Dysfunctional cGMP Signaling Leads to Age-Related Retinal Vascular Alterations and Astrocyte Remodeling in Mice.

The nitric oxide-guanylyl cyclase-1-cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway is integral to the control of vascular tone and morphology. Mice lacking the alpha catalytic domain of guanylate cyclase (GC1-/-) develop retinal ganglion cell (RGC) degeneration with age, with only modest fluctuations in intraocular pressure (IOP). Increasing the bioavailability of cGMP in GC1-/- mice prevents neurodegeneration independently of IOP, suggesting alternative mechanisms of retinal neurodegeneration. In continuation to these studies, we explored the hypothesis that dysfunctional cGMP signaling leads to changes in the neurovascular unit that may contribute to RGC degeneration. We assessed retinal vasculature and astrocyte morphology in young and aged GC1-/- and wild type mice. GC1-/- mice exhibit increased peripheral retinal vessel dilation and shorter retinal vessel branching with increasing age compared to Wt mice. Astrocyte cell morphology is aberrant, and glial fibrillary acidic protein (GFAP) density is increased in young and aged GC1-/- mice, with areas of dense astrocyte matting around blood vessels. Our results suggest that proper cGMP signaling is essential to retinal vessel morphology with increasing age. Vascular changed are preceded by alterations in astrocyte morphology which may together contribute to retinal neurodegeneration and loss of visual acuity observed in GC1-/- mice.

Intravitreal injection worsens outcomes in a mouse model of indirect traumatic optic neuropathy from closed globe injury.

It is well established that an intravitreal needle poke or injection of buffer is protective to the retina in models of photoreceptor degeneration due to release of endogenous neurotrophic factors. Here we assess the effect of intravitreal injection of buffer in a model of closed globe trauma that causes air-blast induced indirect traumatic optic neuropathy (bITON). We injected animals 1-day after the last bITON or sham procedure and performed assessments 1-month later. Surprisingly, we detected a lower electroretinogram (ERG), greater optic nerve damage, and increased levels of pro-inflammatory cytokines in animals given an intravitreal injection. The effect was sometimes independent of bITON and sometimes exacerbated by the injury. Retina histology appeared normal, however the total number of axons in the optic nerve was lower even in uninjured animals that were injected. The number of degenerative axons was further increased in injured animals that were injected. In contrast, we detected a decrease in the ERG a wave and b wave amplitudes, but no effect on the visual evoked potential. Levels of the pro-inflammatory cytokines, IL-1α and IL-1ß were elevated in the mice that received an intravitreal injection. This increase was even greater in animals that also had a bITON. This suggests that intravitreal injections may be injurious to the optic nerve particularly during the acute stage of optic nerve injury. In addition, the data suggests a role for IL-1α and IL-1ß in this response.

Galantamine protects against synaptic, axonal, and vision deficits in experimental neurotrauma.

Our goal was to investigate the neuroprotective effects of galantamine in a mouse model of blast-induced indirect traumatic optic neuropathy (bITON). Galantamine is an FDA-approved acetylcholinesterase inhibitor used to treat mild-moderate Alzheimer's disease. We exposed one eye of an anesthetized mouse to repeat bursts of over-pressurized air to induce traumatic optic neuropathy. Mice were given regular or galantamine-containing water (120 mg/L) ad libitum, beginning immediately after blast and continuing for one month. Electroretinograms and visual evoked potentials were performed just prior to endpoint collection. Histological and biochemical assessments were performed to assess activation of sterile inflammation, axon degeneration, and synaptic changes. Galantamine treatment mitigated visual function deficits induced by our bITON model via preservation of the b-wave of the electroretinogram and the N1 of the visual evoked potential. We also observed a reduction in axon degeneration in the optic nerve as well as decreased rod bipolar cell dendritic retraction. Galantamine also showed anti-inflammatory and antioxidant effects. Galantamine may be a promising treatment for blast-induced indirect traumatic optic neuropathy as well as other optic neuropathies.

Assessment of necroptosis in the retina in a repeated primary ocular blast injury mouse model.

