How the absence of gravity affects the physiology of human beings is generating global research interest as space exploration, including missions aboard the International Space Station, continues to push boundaries. Here, we examined changes in retinal microcirculation and visual electrophysiology in mice suspended by their tails to simulate the cephalad movement of blood that occurs under microgravity conditions. Tail suspension was performed with a head-down tilt with a recommended angle of 30°. Mice in the control groups were similarly attached to a tether but could maintain a normal position. Morphologically, the 15-day tail-suspended mice showed retinal microvascular dilation, tortuosity, and a relatively long fluorescence retention; however, the average diameter of the major retinal vessels was not notably changed. In addition, optical coherence tomography showed their optic nerve head had an increased diameter. However, the mice could adapt to the change, with microcirculation and the optic nerve head recovering following 30-day tail suspension. Expression of rhodopsin and cone-opsins was not notably changed, and no retinal apoptotic-positive cells were detected between 15- and 30-day tail suspensions. Moreover, the three experimental groups of suspended mice showed normal retinal layers and thickness. Functionally, following 15-day tail suspension, scotopic electroretinograms showed a decline in the oscillatory potentials (OPs), but not in the b wave; simultaneously, the peak time of flash visual evoked potential component N1 was delayed compared to its baseline and the time-matched control. Following 30-day tail suspension, the OPs (O2) amplitude recovered to approximately 97% of its baseline or 86% of the time-matched control level. By simulating cephalad shifting of blood, short-term tail suspension can affect rodent retinal microcirculation, the optic nerve head, and disturb visual electrophysiology. However, the change is reversible with no permanent injury observed in the retina. The mice could adapt to the short-term change of retinal microcirculation, indicating new conditions that could be combined with, or could enhance, simulated microgravity for further studying the impact of short- or long-term outer space conditions on the retina.

We investigated the effects of hypercholesterolemia on optic nerve head (ONH) blood flow, visual function, and retinal histology in a rabbit model. Hypercholesterolemia was induced in rabbits by feeding them a high cholesterol (1%) diet for 12 weeks. Changes in blood pressure, intraocular pressure (IOP), and ONH blood flow were monitored at 6 and 12 weeks after treatment. The autoregulation of ONH blood flow as detected by laser speckle flowgraphy was verified by an artificial elevation of IOP at 12 weeks. Visually evoked potentials (VEPs) were also recorded and analyzed at 6 and 12 weeks. Finally, a histological examination as well as immunohistochemistry to endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was performed. In the hypercholesterolemic rabbits, blood pressure, IOP, and ONH blood flow did not alter significantly throughout this study. The autoregulation of ONH blood flow against IOP elevation was found to be impaired at 12 weeks. The amplitudes of the first negative peak of VEPs were diminished. Both the density of the retinal ganglion cells and the thickness of the inner nuclear layer and photoreceptor cell layer were reduced. Immunoreactivity to eNOS was reduced and that to iNOS was enhanced in the hypercholesterolemic rabbits compared to those in the normal control rabbits. The results of this study show that hypercholesterolemia induces impairment in the autoregulation of ONH blood flow and deterioration in visual function and histology. Downregulation of eNOS activity might be one of the causes for impairment of the autoregulation. Enhanced activity of iNOS might be involved in the impaired visual function and histology.

Porcine models of ophthalmological diseases are often used in pre-clinical translational studies due to pigs' similarities to humans. In particular, the iodoacetic acid (IAA) model of photoreceptor degeneration seems to mimic well the endstage phenotype of human pathologies as retinitis pigmentosa and age-related macular degeneration, with high potential for prosthesis/retinal devices testing. IAA is capable of inducing photoreceptor death by blockage of glycolysis, and its effects on the retina have been described. Nonetheless, up to date, literature lacks of a comprehensive morpho-functional characterization of the entire visual system of this model. This gap is particularly critical for prosthesis testing as inner retinal structures and optic pathways must be preserved to elicit cortical responses and restore vision. In this study, we investigated the functional and anatomical features of the visual system of IAA-treated pigs and compared them to control animals. IAA was administered intravenously at 12 mg/kg; control animals received saline solution (NaCl 0.9% w/v). Electrophysiological analyses included full-field (ffERGs) and pattern (PERGs) electroretinograms and flash visually evoked potentials (fVEPs). Histological evaluations were performed on the retina and the optic pathways and included thickness of the different retinal layers, ganglion cells count, and immunohistochemistry for microglial cells, macroglial cells, and oligodendrocytes. The histological results indicate that IAA treatment does not affect the morphology of the inner retina and optic pathways. Electrophysiology confirms the selective rod and partial cone degeneration, but is ambiguous as to the functionality of the optic pathways, seemingly preserved as indicated by the still detectable fVEPs. Overall, the work ameliorates the characterization of such rapid and cost-effective model, providing more strength and reliability for future pre-clinical translational trials.

