Contribution of chemical and electrical transmission to the low delta-like intrinsic retinal oscillation in mice: A role for daylight-activated neuromodulators.

Basal electroretinogram (ERG) oscillations have shown predictive value for modifiable risk factors for type 2 diabetes. However, their origin remains unknown. Here, we seek to establish the pharmacological profile of the low delta-like (δ1) wave in the mouse because it shows light sensitivity in the form of a decreased peak frequency upon photopic exposure. Applying neuropharmacological drugs by intravitreal injection, we eliminated the δ1 wave using lidocaine or by blocking all chemical and electrical synapses. The δ1 wave was insensitive to the blockade of photoreceptor input, but was accelerated when all inhibitory or ionotropic inhibitory receptors in the retina were antagonized. The sole blockade of GABAA, GABAB, GABAC, and glycine receptors also accelerated the δ1 wave. In contrast, the gap junction blockade slowed the δ1 wave. Both GABAA receptors and gap junctions contribute to the light sensitivity of the δ1 wave. We further found that the day light-activated neuromodulators dopamine and nitric oxide donors mimicked the effect of photopic exposure on the δ1 wave. All drug effects were validated through light flash-evoked ERG responses. Our data indicate that the low δ-like intrinsic wave detected by the non-photic ERG arises from an inner retinal circuit regulated by inhibitory neurotransmission and nitric oxide/dopamine-sensitive gap junction-mediated communication.

Preventable risk factors for type 2 diabetes can be detected using noninvasive spontaneous electroretinogram signals.

Given the ever-increasing prevalence of type 2 diabetes and obesity, the pressure on global healthcare is expected to be colossal, especially in terms of blindness. Electroretinogram (ERG) has long been perceived as a first-use technique for diagnosing eye diseases, and some studies suggested its use for preventable risk factors of type 2 diabetes and thereby diabetic retinopathy (DR). Here, we show that in a non-evoked mode, ERG signals contain spontaneous oscillations that predict disease cases in rodent models of obesity and in people with overweight, obesity, and metabolic syndrome but not yet diabetes, using one single random forest-based model. Classification performance was both internally and externally validated, and correlation analysis showed that the spontaneous oscillations of the non-evoked ERG are altered before oscillatory potentials, which are the current gold-standard for early DR. Principal component and discriminant analysis suggested that the slow frequency (0.4-0.7 Hz) components are the main discriminators for our predictive model. In addition, we established that the optimal conditions to record these informative signals, are 5-minute duration recordings under daylight conditions, using any ERG sensors, including ones working with portative, non-mydriatic devices. Our study provides an early warning system with promising applications for prevention, monitoring and even the development of new therapies against type 2 diabetes.

Potential contributions of the intrinsic retinal oscillations recording using non-invasive electroretinogram to bioelectronics.

Targeted electric signal use for disease diagnostics and treatment is emerging as a healthcare game-changer. Besides arrhythmias, treatment-resistant epilepsy and chronic pain, blindness, and perhaps soon vision loss, could be among the pathologies that benefit from bioelectronic medicine. The electroretinogram (ERG) technique has long demonstrated its role in diagnosing eye diseases and early stages of neurodegenerative diseases. Conspicuously, ERG applications are all based on light-induced responses. However, spontaneous, intrinsic activity also originates in retinal cells. It is a hallmark of degenerated retinas and its alterations accompany obesity and diabetes. To the extent that variables extracted from the resting activity of the retina measured by ERG allow the predictive diagnosis of risk factors for type 2 diabetes. Here, we provided a comparison of the baseline characteristics of intrinsic oscillatory activity recorded by ERGs in mice, rats, and humans, as well as in several rat strains, and explore whether zebrafish exhibit comparable activity. Their pattern was altered in neurodegenerative models including the cuprizone-induced demyelination model in mice as well as in the Royal College of Surgeons (RCS-/-) rats. We also discuss how the study of their properties may pave the way for future research directions and treatment approaches for retinopathies, among others.

Histological and diffusion-weighted magnetic resonance imaging data from normal and degenerated optic nerve and chiasm of the rat.

Diffusion-weighted magnetic resonance imaging (dMRI) is widely used to infer microstructural characteristics of tissue, particularly in cerebral white matter. Histological validation of the metrics derived from dMRI methods are needed to fully characterize their ability to capture biologically-relevant histological features non-invasively. The data described here were used to correlate metrics derived from dMRI and quantitative histology in an animal model of axonal degeneration ("Histological validation of per-bundle water diffusion metrics within a region of fiber crossing following axonal degeneration" [1]). Unilateral retinal ischemia/reperfusion was induced in 10 rats, by the elevation of pressure of the anterior chamber of the eye for 90 min. Five rats were used as controls. After five weeks, injured animals were intracardially perfused to analyze the optic nerves and chiasm with dMRI and histology. This resulted in 15 brain scans, each with 80 diffusion-sensitizing gradient directions with b = 2000 and 2500 s/mm2 and 20 non-diffusion-weighted images (b = 0 s/mm2), with isometric voxel resolution of 125 µm3. Histological sections were obtained after dMRI. Optical microscopy photomicrographs of the optic nerves (stained with toluidine blue) are available, as well as their corresponding automatic segmentations of axons and myelin.

Histological validation of per-bundle water diffusion metrics within a region of fiber crossing following axonal degeneration.