Primary blast injury (caused by the initial rapid increase in pressure following an explosive blast) to the retina and optic nerve (ON) causes progressive visual loss and neurodegeneration. Military personnel are exposed to multiple low-overpressure blast waves, which may be in quick succession, such as during breacher training or in combat. We investigated the necroptotic cell death pathway in the retina in a mouse repeated primary ocular blast injury (rPBI) model using immunohistochemistry. We further evaluated whether intravitreal injections of a potent necroptosis inhibitor, Necrostatin-1s (Nec-1s), protects the retina and ON axons by retinal ganglion cells (RGC) counts, ON axonal counting and optical coherence tomography (OCT) analysis of vitreous haze. Receptor interacting protein kinase (RIPK) 3, increased in the inner plexiform layer 2 days post injury (dpi) and persisted until 14 dpi, whilst RIPK1 protein expression did not change after injury. The number of degenerating ON axons was increased at 28 dpi but there was no evidence of a reduction in the number of intact ON axons or RNA-binding protein with multiple splicing (RBPMS)+ RGC in the retina by 28 dpi in animals not receiving any intravitreal injections. But, when intravitreal injections (vehicle or Nec-1s) were given there was a significant reduction in RBPMS+ RGC numbers, suggesting that rPBI with intraocular injections is damaging to RGC. There were fewer RGC lost after Nec-1s than vehicle injection, but there was no effect of Nec-1s or vehicle treatment on the number of degenerating axons. OCT analysis demonstrated no effect of rPBI on vitreous haze, but intravitreal injection combined with rPBI increased vitreous haze (P = 0.004). Whilst necroptosis may be an active cell death signalling pathway after rPBI, its inhibition did not prevent cell death, and intravitreal injections in combination with rPBI increased vitreous inflammation and reduced RBPMS+ RGC numbers, implying intravitreal injection is not an ideal method for drug delivery after rPBI.

Phenotypes of primary retinal macroglia: Implications for purification and culture conditions.

Many neurodegenerations, including those of the visual system, have complex etiologies that include roles for both neurons and glia. In the retina there is evidence that retinal astrocytes play an important role in neurodegeneration. There are several approaches for isolating and growing primary retinal astrocytes, however, they often lead to different results. In this study, we examined the influence of culture conditions on phenotypic maturation of primary, purified retinal glia. We compared retinal astrocytes and Müller glia purified by immunomagnetic separation, as differentiation between these astrocyte subtypes is critical and immuno-based methods are the standard practice of purification. We found that while time in culture impacts the health and phenotype of both astrocytes and Müller glia, the phenotypic maturation of retinal astrocytes was most impacted by serum factors. These factors appeared to actively regulate intermediate filament phenotypes in a manner consistent with the induction of astrocyte-mesenchymal transition (AMT). This propensity for retinal astrocytes to shift along an AMT continuum should be considered when interpreting resulting data. Our goal is that this study will help standardize the field so that studies are replicable, comparable, and as accurate as possible for subsequent interpretation of findings.

Virus-mediated EpoR76E Therapy Slows Optic Nerve Axonopathy in Experimental Glaucoma.

Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)-lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus-mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment.

Erythropoietin either Prevents or Exacerbates Retinal Damage from Eye Trauma Depending on Treatment Timing.

PURPOSE: Erythropoietin (EPO) is a promising neuroprotective agent and is currently in Phase III clinical trials for the treatment of traumatic brain injury. The goal of this study was to determine if EPO is also protective in traumatic eye injury. METHODS: The left eyes of anesthetized DBA/2J or Balb/c mice were exposed to a single 26 psi overpressure air-wave while the rest of the body was shielded. DBA/2J mice were given intraperitoneal injections of EPO or buffer and analyses were performed at 3 or 7 days post-blast. Balb/c mice were given intramuscular injections of rAAV.EpoR76E or rAAV.eGFP either pre- or post-blast and analyses were performed at 1 month post-blast. RESULTS: EPO had a bimodal effect on cell death, glial reactivity, and oxidative stress. All measures were increased at 3 days post-blast and decreased at 7-days post-blast. Increased retinal ferritin and NADPH oxygenases were detected in retinas from EPO-treated mice. The gene therapy approach protected against axon degeneration, cell death, and oxidative stress when given after blast, but not before. CONCLUSIONS: Systemic, exogenous EPO and EPO-R76E protects the retina after trauma even when initiation of treatment is delayed by up to 3 weeks. Systemic treatment with EPO or EPO-R76E beginning before or soon after trauma may exacerbate protective effects of EPO within the retina as a result of increased iron levels from erythropoiesis and, thus, increased oxidative stress within the retina. This is likely overcome with time as a result of an increase in levels of antioxidant enzymes. Either intraocular delivery of EPO or treatment with non-erythropoietic forms of EPO may be more efficacious.