Synaptotagmins are the primary Ca2+ sensors for synaptic exocytosis. Previous work suggested synaptotagmin-1 (Syt1) mediates evoked vesicle release from cone photoreceptor cells in the vertebrate retina whereas release from rods may involve another sensor in addition to Syt1. We found immunohistochemical evidence for syntaptotagmin-7 (Syt7) in mouse rod terminals and so performed electroretinograms (ERG) and single-cell recordings using mice in which Syt1 and/or Syt7 were conditionally removed from rods and/or cones. Synaptic release was measured in mouse rods by recording presynaptic anion currents activated during glutamate re-uptake and from exocytotic membrane capacitance changes. Deleting Syt1 from rods reduced glutamate release evoked by short depolarizing steps but not long steps whereas deleting Syt7 from rods reduced release evoked by long but not short steps. Deleting both sensors completely abolished depolarization-evoked release from rods. Effects of various intracellular Ca2+ buffers showed that Syt1-mediated release from rods involves vesicles close to ribbon-associated Ca2+ channels whereas Syt7-mediated release evoked by longer steps involves more distant release sites. Spontaneous release from rods was unaffected by eliminating Syt7. While whole animal knockout of Syt7 slightly reduced ERG b-waves and oscillatory potentials, selective elimination of Syt7 from rods had no effect on ERGs. Furthermore, eliminating Syt1 from rods and cones abolished ERG b-waves and additional elimination of Syt7 had no further effect. These results show that while Syt7 contributes to slow non-ribbon release from rods, Syt1 is the principal sensor shaping rod and cone inputs to bipolar cells in response to light flashes.

The DBA/2J mouse is a common animal model of glaucoma. The intraocular pressure increases with age, and retinal ganglion cells (RGC) degenerate, usually starting at an age of approximately six months. In this study, we used two-year-old DBA/2J mice presuming an end-point of RGC degeneration. We investigated visual function in these animals using electroretinography (ERG) and visual evoked potentials (VEP), and we checked the number of remaining RGC by retrograde staining. Almost no RGC were left in the retina, and VEP were hardly recordable. Surprisingly, also ERG amplitudes of scotopic a-waves and b-waves, photopic b-waves and oscillatory potentials were decreased significantly by approximately 40% compared to amplitudes measured in age-matched C57BL/6J mice. The latencies were not changed in DBA/2J mice compared to C57BL/6J mice, and so were the ratios between amplitudes of a-waves, b-waves and oscillatory potentials. Our results indicate that, in addition to degeneration of RGC, also photoreceptors are affected by pathological processes in the eye caused by the mutations present in DBA/2J mice.

We investigated the feasibility and efficacy of using a specific sphingosine 1-phosphate (S1P1) receptor agonist, CYM-5442, to slow or block retinal ganglion cell (RGC) loss in endothelin-1 (ET-1) induced RGC loss. A single intravitreal injection of ET-1 (20pmol/ul), a potent vasoactive peptide that produces retinal vessels vasoconstriction, was used to induce and characterize RGC-specific cell death. CYM-5442 (1 mgr/kg) or vehicle was administered intraperitoneally for five consecutive days after ET-1-induced RGC loss. The functional extent of RGC loss injury was evaluated with pattern visual evoked potentials (VEP) and electroretinography. RGCs and retinal nerve fiber layer (RNFL) thickness were assessed in vivo using optical coherence tomography and ex vivo using Brn3a immunohistochemistry in flat-mounted retinas. ET-1 caused significant RGC loss and function loss one week after intravitreal injection. VEP showed preserved visual function after CYM-5442 administration compared to vehicle-treated animals (11.95 ± 0.86 μV vs 3.47 ± 1.20 μV, n = 12) (p < 0.05). RNFL was significantly thicker in the CYM treated-animals compared to the vehicle (93.62 ± 3.22 μm vs 77.72 ± 0.35 μm, n = 12) (p < 0.05). Furthermore, Brn3a immunohistochemistry validated this observation, showing significantly higher RGCs numbers in CYM treated rats than in the vehicle group (76,540 ± 303 vs 52,426 ± 1,932 cells/retina, n = 9) (p = 0.05). CYM-5442 administration was associated with significant retinal cleaved caspase-3 deactivation, indicating reduced apoptotic levels. The results of the present study further demonstrate the important role of S1P1 receptor agonists to lessen intravitreal ET-1 induced RGC loss.

Secondary degeneration is a common event in traumatic central nervous system disorders, which involves neuronal apoptosis and mitochondrial dysfunction. Exogenous methane exerts the therapeutic effects in many organ injury. Our study aims to investigate the potential neuroprotection of methane in a rat model of optic nerve crush (ONC). Adult male Sprague-Dawley rats were subjected to ONC and administrated intraperitoneally with methane-saturated or normal saline (10 ml/kg) once per day for one week after ONC. The retinal ganglion cells (RGCs) density was assessed by hematoxylin and eosin staining and Fluoro-Gold retrogradely labeling. Visual function was evaluated by flash visual evoked potentials (FVEP). The retinal apoptosis was measured by terminal-deoxy-transferase-mediated dUTP nick end labeling (TUNEL) assay and the expression of apoptosis-related factors, such as phosphorylated Bcl-2-associated death promoter (pBAD), phosphorylated glycogen synthase kinase-3β (pGSK-3β), Bcl-2 associated X protein (Bax) and Bcl-2 extra large (Bcl-xL). Retinal mitochondrial function was assessed by the mRNA expressions of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), the mitochondrial DNA (mtDNA) copy number, citrate synthase activity and ATP content. Methane treatment significantly improved the RGC loss and visual dysfunction following ONC. As expected, methane also remarkably inhibited the retinal neural apoptosis, such as the fewer TUNEL-positive cells in ganglion cell layer, accompanied by the up-regulations of anti-apoptotic factors (pGSK-3β, pBAD, Bcl-xL) and the down-regulation of pro-apoptotic factor (Bax). Furthermore, methane treatment suppressed up-regulations of critical mitochondrial components (PGC-1α, NRF1 and TFAM) mRNA and mtDNA copy number, as well as improved the reduction of functional mitochondria markers, including citrate synthase activity and ATP content, in retinas with ONC. Taken together, methane treatment promotes RGC survival and limits retinal mitochondrial dysfunction against ONC insult. Methane can be a potential neuroprotective agent for traumatic and glaucomatous neurodegeneration.

The loss of retinal ganglion cells (RGCs) is one of the common pathological features associated with optic nerve diseases leading to blindness. The aims of our study were to compare the neuroprotection of two forms of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on RGCs and axon regeneration after optic nerve crush (ONC) in vivo, and to investigate the molecular mechanism. The effects of intravitreally transplanted hUCB-MSCs cultured in two-dimensional (2D-MSCs) and spheroids (3D-MSCs) were assessed by the survival of RGCs, regenerating axons, and flash visual evoked potentials (fVEP); the level of signal factors secreted by transplanted MSCs in vitreous and the marker protein levels of JAK/STAT3, PI3K/Akt/mTOR and MAPK/ERK pathways were detected using Bead-Based analysis and Western blot, respectively. We found that RGCs began to lose at day 3 after ONC, rapidly decreased at day 7, and flattened at day 14. The neuroprotection of transplanted 2D-MSCs was much stronger than that of 3D-MSCs. The transplanted 2D-MSCs could survive at least 2 weeks without differentiation and keep the characters of MSCs, which secreted multiple tropic factors and accompanied by activation of JAK/STAT3 and MAPK/ERK signaling pathways, top three most abundant factors: stem cell growth factor- β (SCGF-β), hepatocyte growth factor (HGF), and monocyte chemoattractant protein-1 (MCP-1). These results indicate that intravitreal injection of 2D-MSCs is a promising therapeutic strategy for retinal pathological diseases characterized by the loss of RGCs and open the door for the application of SCGF-β, HGF, and MCP-1 in the treatment of optic nerve diseases.