Micro-architectural characteristics of white matter can be inferred through analysis of diffusion-weighted magnetic resonance imaging (dMRI). The diffusion-dependent signal can be analyzed through several methods, with the tensor model being the most frequently used due to its straightforward interpretation and low requirements for acquisition parameters. While valuable information can be gained from the tensor-derived metrics in regions of homogeneous tissue organization, this model does not provide reliable microstructural information at crossing fiber regions, which are pervasive throughout human white matter. Several multiple fiber models have been proposed that seem to overcome the limitations of the tensor, with few providing per-bundle dMRI-derived metrics. However, biological interpretations of such metrics are limited by the lack of histological confirmation. To this end, we developed a straightforward biological validation framework. Unilateral retinal ischemia was induced in ten rats, which resulted in axonal (Wallerian) degeneration of the corresponding optic nerve, while the contralateral was left intact; the intact and injured axonal populations meet at the optic chiasm as they cross the midline, generating a fiber crossing region in which each population has different diffusion properties. Five rats served as controls. High-resolution ex vivo dMRI was acquired five weeks after experimental procedures. We correlated and compared histology to per-bundle descriptors derived from three methodologies for dMRI analysis (constrained spherical deconvolution and two multi-tensor representations). We found a tight correlation between axonal density (as evaluated through automatic segmentation of histological sections) with per-bundle apparent fiber density and fractional anisotropy (derived from dMRI). The multi-fiber methods explored were able to correctly identify the damaged fiber populations in a region of fiber crossings (chiasm). Our results provide validation of metrics that bring substantial and clinically useful information about white-matter tissue at crossing fiber regions. Our proposed framework is useful to validate other current and future dMRI methods.

TRPV4 inhibition prevents increased water diffusion and blood-retina barrier breakdown in the retina of streptozotocin-induced diabetic mice.

A better understanding of the molecular and cellular mechanisms involved in retinal hydro-mineral homeostasis imbalance during diabetic macular edema (DME) is needed to gain insights into retinal (patho-)physiology that will help elaborate innovative therapies with lower health care costs. Transient receptor potential cation channel subfamily vanilloid member 4 (TRPV4) plays an intricate role in homeostatic processes that needs to be deciphered in normal and diabetic retina. Based on previous findings showing that TRPV4 antagonists resolve blood-retina barrier (BRB) breakdown in diabetic rats, we evaluated whether TRPV4 channel inhibition prevents and reverts retinal edema in streptozotocin(STZ)-induced diabetic mice. We assessed retinal edema using common metrics, including retinal morphology/thickness (histology) and BRB integrity (albumin-associated tracer), and also by quantifying water mobility through apparent diffusion coefficient (ADC) measures. ADC was measured by diffusion-weighted magnetic resonance imaging (DW-MRI), acquired ex vivo at 4 weeks after STZ injection in diabetes and control groups. DWI images were also used to assess retinal thickness. TRPV4 was genetically ablated or pharmacologically inhibited as follows: left eyes were used as vehicle control and right eyes were intravitreally injected with TRPV4-selective antagonist GSK2193874, 24 h before the end of the 4 weeks of diabetes. Histological data show that retinal thickness was similar in nondiabetic and diabetic wt groups but increased in diabetic Trpv4-/- mice. In contrast, DWI shows retinal thinning in diabetic wt mice that was absent in diabetic Trpv4-/- mice. Disorganized outer nuclear layer was observed in diabetic wt but not in diabetic Trpv4-/- retinas. We further demonstrate increased water diffusion, increased distances between photoreceptor nuclei, reduced nuclear area in all nuclear layers, and BRB hyperpermeability, in diabetic wt mice, effects that were absent in diabetic Trpv4-/- mice. Retinas of diabetic mice treated with PBS showed increased water diffusion that was not normalized by GSK2193874. ADC maps in nondiabetic Trpv4-/- mouse retinas showed restricted diffusion. Our data provide evidence that water diffusion is increased in diabetic mouse retinas and that TRPV4 function contributes to retinal hydro-mineral homeostasis and structure under control conditions, and to the development of BRB breakdown and increased water diffusion in the retina under diabetes conditions. A single intravitreous injection of TRPV4 antagonist is however not sufficient to revert these alterations in diabetic mouse retinas.

Author Correction: Dual contribution of TRPV4 antagonism in the regulatory effect of vasoinhibins on blood-retinal barrier permeability: diabetic milieu makes a difference.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Dual contribution of TRPV4 antagonism in the regulatory effect of vasoinhibins on blood-retinal barrier permeability: diabetic milieu makes a difference.

Breakdown of the blood-retinal barrier (BRB), as occurs in diabetic retinopathy and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing vision loss. Vasoinhibins are N-terminal fragments of prolactin that prevent BRB breakdown during diabetes. They modulate the expression of some transient receptor potential (TRP) family members, yet their role in regulating the TRP vanilloid subtype 4 (TRPV4) remains unknown. TRPV4 is a calcium-permeable channel involved in barrier permeability, which blockade has been shown to prevent and resolve pulmonary edema. We found TRPV4 expression in the endothelium and retinal pigment epithelium (RPE) components of the BRB, and that TRPV4-selective antagonists (RN-1734 and GSK2193874) resolve BRB breakdown in diabetic rats. Using human RPE (ARPE-19) cell monolayers and endothelial cell systems, we further observed that (i) GSK2193874 does not seem to contribute to the regulation of BRB and RPE permeability by vasoinhibins under diabetic or hyperglycemic-mimicking conditions, but that (ii) vasoinhibins can block TRPV4 to maintain BRB and endothelial permeability. Our results provide important insights into the pathogenesis of diabetic retinopathy that will further guide us toward rationally-guided new therapies: synergistic combination of selective TRPV4 blockers and vasoinhibins can be proposed to mitigate diabetes-evoked BRB breakdown.

Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death.

The identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca(2+) rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr(-/-)) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.