Virus-mediated EpoR76E gene therapy preserves vision in a glaucoma model by modulating neuroinflammation and decreasing oxidative stress.

BACKGROUND: Glaucoma is a complex neurodegeneration and a leading cause of blindness worldwide. Current therapeutic strategies, which are all directed towards lowering the intraocular pressure (IOP), do not stop progression of the disease. We have demonstrated that recombinant adeno-associated virus (rAAV) gene delivery of a form of erythropoietin with attenuated erythropoietic activity (EpoR76E) can preserve retinal ganglion cells, their axons, and vision without decreasing IOP. The goal of this study was to determine if modulation of neuroinflammation or oxidative stress played a role in the neuroprotective activity of EPO.R76E. METHODS: Five-month-old DBA/2J mice were treated with either rAAV.EpoR76E or a control vector and collected at 8 months of age. Neuroprotection was assessed by quantification of axon transport and visual evoked potentials. Microglia number and morphology and cytokine and chemokine levels were quantified. Message levels of oxidative stress-related proteins were assessed. RESULTS: Axon transport and visual evoked potentials were preserved in rAAV.EpoR76E-treated mice. The number of microglia was decreased in retinas from 8-month-old rAAV.EpoR76E-treated mice, but proliferation was unaffected. The blood-retina barrier was also unaffected by treatment. Levels of some pro-inflammatory cytokines were decreased in retinas from rAAV.EpoR76E-treated mice including IL-1, IL-12, IL-13, IL-17, CCL4, and CCL5. TNFα messenger RNA (mRNA) was increased in retinas from 8-month-old mice compared to 3-month-old controls regardless of treatment. Expression of several antioxidant proteins was increased in retinas of rAAV.EpoR76E-treated 8-month-old mice. CONCLUSIONS: Treatment with rAAV.EpoR76E preserves vision in the DBA/2J model of glaucoma at least in part by decreasing infiltration of peripheral immune cells, modulating microglial reactivity, and decreasing oxidative stress.

Exacerbation of blast-induced ocular trauma by an immune response.

BACKGROUND: Visual prognosis after an open globe injury is typically worse than after a closed globe injury due, in part, to the immune response that ensues following open globe trauma. There is a need for an animal model of open globe injury in order to investigate mechanisms of vision loss and test potential therapeutics. METHODS: The left eyes of DBA/2 J mice were exposed to an overpressure airwave blast. This strain lacks a fully functional ocular immune privilege, so even though the blast wave does not rupture the globe, immune infiltrate and neuroinflammation occurs as it would in an open globe injury. For the first month after blast wave exposure, the gross pathology, intraocular pressure, visual function, and retinal integrity of the blast-exposed eyes were monitored. Eyes were collected at three, seven, and 28 days to study the histology of the cornea, retina, and optic nerve, and perform immunohistochemical labeling with markers of cell death, oxidative stress, and inflammation. RESULTS: The overpressure airwave caused anterior injuries including corneal edema, neovascularization, and hyphema. Immune infiltrate was detected throughout the eyes after blast wave exposure. Posterior injuries included occasional retinal detachments and epiretinal membranes, large retinal pigment epithelium vacuoles, regional photoreceptor cell death, and glial reactivity. Optic nerve degeneration was evident at 28 days post-blast wave exposure. The electroretinogram (ERG) showed an early deficit in the a wave that recovered over time. Both visual acuity and the ERG b wave showed an early decrease, then a transient improvement that was followed by further decline at 28 days post-blast wave exposure. CONCLUSIONS: Ocular blast injury in the DBA/2 J mouse recapitulates damage that is characteristic of open globe injuries with the advantage of a physically intact globe that prevents complications from infection. The injury was more severe in DBA/2 J mice than in C57Bl/6 J mice, which have an intact ocular immune privilege. Early injury to the outer retina mostly recovers over time. In contrast, inner retinal dysfunction seems to drive later vision loss.

Comprehensive behavioral and physiologic assessment of peripheral and central auditory function in individuals with mild traumatic brain injury.

