A ginger extract improves ocular blood flow in rats with endothelin-induced retinal blood flow dysfunction.

The aim of this study was to investigate the effect of a ginger extract on optic nerve head blood flow (ONH BF) under endothelin-1 (ET-1) stimulation. Using laser speckle flowgraphy, we measured ONH BF in brown Norway rats. To establish the ONH BF impairment profile under ET-1 stimulation, we administered an intravitreal injection of ET-1 under anesthesia. We then gave the ginger extract sublingually to assess its effect on ONH BF in both normal and ET-1-induced ischemic conditions. Post ET-1 injection, there were no significant changes in parameters including intraocular pressure or systemic factors. ONH BF showed a dose-dependent decline after ET-1 injection, with a significant reduction after a 2.50 pmol ET-1 dose. Sublingual administration of the ginger extract significantly improved ONH BF in both normal and ET-1-stimulated rats. This suggests that our newly developed supplement for improving ONH BF has a potential role in retinal ischemic diseases, including glaucoma.

Cyclin-Dependent Kinase Inhibitor 2b Mediates Excitotoxicity-Induced Death of Retinal Ganglion Cells.

Purpose: Glutamate excitotoxicity seems to contribute to retinal ganglion cell (RGC) death in various eye diseases, but the underlying molecular mechanisms are not fully understood. We studied the roles of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, known as cellular stress-related senescence markers, in N-methyl-d-aspartate (NMDA)-induced RGC death. Methods: Gene expression was analyzed using quantitative reverse transcription (qRT)-PCR, in situ hybridization, and immunochemistry. Cdkn2a and Cdkn2b gain- and loss-of-function experiments were performed using the adeno-associated virus type 2 (AAV2)-mediated gene delivery system. AAV2-CRISPR-Cas9-mediated knockout of Cdkn2a or Cdkn2b was validated using cultured cells by T7 endonuclease I assay and Western blot analysis. The effects of altered expression of Cdkn2a and Cdkn2b on NMDA-induced RGC death were evaluated by quantification of RNA binding protein with multiple splicing (Rbpms)-immunoreactive RGCs. Results: Intravitreal NMDA injection resulted in upregulation of Cdkn2a and Cdkn2b expression in RGCs of the mouse retina. AAV2-mediated overexpression of Cdkn2b led to increased expression of Cdkn2a in RGCs, but not vice versa. Overexpression of Cdkn2b, but not Cdkn2a, resulted in a further reduction in RGC viability in NMDA-injected retinas. However, excessive levels of Cdkn2a or Cdkn2b had no effect on RGC viability in healthy mice. AAV2-CRISPR-Cas9-mediated knockout of either Cdkn2a or Cdkn2b attenuated NMDA-induced RGC death. Conclusions: Cdkn2a and Cdkn2b have pivotal roles in the regulation of excitotoxic RGC degeneration under NMDA-induced pathologic conditions. Our findings imply that Cdkn2a and Cdkn2b are novel therapeutic targets for ocular diseases displaying excitotoxicity-induced neuronal degeneration.

Ecel1 Knockdown With an AAV2-Mediated CRISPR/Cas9 System Promotes Optic Nerve Damage-Induced RGC Death in the Mouse Retina.

Purpose: To assess the therapeutic potential of endothelin-converting enzyme-like 1 (Ecel1) in a mouse model of optic nerve crush. Methods: Ecel1 expression was evaluated with real time quantitative (qRT)-PCR, Western blotting, and immunohistochemistry in mouse retinas after optic nerve crush. Vinblastine administration to the optic nerve and the intravitreal injection of N-methyl-d-aspartate (NMDA) were used to assess Ecel1 gene expression. Ecel1 was deleted with an adeno-associated viral (AAV) clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 system, and retinal ganglion cell (RGC) survival was investigated with retrograde labeling, qRT-PCR, and visual evoked potential. Results: Optic nerve crush induced Ecel1 expression specifically in the RGCs, peaking on day 4 after optic nerve crush. Ecel1 gene expression was induced by the vinblastine-induced inhibition of axonal flow, but not by NMDA-induced excitotoxicity, even though both are triggers of RGC death. Knockdown of Ecel1 promoted the loss of RGCs after optic nerve crush. Conclusions: Our data suggest that Ecel1 induction is part of the retinal neuroprotective response to axonal injury in mice. These findings might provide insight into novel therapeutic targets for the attenuation of RGC damage, such as occurs in traumatic optic neuropathy.

Bilberry extract administration prevents retinal ganglion cell death in mice via the regulation of chaperone molecules under conditions of endoplasmic reticulum stress.

