the rabbit retina.

Optic nerve section in mammals induces apoptotic death of retinal ganglion cells (RGCs). However, a small population of RGCs survives for a relatively long time. These cells experience significant morphological changes due to the apoptotic process, but some of these changes are not clearly differentiated from those experienced in necrotic cells. In the present work, rabbit RGCs were studied 1 month after optic nerve section using light microscopy after neurobiotin injection, transmission electron microscopy (EM) and scanning electron microscopy (SEM). Apoptosis was identified by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling and characteristic signs of apoptosis were observed in the EM images. Ultrastructural analyses showed vacuolar degeneration in the cytoplasm and normal cellular structure loss. Signs of membrane changes were observed in axotomized RGCs by SEM. Early changes seen in the cell membrane suggest that axotomy may cause important changes in the cytoskeleton. We conclude that characteristic signs of apoptosis at the cell membrane level are clearly observed in rabbit RGCs after axotomy and they may be responsible for the cellular death.

Effect of diabetes mellitus on Corvis ST measurement process

Diabetes mellitus (DM) affects corneal biomechanical parameters. We compared analyses using ORA (Ocular response analyser) and Corvis ST to determine the influence of disease duration, hyperglycaemia and haemoglobin A1c (HbA1c) levels on these parameters.

Morphometrical analysis of dendritic arborization in axotomized retinal ganglion cells

It has been reported that section of the optic nerve in mammals causes death in >90% of the retinal ganglion cells (RGCs). The cells which survive the section experience an irreparable loss of many of their dendritic segments and a rapid retraction of the dendritic tree. However, some growth cones and abnormal processes have been also reported. Our aim was to make a quantitative study of the morphological changes found in rabbit RGCs after optic nerve section. The morphometrical analysis of the RGCs which survived the axotomy showed an increase in the diameter of the soma and a significant increase in the area of the dendritic field; also, the length of the dendritic segments was significantly longer in axotomized RGCs than in control cells. Terminal dendritic segments (T) and preterminal segments (PT) were both measured in control and axotomized cells; the length ratio of T : PT segments was significantly greater in the axotomized cells than in the controls. We conclude that RGCs which survived the axotomy experienced a significant growth of their terminal dendritic branches.

Effect of intravitreal ranibizumab on corneal endothelium in age-related macular degeneration.

Purpose: To determine the effect of intravitreal injection of ranibizumab on the corneal endothelium in patients with choroidal neovascularization in age-related macular degeneration. Methods: Observational prospective case series study. Fifty-two eyes of 52 consecutive patients (29 men, 23 women; age range, 61-80 years) were evaluated. All participants received monthly intravitreal injections of (0.05 mL, 0.5 mg) ranibizumab for 3 consecutive months; the follow-up period was 6 months. Central corneal specular microscopy was performed before injection and at 7 days and 6 months after the first intravitreal injection. The endothelial cell density, coefficient of variation of cell size, and percentage of hexagonal cells were analyzed, and the central corneal thickness was measured. Results: There were no significant differences in the endothelial cell densities, coefficient of variation of cell sizes, and percentage of hexagonal cells values before injection and at 7 days and 6 months after the first intravitreal ranibizumab injection (P = 0.987, P = 0.822, and P = 0.918, respectively). There was also no significant difference in central corneal thickness measurements before injection and at 7 days and 6 months after the first intravitreal ranibizumab injection (P = 0.325). Conclusion: Repeated intravitreal injections of 0.5 mg of ranibizumab do not seem to cause substantial changes in the corneal endothelium at 6 months.

Intraoperative mitomycin C and corneal endothelium after pterygium surgery.