Auditory complaints are frequently reported by individuals with mild traumatic brain injury (mTBI) yet remain difficult to detect in the absence of clinically significant hearing loss. This highlights a growing need to identify sensitive indices of auditory-related mTBI pathophysiology beyond pure-tone thresholds for improved hearing healthcare diagnosis and treatment. Given the heterogeneity of mTBI etiology and the diverse peripheral and central processes required for normal auditory function, the present study sought to determine the audiologic assessments sensitive to mTBI pathophysiology at the group level using a well-rounded test battery of both peripheral and central auditory system function. This test battery included pure-tone detection thresholds, word understanding in quiet, sentence understanding in noise, distortion product otoacoustic emissions (DPOAEs), middle-ear muscle reflexes (MEMRs), and auditory evoked potentials (AEPs), including auditory brainstem responses (ABRs), middle latency responses (MLRs), and late latency responses (LLRs). Each participant also received magnetic resonance imaging (MRI). Compared to the control group, we found that individuals with mTBI had reduced DPOAE amplitudes that revealed a compound effect of age, elevated MEMR thresholds for an ipsilateral broadband noise elicitor, longer ABR Wave I latencies for click and 4 kHz tone burst elicitors, longer ABR Wave III latencies for 4 kHz tone bursts, larger MLR Na and Nb amplitudes, smaller MLR Pb amplitudes, longer MLR Pa latencies, and smaller LLR N1 amplitudes for older individuals with mTBI. Further, mTBI individuals with combined hearing difficulty and noise sensitivity had a greater number of deficits on thalamic and cortical AEP measures compared to those with only one/no self-reported auditory symptoms. This finding was corroborated with MRI, which revealed significant structural differences in the auditory cortical areas of mTBI participants who reported combined hearing difficulty and noise sensitivity, including an enlargement of left transverse temporal gyrus (TTG) and bilateral planum polare (PP). These findings highlight the need for continued investigations toward identifying individualized audiologic assessments and treatments that are sensitive to mTBI pathophysiology.

Ultrasound Modulates Calcium Activity in Cultured Neurons, Glial Cells, Endothelial Cells and Pericytes.

OBJECTIVE: Ultrasound is being researched as a method to modulate the brain. Studies of the interaction of sound with neurons support the hypothesis that mechanosensitive ion channels play an important role in ultrasound neuromodulation. The response of cells other than neurons (e.g., astrocytes, pericytes and endothelial cells) have not been fully characterized, despite playing an important role in brain function. METHODS: To address this gap in knowledge, we examined cultured murine primary cortical neurons, astrocytes, endothelial cells and pericytes in an in vitro widefield microscopy setup during application of a 500 ms burst of 250 kHz focused ultrasound over a pressure range known to elicit neuromodulation. We examined cell membrane health in response to a range of pulses and used optical calcium indicators in conjunction with pharmacological antagonists to selectively block different groups of thermo- and mechanosensitive ion channels known to be responsive to ultrasound. RESULTS: All cell types experienced an increase in calcium fluorescence in response to ultrasound. Gadolinium (Gad), 2-aminoethoxydiphenyl borate (2-APB) and ruthenium red (RR) reduced the percentage of responding neurons and magnitude of response. The percentage of astrocytes responding was significantly lowered only by Gad, whereas both 2-APB and Gad decreased the amplitude of the fluorescence response. 2-APB decreased the percentage of responding endothelial cells, whereas only Gad reduced the magnitude of responses. Pericytes exposed to RR or Gad were less likely to respond to stimulation. RR had no detectable effect on the magnitude of the pericyte responses while 2-APB and Gad significantly decreased the fluorescence intensity, despite not affecting the percentage responding. CONCLUSION: Our study highlights the role of non-neuronal cells during FUS neuromodulation. All of the investigated cell types are sensitive to mechanical ultrasound stimulation and rely on mechanosensitive ion channels to undergo ultrasound neuromodulation.

An Open-Source Mouse Chronic EEG Array System with High-Density MXene-Based Skull Surface Electrodes.

Electroencephalography (EEG) is an indispensable tool in epilepsy, sleep, and behavioral research. In rodents, EEG recordings are typically performed with metal electrodes that traverse the skull into the epidural space. In addition to requiring major surgery, intracranial EEG is difficult to perform for more than a few electrodes, is time-intensive, and confounds experiments studying traumatic brain injury. Here, we describe an open-source cost-effective refinement of this technique for chronic mouse EEG recording. Our alternative two-channel (EEG2) and sixteen-channel high-density EEG (HdEEG) arrays use electrodes made of the novel, flexible 2D nanomaterial titanium carbide (Ti3C2T x ) MXene. The MXene electrodes are placed on the surface of the intact skull and establish an electrical connection without conductive gel or paste. Fabrication and implantation times of MXene EEG electrodes are significantly shorter than the standard approach, and recorded resting baseline and epileptiform EEG waveforms are similar to those obtained with traditional epidural electrodes. Applying HdEEG to a mild traumatic brain injury (mTBI) model in mice of both sexes revealed that mTBI significantly increased spike-wave discharge (SWD) preictal network connectivity with frequencies of interest in the ß-spectral band (12-30 Hz). These findings indicate that the fabrication of MXene electrode arrays is a cost-effective, efficient technology for multichannel EEG recording in mice that obviates the need for skull-penetrating surgery. Moreover, increased preictal ß-frequency network connectivity may contribute to the development of early post-mTBI SWDs.

NRF2/ARE mediated antioxidant response to glaucoma: role of glia and retinal ganglion cells.

Glaucoma, the second leading cause of irreversible blindness worldwide, is associated with age and sensitivity to intraocular pressure (IOP). We have shown that elevated IOP causes an early increase in levels of reactive oxygen species (ROS) in the microbead occlusion mouse model. We also detected an endogenous antioxidant response mediated by Nuclear factor erythroid 2-Related Factor 2 (NRF2), a transcription factor that binds to the antioxidant response element (ARE) and increases transcription of antioxidant genes. Our previous studies show that inhibiting this pathway results in earlier and greater glaucoma pathology. In this study, we sought to determine if this endogenous antioxidant response is driven by the retinal ganglion cells (RGCs) or glial cells. We used Nrf2fl/fl mice and cell-type specific adeno-associated viruses (AAVs) expressing Cre to alter Nrf2 levels in either the RGCs or glial cells. Then, we quantified the endogenous antioxidant response, visual function and optic nerve histology after IOP elevation. We found that knock-down of Nrf2 in either cell type blunts the antioxidant response and results in earlier pathology and vision loss. Further, we show that delivery of Nrf2 to the RGCs is sufficient to provide neuroprotection. In summary, both the RGCs and glial cells contribute to the antioxidant response, but treatment of the RGCs alone with increased Nrf2 is sufficient to delay onset of vision loss and axon degeneration in this induced model of glaucoma.

Intraocular Sustained Release of EPO-R76E Mitigates Glaucoma Pathogenesis by Activating the NRF2/ARE Pathway.

Erythropoietin (EPO) is neuroprotective in multiple models of neurodegenerative diseases, including glaucoma. EPO-R76E retains the neuroprotective effects of EPO but diminishes the effects on hematocrit. Treatment with EPO-R76E in a glaucoma model increases expression of antioxidant proteins and is neuroprotective. A major pathway that controls the expression of antioxidant proteins is the NRF2/ARE pathway. This pathway is activated endogenously after elevation of intraocular pressure (IOP) and contributes to the slow onset of pathology in glaucoma. In this study, we explored if sustained release of EPO-R76E in the eye would activate the NRF2/ARE pathway and if this pathway was key to its neuroprotective activity. Treatment with PLGA.EPO-E76E prevented increases in retinal superoxide levels in vivo, and caused phosphorylation of NRF2 and upregulation of antioxidants. Further, EPO-R76E activates NRF2 via phosphorylation by the MAPK pathway rather than the PI3K/Akt pathway, used by the endogenous antioxidant response to elevated IOP.

Dose-dependent treatment of optic nerve crush by exogenous systemic mutant erythropoietin.

The goal of the present study was to determine the minimum concentration of systemic erythropoietin-R76E required for neuroprotection in the retina. Erythropoietin (EPO) exhibits neuroprotective effects in both in vitro and in vivo models of neuronal cell death although its classical function is the regulation of red blood cell production. It can cross the blood brain barrier and therefore can be delivered systemically to affect the retina. However, long-term treatment with exogenous erythropoietin causes polycythemia. To decrease this potentially lethal effect, we generated and tested a modified form that contains a single arginine to glutamate mutation at the 76th position (EPO-R76E). In previous studies, this mutant protected retinal neurons in mouse models of retinal degeneration and glaucoma with similar efficacy as wild-type EPO. However, EPO-R76E has attenuated erythropoietic activity, therefore, neuroprotection can be achieved without causing a significant rise in hematocrit. BALB/cByJ mice received a single intramuscular injection of recombinant adeno-associated virus carrying enhanced green fluorescent protein, Epo, or Epo-R76E. To result in continuous production of four different doses of EPO-R76E, two doses of two different serotypes (2/5 and 2/8) were used. Mice were subjected to optic nerve crush and analysis was performed thirty days later. EPO-R76E showed dose-dependent protection of the retinal ganglion cell bodies, but was unable to prevent axonal degeneration. Furthermore, EPO-R76E induced a dose-dependent rise in the hematocrit that was still attenuated as compared to wild-type EPO.

A mouse model of ocular blast injury that induces closed globe anterior and posterior pole damage.

We developed and characterized a mouse model of primary ocular blast injury. The device consists of: a pressurized air tank attached to a regulated paintball gun with a machined barrel; a chamber that protects the mouse from direct injury and recoil, while exposing the eye; and a secure platform that enables fine, controlled movement of the chamber in relation to the barrel. Expected pressures were calculated and the optimal pressure transducer, based on the predicted pressures, was positioned to measure output pressures at the location where the mouse eye would be placed. Mice were exposed to one of three blast pressures (23.6, 26.4, or 30.4 psi). Gross pathology, intraocular pressure, optical coherence tomography, and visual acuity were assessed 0, 3, 7, 14, and 28 days after exposure. Contralateral eyes and non-blast exposed mice were used as controls. We detected increased damage with increased pressures and a shift in the damage profile over time. Gross pathology included corneal edema, corneal abrasions, and optic nerve avulsion. Retinal damage was detected by optical coherence tomography and a deficit in visual acuity was detected by optokinetics. Our findings are comparable to those identified in Veterans of the recent wars with closed eye injuries as a result of blast exposure. In summary, this is a relatively simple system that creates injuries with features similar to those seen in patients with ocular blast trauma. This is an important new model for testing the short-term and long-term spectrum of closed globe blast injuries and potential therapeutic interventions.

cGMP Signaling in the Neurovascular Unit-Implications for Retinal Ganglion Cell Survival in Glaucoma.

Glaucoma is a progressive age-related disease of the visual system and the leading cause of irreversible blindness worldwide. Currently, intraocular pressure (IOP) is the only modifiable risk factor for the disease, but even as IOP is lowered, the pathology of the disease often progresses. Hence, effective clinical targets for the treatment of glaucoma remain elusive. Glaucoma shares comorbidities with a multitude of vascular diseases, and evidence in humans and animal models demonstrates an association between vascular dysfunction of the retina and glaucoma pathology. Integral to the survival of retinal ganglion cells (RGCs) is functional neurovascular coupling (NVC), providing RGCs with metabolic support in response to neuronal activity. NVC is mediated by cells of the neurovascular unit (NVU), which include vascular cells, glial cells, and neurons. Nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling is a prime mediator of NVC between endothelial cells and neurons, but emerging evidence suggests that cGMP signaling is also important in the physiology of other cells of the NVU. NO-cGMP signaling has been implicated in glaucomatous neurodegeneration in humans and mice. In this review, we explore the role of cGMP signaling in the different cell types of the NVU and investigate the potential links between cGMP signaling, breakdown of neurovascular function, and glaucoma pathology.

Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice.

Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4(-/-) mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system.

Systemic gene delivery protects the photoreceptors in the retinal degeneration slow mouse.

The retinal degeneration slow (rds/rds) mouse was used to test photoreceptor protection by systemic gene delivery of non-erythropoietic forms of erythropoietin (EPO). Two Epo mutants were generated and packaged into recombinant adeno-associated virus (rAAV) serotype 2/5, controls included rAAV2/5.Epo and rAAV2/5.enhanced green fluorescent protein (eGFP). Mice were injected in the quadriceps at postnatal day seven and analyses were performed at postnatal day 90. Hematocrit, serum EPO levels, and outer nuclear layer (ONL) thickness were quantified. Hematocrit and serum EPO levels in rAAV2/5.eGFP, rAAV2/5.Epo, and rAAV2/5.EpoR103E treated mice were: 46%, 8 mU/ml; 63%, 117 mU/ml; and 52%, 332 mU/ml, respectively. The ONL from rds/rds mice treated with the Epo vectors were approximately twice as thick as the negative controls. This demonstrates that the photoreceptors can be protected without performing an intraocular injection and without increasing the hematocrit to unsafe levels. Intramuscular delivery of rAAV.EpoR103E is an attractive treatment for retinal degenerative diseases.