PURPOSE: To investigate the effect of bilberry extract anthocyanins on retinal ganglion cell (RGC) survival after optic nerve crush. Additionally, to determine details of the mechanism of the neuroprotective effect of bilberry extract anthocyanins and the involvement of endoplasmic reticulum stress suppression in the mouse retina. MATERIALS AND METHODS: Anthocyanins in bilberry extract (100 mg/kg/day or 500 mg/kg/day) were administrated orally to C57BL/6J mice. The expression levels of various molecular chaperones were assessed with quantitative reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry. RGC survival was evaluated by measuring the gene expression of RGC markers and counting retrogradely labeled RGCs after optic nerve crush. RESULTS: The protein levels of Grp78 and Grp94 increased significantly in mice after bilberry extract administration. Increased Grp78 and Grp94 levels were detected in the inner nuclear layer and ganglion cell layer of the retina, surrounding the RGCs. Gene expression of Chop, Bax, and Atf4 increased in mice after optic nerve crush and decreased significantly after oral bilberry extract administration. RGC survival after nerve crush also increased with bilberry extract administration. CONCLUSION: These results indicate that oral bilberry extract administration suppresses RGC death. Bilberry extract administration increased Grp78 and Grp94 protein levels, an effect which may underlie the neuroprotective effect of bilberry extract after optic nerve crush. Thus, bilberry extract has a potential role in neuroprotective treatments for retinal injuries, such as those which occur in glaucoma.

The neuroprotective effect of hesperidin in NMDA-induced retinal injury acts by suppressing oxidative stress and excessive calpain activation.

We found that hesperidin, a plant-derived bioflavonoid, may be a candidate agent for neuroprotective treatment in the retina, after screening 41 materials for anti-oxidative properties in a primary retinal cell culture under oxidative stress. We found that the intravitreal injection of hesperidin in mice prevented reductions in markers of the retinal ganglion cells (RGCs) and RGC death after N-methyl-D-aspartate (NMDA)-induced excitotoxicity. Hesperidin treatment also reduced calpain activation, reactive oxygen species generation and TNF-α gene expression. Finally, hesperidin treatment improved electrophysiological function, measured with visual evoked potential, and visual function, measured with optomotry. Thus, we found that hesperidin suppressed a number of cytotoxic factors associated with NMDA-induced cell death signaling, such as oxidative stress, over-activation of calpain, and inflammation, thereby protecting the RGCs in mice. Therefore, hesperidin may have potential as a therapeutic supplement for protecting the retina against the damage associated with excitotoxic injury, such as occurs in glaucoma and diabetic retinopathy.

Metabolomic profiling of reactive persulfides and polysulfides in the aqueous and vitreous humors.

We investigate the metabolomic profile of reactive persulfides and polysulfides in the aqueous and vitreous humors. Eighteen eyes of 18 consecutive patients with diabetes mellitus (DM) and diabetic retinopathy underwent microincision vitrectomy combined with cataract surgery. Samples of the aqueous and vitreous humors were collected and underwent mass spectrometry-based metabolomic profiling of reactive persulfides and polysulfides (polysulfidomics). The effect of reactive polysulfide species on the viability of immortalized retinal cells (the RGC-5 cell line) under oxidative stress (induced with H2O2) was also evaluated with an Alamar Blue assay. The experiments showed that cysteine persulfides (CysSSH), oxidized glutathione trisulfide (GSSSG) and cystine were elevated in the aqueous humor, and CysSSH, Cys, and cystine were elevated in the vitreous. Furthermore, GSSSG, cystine, and CysSSH levels were correlated in the aqueous and vitreous humors. A comparison, in DM and control subjects, of plasma levels of reactive persulfides and polysulfides showed that they did not differ. In vitro findings revealed that reactive polysulfide species increased cell viability under oxidative stress. Thus, various reactive persulfides and polysulfides appear to be present in the eye, and some reactive sulfide species, which have a protective effect against oxidative stress, are upregulated in the aqueous and vitreous humors of DM eyes.

Restoration of the majority of the visual spectrum by using modified Volvox channelrhodopsin-1.

