PathFinder 2014

担当教員:小林聡子平成26年度(2014)教養展開科目(国際性を高める),授業コード:G15N6220

Seizure-Related Gene 6 (Sez-6) in Amacrine Cells of the Rodent Retina and the Consequence of Gene Deletion

Background: Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development. Methodology/Principal Findings: The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in All glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear "bright spot" similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG). Conclusions: In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected

Physiological Effects of Superoxide Dismutase on Altered Visual Function of Retinal Ganglion Cells in db/db Mice

Background: The C57BLKS/J db/db (db/db) mouse is a widely used type 2 diabetic animal model, and this model develops early inner retinal neuronal dysfunction beginning at 24 weeks. The neural mechanisms that mediate early stage retinal dysfunction in this model are unknown. We evaluated visual response properties of retinal ganglion cells (RGCs) during the early stage of diabetic insult (8, 12, and 20 wk) in db/db mice and determined if increased oxidative stress plays a role in impaired visual functions of RGCs in 20 wk old db/db mice. Methodology/Principal Findings: In vitro extracellular single-unit recordings from RGCs in wholemount retinas were performed. The receptive field size, luminance threshold, and contrast gain of the RGCs were investigated. Although ONand OFF-RGCs showed a different time course of RF size reduction, by 20 wk, the RF of ON- and OFF-RGCs were similarly affected. The LT of ON-RGCs was significantly elevated in 12 and 20 wk db/db mice compared to the LT of OFF-RGCs. The diabetic injury also affected contrast gains of ON- and OFF-RGCs differently. The generation of reactive oxidative species (ROS) in fresh retina was estimated by dihydroethidium. Superoxide dismutase (SOD) (300 unit/ml) was applied in Ames medium to the retina, and visual responses of RGCs were recorded for five hours. ROS generation in the retinas of db/db mice increased at 8wk and continued to progress at 20 wk of ages. In vitro application of SOD improved visual functions in 20 wk db/db mice but the SOD treatment affected ON- and OFF-RGCs differently in db/m retina

Morphological and Functional Changes in the Retina after Chronic Oxygen-Induced Retinopathy

The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment

Evidence for Diffuse Central Retinal Edema In Vivo in Diabetic Male Sprague Dawley Rats

Background: Investigations into the mechanism of diffuse retinal edema in diabetic subjects have been limited by a lack of animal models and techniques that co-localized retinal thickness and hydration in vivo. In this study we test the hypothesis that a previously reported supernormal central retinal thickness on MRI measured in experimental diabetic retinopathy in vivo represents a persistent and diffuse edema. Methodology/Principal Findings: In diabetic and age-matched control rats, and in rats experiencing dilutional hyponatremia (as a positive edema control), whole central retinal thickness, intraretinal water content and apparent diffusion coefficients (ADC, ‘water mobility’) were measured in vivo using quantitative MRI methods. Glycated hemoglobin and retinal thickness ex vivo (histology) were also measured in control and diabetic groups. In the dilutional hyponatremia model, central retinal thickness and water content were supernormal by quantitative MRI, and intraretinal water mobility profiles changed in a manner consistent with intracellular edema. Groups of diabetic (2, 3, 4, 6, and 9 mo of diabetes), and age-matched controls were then investigated with MRI and all diabetic rats showed supernormal whole central retinal thickness. In a separate study in 4 mo diabetic rats (and controls), MRI retinal thickness and water content metrics were significantly greater than normal, and ADC was subnormal in the outer retina; the increase in retinal thickness was not detected histologically on sections of fixed and dehydrated retinas from these rats

Reactivity in the human retinal microvasculature measured during acute gas breathing provocations

Although changes in vessel diameter following gas perturbation have been documented in retinal arterioles and venules, these responses have yet to be quantified in the smallest vessels of the human retina. Here, using in vivo adaptive optics, we imaged 3-25 µm diameter vessels of the human inner retinal circulation and monitored the effects of altered gas-breathing conditions. During isocapnic hyperoxia, definite constrictions were seen in 51% of vessel segments (mean ± SD for pre-capillary arterioles -9.5 ± 3.0%; capillaries -11.8 ± 3.3%; post-capillary venules -6.3 ± 2.8%); these are comparable with responses previously reported in larger vessels. During isoxic hypercapnia, definite dilations were seen in 47% of vessel segments (mean ± SD for pre-capillary arterioles +9.8 ± 1.5%; capillaries +13.7 ± 3.8%; post-capillary venules +7.5 ± 4.2%); these are proportionally greater than responses previously reported in larger vessels. The magnitude of these proportional changes implies that the capillary beds themselves play an important role in the retinal response to changes in carbon dioxide levels. Interestingly, the distribution of microvascular responses shown here differs from our previously reported responses to flicker stimulation, suggesting differences in the way blood supply is coordinated following gas perturbation and altered neural activity