Purpose: To determine the effect of mitomycin C (MMC) on the corneal endothelium after primary pterygium surgery. Methods: This prospective, interventional, nonrandomized, observer-masked study included 46 consecutive patients (51 eyes) with primary pterygium. The bare sclera technique with 1-minute application of 0.02% MMC intraoperatively was used in all cases. The follow-up period was 3 months. Preoperative and postoperative central corneal specular microscopy was performed. The endothelial cell density, coefficient of variation of cell size, and percentage of hexagonal cells were analyzed, and the corneal thickness was measured. Results: The mean endothelial cell densities preoperatively and 3 months postoperatively were 2382.35 ± 342.07 cells per square millimeter (range, 1020-3129) and 2385.02 ± 356.83 cells per square millimeter (range, 1001-3151), respectively (P = 0.96). The mean coefficients of variation of cell size preoperatively and 3 months postoperatively were 34.31 ± 5.62 (range, 22-49) and 35.29 ± 7.50 (range, 22-55), respectively (P = 0.17). The mean percentages of hexagonal cells values preoperatively and 3 months postoperatively were 52.98 ± 7.32 (range, 32-71) and 51.61 ± 8.98 (range, 32-67), respectively (P = 0.48). The mean pachymetry measurements preoperatively and 3 months postoperatively were 506.65 ± 36.87 μm (range, 411-583) and 502.08 ± 41.33 μm (range, 411-593), respectively (P = 0.99). Conclusions: One intraoperative application of 0.02% MMC for 1 minute after primary pterygium surgery does not seem to cause substantial changes in the corneal endothelium at 3 months.

Effect of topical 0.05% cyclosporine A on corneal endothelium in patients with dry eye disease.

AIM To determine the effect of topical 0.05% cyclosporine A (CsA) on corneal endothelium in patients with dry eye disease. METHODS Observational, prospective, case series study. Fifty-five eyes of 29 consecutive patients (9 males and 20 females; median age: 66.8 years, interquartile range: 61-73.2 years) with moderate-severe dry eye disease were evaluated. All patients were treated with topical 0.05% CsA ophthalmic emulsion twice a day in addition to lubricant eyedrops 5 times a day. The follow-up period was 12 months. Before treatment and at 3 and 12 months post-treatment central corneal specular microscopy was performed. The endothelial cell density (ECD), coefficient of variation of cell size (CoV), and percentage of hexagonal cells (Hex %) were analyzed. RESULTS The median ECDs pre-treatment and at 3 and 12 months post-treatment were 2 352.5/mm(2) (interquartile range, 2 178-2 548.5), 2364/mm(2) (interquartile range, 2 174.25-2 657.5), and 2366 cells/mm(2) (interquartile range, 2 174.75-2 539.75), respectively (P=0.927, one way ANOVA). The median CoVs pre-treatment and at 3 and 12 months post-treatment were 34.5 (interquartile range, 30-37), 35 (interquartile range, 30-38), and 34 (interquartile range, 30.75-38.25), respectively (P=0.7193, one way ANOVA). The median Hex % values pre-treatment and at 3 and 12 months post-treatment were 53 (interquartile range, 47-58), 54 (interquartile range, 45.75-59), and 50.5 (interquartile range, 45.75-58), respectively (P=0.824, one way ANOVA). CONCLUSION Treatment of patients with dry eye disease for 12 months with topical 0.05% CsA does not seem to cause substantial changes on corneal endothelium.

The retina of the PCD/PCD mouse as a model of photoreceptor degeneration. A structural and functional study.

In this work, we used the pcd (Purkinje cell degeneration) mutant mouse with a slow temporal progression of photoreceptor degeneration in order to analyze the structural and functional modifications in the neuronal populations of the outer and inner retina. Retinal immunocytochemistry and functional electroretinography were performed on the pcd/pcd mutant mice and control wild type animals of the C57/DBA strain at 45, 90, 180 and 270 post-natal days. Immunohistochemical studies were performed for a series of protein markers: calbindin, calretinin, PKCα, bassoon, synapsin, syntaxin and islet1. Full field electroretinography recordings were performed on control and dystrophic mice. Rod and mixed responses, and oscillatory potentials, were recorded in dark adapted conditions; cone and flicker responses were recorded under light adaptation. Our results show significant structural modifications in the photoreceptor populations and neurons of the inner retina. Changes in cell morphology affect mainly to the bipolar cells, which gradually lose their dendritic tufts. The electroretinography records reveal that in the pcd retinas the rod and cone systems show a reduction in the amplitude of the electrical signals. This decrease progresses slowly with the passage of time, although for the most advanced stage of photoreceptor degeneration considered, 270 post-natal days, it is still possible to record light induced responses. We conclude that pcd mice experience a loss of retinal function in correlation with the loss of photoreceptors with age, and significant changes in retinal synaptic processes.

Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration.

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.