We previously showed that blind rats whose vision was restored by gene transfer of Chlamydomonas channelrhodopsin-2 (ChR2) could only detect wavelengths less than 540 nm because of the action spectrum of the transgene product. Volvox-derived channelrhodopsin-1, VChR1, has a broader spectrum than ChR2. However, the VChR1 protein was mainly localized in the cytoplasm and showed weak ion channel properties when the VChR1 gene was transfected into HEK293 cells. We generated modified Volvox channelrhodopsin-1 (mVChR1), which is a chimera of Volvox channelrhodopsin-1 and Chlamydomonas channelrhodopsin-1 and demonstrated increased plasma membrane integration and dramatic improvement in its channel properties. Under whole-cell patch clamp, mVChR1-expressing cells showed a photo-induced current upon stimulation at 468-640 nm. The evoked currents in mVChR1-expressing cells were ~30 times larger than those in VChR1-expressing cells. Genetically, blind rats expressing mVChR1 via an adeno-associated virus vector regained their visual responses to light with wavelengths between 468 and 640 nm and their recovered visual responses were maintained for a year. Thus, mVChR1 is a candidate gene for gene therapy for restoring vision, and gene delivery of mVChR1 may provide blind patients access to the majority of the visible light spectrum.

Essential role of thioredoxin 2 in mitigating oxidative stress in retinal epithelial cells.

The retina is constantly subjected to oxidative stress, which is countered by potent antioxidative systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems leads to the development of age-related macular degeneration. Thioredoxin 2 (Trx2) is a potent antioxidant, which acts directly on mitochondria. In the present study, oxidative stress was induced in the human RPE cell line (ARPE-19) using 4-hydroxynonenal (4-HNE) or C2-ceramide. The protective effect of Trx2 against oxidative stress was investigated by assessing cell viability, the kinetics of cell death, mitochondrial metabolic activity, and expression of heat shock proteins (Hsps) in Trx2-overexpressing cell lines generated by transfecting ARPE cells with an adeno-associated virus vector encoding Trx2. We show that overexpression of Trx2 reduced cell death induced by both agents when they were present in low concentrations. Moreover, early after the induction of oxidative stress Trx2 played a key role in the maintenance of the cell viability through upregulation of mitochondrial metabolic activity and inhibition of Hsp70 expression.

Establishment of monocular-limited photoreceptor degeneration models in rabbits.

BACKGROUND: Numerous rodent models of photoreceptor degeneration have been developed for the study of visual function. However, no viable model has been established in a species that is more closely related to Homo sapiens. Here, we present a rabbit model of monocular photoreceptor degeneration. METHODS: We tested 2 chemicals, verteporfin and sodium nitroprusside (SNP), for developing a 1-eye limited photoreceptor degeneration model in pigmented rabbits. After the intravenous injection of verteporfin, the retina was exposed to light from a halogen lamp for 0, 10, 30, or 60 min. Alternately, 100 µL of various concentrations of sodium nitroprusside (0.1 mM, 0.5 mM, and 1 mM) were intravitreously injected into the rabbit eye. Retinal degeneration was evaluated by fundus photography, electroretinogram (ERG), and histological examinations. RESULTS: Fundus photographs of animals in the verteporfin- or SNP-treated groups showed evidence of retinal degeneration. The severity of this degradation depended on the duration of light exposure and the concentration of SNP administered. The degeneration was clearly limited to the light-exposed areas in the verteporfin-treated groups. Extensive retinal atrophy was observed in the SNP-treated groups. The a- and b-wave amplitudes were dramatically decreased on the ERGs from SNP-treated groups. Histological examination revealed that either verteporfin or SNP induced severe photoreceptor degeneration. High-dose SNP treatment (1 mM) was also associated with inner retinal layer degeneration. CONCLUSIONS: Both SNP and verteporfin clearly caused photoreceptor degeneration without any effect on the contralateral eye. These compounds therefore represent valuable tools for the empirical investigation of visual function recovery. The findings will inform guidelines for clinical applications such as retinal prostheses, cell-based therapy, and gene therapy.

Different anti-oxidant effects of thioredoxin 1 and thioredoxin 2 in retinal epithelial cells.

Age-related macular degeneration (AMD) affects the retina and is the most common cause of blindness in elderly persons in developed countries. The retina is constantly subjected to oxidative stress; to avoid the effects of oxidative stress, retinal pigment epithelial (RPE) cells possess potent anti-oxidant systems. Disruption of these systems leads to dysfunction of RPE cells, which then accelerates the development of AMD. Here, we investigated the role of thioredoxins (TRXs), scavengers of intracellular reactive oxygen species, by assessing the effect of TRX overexpression on cell viability, morphology, NF-κB expression, and mitochondrial membrane potential, in RPE cells. TRX-overexpressing cell lines were generated by infection of an established human RPE cell line (ARPE) with adeno-associated virus vectors encoding either TRX1 or TRX2. We showed that overexpression of TRXs reduced cell death caused by 4-hydroxynonenal (4-HNE)-induced oxidative stress; TRX2 was more effective than TRX1 in promoting cell survival. 4-HNE caused perinuclear NF-κB accumulation, which was absent in TRX-overexpressing cells. Moreover, overexpression of TRXs prevented depolarization of mitochondrial membranes; again, TRX2 was more effective than TRX1 in maintaining the membrane potential. The difference in the protective effects of these TRXs against oxidative stress may be due to their expression profile. TRX2 was expressed in the mitochondria, while TRX1 was expressed in the cytoplasm. Thus, TRX2 may directly protect mitochondria by preventing depolarization. These results demonstrate that TRXs are potent antioxidant proteins in RPE cells and their direct effect on mitochondria may be a key to prevent oxidative stress.

Age-dependent differences in recovered visual responses in Royal College of Surgeons rats transduced with the Channelrhodopsin-2 gene.

The objective of this study is to investigate age-related differences in recovered visual function in Royal College of Surgeons (RCS) rats transduced with the Channelrhodopsin-2 (ChR2) gene. An adeno-associated virus vector that contained ChR2 was injected intravitreously into young or aged RCS rats. After 4 months, visual evoked potentials were recorded. To estimate the transduction efficiencies, ChR2V-expressing cells and retrograde labeled retinal ganglion cells (RGCs) were counted. After photoreceptor degradation, immunohistochemistry was used to detect glial fibrillary acidic protein (GFAP) in the retinas. The amplitudes and latencies from young RCS rats were higher and shorter, respectively, than those from aged RCS rats. ChR2V was expressed in the RGCs of both groups of rats; there was no significant difference in the transduction efficiency of either group. However, the number of RGCs in aged RCS rats was significantly less than that in young RCS rats. In addition, strong GFAP immunoreactivity was observed after photoreceptor degeneration, whereas it was weaker in ChR2V-expressing RGCs. ChR2 transduction produced photosensitive RGCs in both young and aged rats. However, the degree of recovery depended on the age at the time of transduction.

TRPV2 enhances axon outgrowth through its activation by membrane stretch in developing sensory and motor neurons.

Thermosensitive TRP (thermo TRP) channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, and osmolarity. However, the precise roles for the thermo TRP channels during development have not been determined. To explore the functional importance of thermo TRP channels during neural development, the temporal expression was determined in embryonic mice. Interestingly, TRPV2 expression was detected in spinal motor neurons in addition to the dorsal root ganglia from embryonic day 10.5 and was localized in axon shafts and growth cones, suggesting that the channel is important for axon outgrowth regulation. We revealed that endogenous TRPV2 was activated in a membrane stretch-dependent manner in developing neurons by knocking down the TRPV2 function with dominant-negative TRPV2 and TRPV2-specific shRNA and significantly promoted axon outgrowth. Thus, for the first time we revealed that TRPV2 is an important regulator for axon outgrowth through its activation by membrane stretch during development.

Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCepsilon-mediated phosphorylation at S800.

Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKCepsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKCepsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKCepsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKCepsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.

Regulation mechanisms of vanilloid receptors.

The capsaicin receptor TRPV1 (also known as the vanilloid receptor VR1) is a non-selective cation channel and is activated not only by capsaicin but also by noxious heat or protons. Tissue damage associated with infection, inflammation or ischaemia, produces an array of chemical mediators that activate or sensitize nociceptor terminals. An important component of this pro-algeic response is ATP. In cells expressing TRPV1, ATP increased the currents evoked by capsaicin or protons through activation of P2Y metabotropic receptors in a PKC-dependent manner. In the presence of ATP, the temperature threshold for TRPV1 activation was reduced from 42 degrees C to 35 degrees C, such that normal body temperature could activate TRPV1. Functional interaction between P2Y receptors and TRPV1 was confirmed in a behavioural analysis using TRPV1-deficient mice. Direct phosphorylation of TRPV1 by PKC was confirmed biochemically and the two serine residues involved were identified. Extracellular Ca2+ -dependent desensitization of TRPV1 is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. The Ca2+ -binding protein calmodulin binds to the C-terminus of TRPV1. We found that disruption of the calmodulin binding segment prevented TRPV1 desensitization even in the presence of extracellular Ca2+.

Structural determinant of TRPV1 desensitization interacts with calmodulin.

The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca2+-dependent desensitization of TRPV1 observed in patch-clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. Here, we show that the Ca2+-binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and that disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain.