A new model of axon degeneration in the mouse optic nerve using repeat intraocular pressure challenge.

We characterize a new experimental model for inducing retinal ganglion cell (RGC) dysfunction and degeneration in mice. C57BL/6J mice were subjected to two acute periods of intraocular pressure (IOP) elevation (50 mmHg for 30 min) by cannulation of the anterior chamber. We used full-field electroretinography and visual evoked potentials (VEPs) to measure subsequent changes in retina and optic nerve function, and histochemical techniques to assess RGC survival and optic nerve structure. In 12 month old mice, a single IOP challenge caused loss and subsequent recovery of RGC function over the following 28 days with minimal cell death and no observed axonal damage. A second identical IOP challenge resulted in persistent RGC dysfunction and significant (36%) loss of RGC somas. This was accompanied by a 16.7% delay in the latency and a 27.6% decrease in the amplitude of the VEP. Severe axonal damage was seen histologically with enlargement of axons, myelin disruption, reduced axon density, and the presence of glial scarring. In contrast, younger 3 month old mice when exposed to a single or repeat IOP challenge showed quicker RGC functional recovery after a single challenge and full functional recovery after a repeat challenge with no detectable optic nerve dysfunction. These data demonstrate a highly reproducible and minimally invasive method for inducing RGC degeneration and axonal damage in mice. Resilience of the optic nerve to damage is highly dependent on animal age. The time-defined nature of functional versus structural loss seen in this model stands to facilitate investigation of neuroglial responses in the retina after IOP injury and the associated evaluation of neuroprotective treatment strategies. Further, the model may be used to investigate the impact of aging and the cellular switch between neurorecovery and neurodegeneration.

Photoreceptor laminin drives differentiation of human pluripotent stem cells to photoreceptor progenitors that partially restore retina function.

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors. This chemically defined and xenogen-free method enables reproducible production of photoreceptor progenitors within 32 days. We observed that the transplantation into rd10 mice were able to protect the host photoreceptor outer nuclear layer (ONL) up to 2 weeks post transplantation as measured by full-field electroretinogram. At 4 weeks post transplantation, the engrafted cells were found to survive, mature, and associate with the host's rod bipolar cells. Visual behavioral assessment using the water maze swimming test demonstrated visual improvement in the cell-transplanted rodents. At 20 weeks post transplantation, the maturing engrafted cells were able to replace the loss of host ONL by extensive association with host bipolar cells and synapses. Post-transplanted rabbit model also provided congruent evidence for synaptic connectivity with the degenerated host retina. The results may pave the way for the development of stem cell-based therapeutics for retina degeneration.

The Effect of Aging on Retinal Function and Retinal Ganglion Cell Morphology Following Intraocular Pressure Elevation.

Aging and elevated intraocular pressure (IOP) are two major risk factors for glaucomatous optic neuropathy; a condition characterized by the selective, progressive injury, and subsequent loss of retinal ganglion cells (RGCs). We examined how age modified the capacity for RGCs to functionally recover following a reproducible IOP elevation (50 mmHg for 30 min). We found that RGC functional recovery (measured using electroretinography) was complete by 7 days in 3-month-old mice but was delayed in 12-month-old mice until 14 days. At the 7-day recovery endpoint when RGC function had recovered in young but not older eyes, we examined RGC structural responses to IOP-related stress by analyzing RGC dendritic morphology. ON-RGC cell volume was attenuated following IOP elevation in both young and older mice. We also found that following IOP elevation OFF-RGC dendritic morphology became less complex per cell volume in young mice, an effect that was not observed in older eyes. Our data suggest that adaptations in OFF-RGCs in young eyes were associated with better functional recovery 7 days after IOP elevation. Loss of RGC cellular adaptations may account for delayed functional recovery in older eyes.

Targeting Diet and Exercise for Neuroprotection and Neurorecovery in Glaucoma.

Glaucoma is a leading cause of blindness worldwide. In glaucoma, a progressive dysfunction and death of retinal ganglion cells occurs, eliminating transfer of visual information to the brain. Currently, the only available therapies target the lowering of intraocular pressure, but many patients continue to lose vision. Emerging pre-clinical and clinical evidence suggests that metabolic deficiencies and defects may play an important role in glaucoma pathophysiology. While pre-clinical studies in animal models have begun to mechanistically uncover these metabolic changes, some existing clinical evidence already points to potential benefits in maintaining metabolic fitness. Modifying diet and exercise can be implemented by patients as an adjunct to intraocular pressure lowering, which may be of therapeutic benefit to retinal ganglion cells in glaucoma.

Effects of age on retinal macrophage responses to acute elevation of intraocular pressure.

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow.

Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina.

Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were initially designed to target Streptococcus pyogenes Cas9 (SpCas9) and after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein [YFP]), in the presence of mCherry. Both constructs were packaged into AAV2 vectors and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of YFP gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (∼85.5% reduction compared with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.

Correction to: Amyloid Precursor Protein Mediates Neuronal Protection from Rotenone Toxicity.

The original version of this article unfortunately contained a mistake in the author name. The family name of Dr. Vanessa A. Johannsen should be written as "Johanssen."

A biocompatible reverse thermoresponsive polymer for ocular drug delivery.

Age-related macular degeneration (AMD) is a leading cause of vision loss, the treatment of which may require monthly intravitreal injections. This is a burden on patients and health services, and new delivery modalities that reduce injection frequency are required. To that end, we investigated the suitability of a novel reverse thermoresponsive polymer (RTP) as an ocular drug-delivery vehicle. In this work, we detail the structure and synthesis of a novel RTP, and determine drug release curves for two drugs commonly used in the treatment of AMD, bevacizumab and aflibercept. Biocompatibility of the RTP was assessed in vitro in human and rat cell lines and in vivo following intravitreal injection in rats. Bevacizumab demonstrated a more appropriate release profile than aflibercept, with 67% released within 14 days and 78% released in total over a 183-day period. No toxic effects of RTP were seen in human or rat cells in up to 14 days of co-culture with RTP. Following intravitreal injection, intraocular pressure was unaffected by the presence of RTP and no changes in retinal function or structure were observed at 1 week or 1 month post-injection. RTP injection did not cause inflammation, gliosis or apoptosis in the retina. This work demonstrates the potential suitability of the novel RTP as a sustained-release vehicle for ocular drug delivery for anti-neovascular therapies. Optimization of polymer chemistry for optimal drug loading and release is needed.

Amyloid Precursor Protein Mediates Neuronal Protection from Rotenone Toxicity.

Mitochondrial complex I dysfunction is the most common respiratory chain defect in human disorders and a hotspot for neurodegenerative diseases. Amyloid precursor protein (APP) and its non-amyloidogenic processing products, in particular soluble APP α (sAPPα), have been shown to provide neuroprotection in models of neuronal injury; however, APP-mediated protection from acute mitochondrial injury has not been previously reported. Here, we use the plant-derived pesticide rotenone, a potent complex I-specific mitochondrial inhibitor, to discover neuroprotective effects of APP and sAPPα in vitro, in neuronal cell lines over-expressing APP, and in vivo, in a retinal neuronal rotenone toxicity mouse model. Our results show that APP over-expression is protective against rotenone toxicity in neurons via sAPPα through an autocrine/paracrine mechanism that involves the Pi3K/Akt pro-survival pathway. APP-/- mice exhibit greater susceptibility to retinal rotenone toxicity, while intravitreal delivery of sAPPα reduces inner retinal neuronal death in wild-type mice following rotenone challenge. We also show a significant decrease in human retinal expression of APP with age. These findings provide insights into the therapeutic potential of non-amyloidogenic processing of APP in complex I-related neurodegeneration.

The coma in glaucoma: Retinal ganglion cell dysfunction and recovery.

Retinal ganglion cell (RGC) degeneration causes vision loss in patients with glaucoma, and this has been generally considered to be irreversible due to RGC death. We question this assertion and summarise accumulating evidence that points to visual function improving in glaucoma patients with treatment, particularly in the early stages of disease. We propose that prior to death, RGCs enter periods of dysfunction but can recover with relief of RGC stress. We first summarise the clinical evidence for vision improvement in glaucoma and then detail our experimental work that points to the underlying processes that underpin clinical improvement. We show that functional recovery can occur following a prolonged course of RGC dysfunction and demonstrate how the capacity for recovery can be modified. Detecting RGC dysfunction and augmenting recovery of such 'comatosed' RGCs holds clinical potential to improve early detection of glaucoma and improve visual function.

A short term high-fat high-sucrose diet in mice impairs optic nerve recovery after injury and this is not reversed by exercise.

The aim of the current work was to test whether increased intake of dietary fat and sucrose in mice modifies the response of retinal ganglion cells (RGCs) of the optic nerve to injury, and whether any effects of diet are influenced by physical activity levels. C57BL/6J mice were given a high-fat high-sucrose (HFS) diet for 7 weeks, with or without exposure to regular exercise by swimming (60 min/day, 5 days/week). Injury to RGCs was subsequently induced by acute elevation of intraocular pressure (IOP) and retinas were assessed for function and structure. We report that mice on a HFS diet had similar body mass and blood glucose levels compared to mice on a control diet but suffered a 30% greater loss of RGC function following injury, as measured in vivo with the electroretinogram. RGC dysfunction in retinas from mice on the HFS diet was accompanied by activation of retinal macroglia but was not associated with neuronal cell loss. Exercising mice by swimming did not prevent HFS-induced RGC dysfunction in response to injury. This study shows for the first time that a short term increase in dietary fat and sucrose enhances the vulnerability of RGCs to dysfunction and cell stress after an acute injury, and that this is independent of obesity or hyperglycemia. Furthermore, our results suggest that detrimental effects of diet predominate over protective effects of exercise.

Exercise reverses age-related vulnerability of the retina to injury by preventing complement-mediated synapse elimination via a BDNF-dependent pathway.

Retinal ganglion cells (RGCs) become increasingly vulnerable to injury with advancing age. We recently showed that this vulnerability can be strongly modified in mice by exercise. However, the characteristics and underlying mechanisms of retinal protection with exercise remain unknown. Hence, the aim of this study was to investigate cellular changes associated with exercise-induced protection of aging retinal cells and the role of local and peripheral trophic signalling in mediating these effects. We focussed on two molecules that are thought to play key roles in mediating beneficial effects of exercise: brain-derived neurotrophic factor (BDNF) and AMP-activated protein kinase (AMPK). In middle-aged (12 months old) C57BL/6J mice, we found that exercise protected RGCs against dysfunction and cell loss after an acute injury induced by elevation of intra-ocular pressure. This was associated with preservation of inner retinal synapses and reduced synaptic complement deposition. Retinal expression of BDNF was not upregulated in response to exercise alone. Rather, exercise maintained BDNF levels in the retina, which were decreased postinjury in nonexercised animals. Confirming a critical role for BDNF, we found that blocking BDNF signalling during exercise by pharmacological means or genetic knock-down suppressed the functional protection of RGCs afforded by exercise. Protection of RGCs with exercise was independent of activation of AMPK in either retina or skeletal muscle. Our data support a previously unidentified mechanism in which exercise prevents loss of BDNF in the retina after injury and preserves neuronal function and survival by preventing complement-mediated elimination of synapses.

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo.

PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

Mini-Review: Impaired Axonal Transport and Glaucoma.

Glaucoma is increasingly recognized as a neurodegenerative disorder, characterized by the accelerated loss of retinal ganglion cells (RGCs) and their axons. Impaired axonal transport has been implicated as a pathogenic mechanism in a number of neurodegenerative diseases, including glaucoma. The long RGC axon, with its high metabolic demand and crucial role in conveying neurotrophic signals, relies heavily on intact axonal transport. In this mini review, we consider the evidence for transport disruption along RGCs in association with glaucoma and other intraocular pressure models. We give a brief overview of the axonal transport process and the methods by which it is assessed. Spatial and temporal patterns of axonal transport disruption are considered as well as the reversibility of these changes. Biomechanical, metabolic and cytoskeletal insults may underlie the development of axonal transport deficits, and there are multiple perspectives on the impact that transport disruption has on the RGC. Eliciting the role of impaired axonal transport in glaucoma pathogenesis may uncover novel therapeutic targets for protecting the optic nerve and preventing vision loss in glaucoma.

An acute intraocular pressure challenge to assess retinal ganglion cell injury and recovery in the mouse.

We describe a model of acute intraocular pressure (IOP) elevation in the mouse eye that induces reversible loss of inner retinal function associated with oxidative stress, glial cell activation and minimal loss of retinal ganglion cell (RGC) number. Young healthy mouse eyes recover inner retinal function within 7-days but more persistent functional loss is seen in older mice. Manipulation of diet and exercise further modify RGC recovery demonstrating the utility of this injury model for investigating lifestyle and therapeutic interventions. We believe that systematic investigation into the characteristics and determinants of RGC recovery following an IOP challenge will shed light on processes that govern RGC vulnerability in the early stages of glaucoma.

Characterization of the retinal pigment epithelium in Friedreich ataxia.

We assessed structural elements of the retina in individuals with Friedreich ataxia (FRDA) and in mouse models of FRDA, as well as functions of the retinal pigment epithelium (RPE) in FRDA using induced pluripotent stem cells (iPSCs). We analyzed the retina of the FRDA mouse models YG22R and YG8R containing a human FRATAXIN (FXN) transgene by histology. We complemented this work with post-mortem evaluation of eyes from FRDA patients. Finally, we derived RPE cells from patient FRDA-iPSCs to assess oxidative phosphorylation (OXPHOS) and phagocytosis. We showed that whilst the YG22R and YG8R mouse models display elements of retinal degeneration, they do not recapitulate the loss of retinal ganglion cells (RGCs) found in the human disease. Further, RPE cells differentiated from human FRDA-iPSCs showed normal OXPHOS and we did not observe functional impairment of the RPE in Humans.

Forced exercise protects the aged optic nerve against intraocular pressure injury.

We have previously shown that the optic nerve of mice becomes increasingly vulnerable to injury with advancing age. Here, we investigated whether regular exercise can modify this age-related vulnerability and improve optic nerve recovery after injury. Aged (12-month-old) C57BL/6J mice were exercised by swimming for 60 min/d, 5 d/wk for 6 weeks. After 5 weeks, injury to the optic nerve was induced by short-term elevation of intraocular pressure. Retinal function was recorded using the electroretinogram and the cellular and biochemical changes induced by injury were assessed using immunohistochemistry and quantitative polymerase chain reaction. We found that exercise almost completely reversed age-related vulnerability of the optic nerve to injury such that exercised aged mice had a similar functional response to injury as non-exercised young (3-month-old) mice. Exercise also abrogated injury-induced astrocytic gliosis and macrophage activation in the aged retina. These data suggest that the known benefits of exercise also extend to the visual system and support further investigation of physical activity as a means of protecting against injury, dysfunction, and degeneration in the aging eye.

The photopic negative response of the mouse electroretinogram: reduction by acute elevation of intraocular pressure.

PURPOSE: To determine the presence and magnitude of the photopic negative response (PhNR) component of the electroretinogram (ERG) in the mouse eye and to test if it is altered by short-term elevation of intraocular pressure (IOP). METHODS: Photopic and scotopic ERGs were recorded from 12-month-old C57BL/6J mice and analyzed for photoreceptoral responses (a-wave), bipolar cell responses (b-wave), scotopic threshold responses (STRs), and PhNRs. Electroretinogram signals were measured before and after short-term subischemic elevation of IOP (50 mm Hg for 30 minutes) induced by cannulation of the anterior chamber. Retinas were subsequently assessed for signs of retinal stress and cell survival using immunohistochemistry and quantitative PCR. RESULTS: The corneal negative PhNR of the photopic ERG was elicited in the mouse eye, and its amplitudes correlated with amplitudes of the positive STR (pSTR). Elevation of IOP significantly reduced amplitudes of both the PhNR and pSTR, while scotopic a-waves, scotopic b-waves, and photopic b-waves were unchanged. Pressure elevation was associated with upregulation of glial fibrillary acidic protein and heme oxygenase 1 expression in retinal macroglia in the absence of retinal cell death. CONCLUSIONS: The PhNR component of the full-field ERG can be recorded in mice and is sensitive to elevation of IOP. Correlation between PhNR and pSTR signals before and after IOP elevation suggests that the PhNR depends on inner retinal integrity and provides a means for evaluating inner retinal function in mouse models.

Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse.

PURPOSE: To assess the retinal macrophage response to cannulation of the anterior chamber (AC) and acute elevation of intraocular pressure (IOP) in adult mice. METHODS: Eyes from 12-month-old C57BL/6J WT mice were subject to IOP increase (50 mm Hg for 30 minutes) by direct cannulation of the AC. Fellow eyes were either cannulated without pressure increase or left untreated. Electroretinography was carried out prior to IOP elevation and 1 week later. Immunofluorescence staining was performed on frozen sections and retinal wholemounts 1 week after sham and IOP elevation. Eyes were assessed by epifluorescence and confocal microscopy and the density of vitreal hyalocytes and subretinal macrophages was calculated. RESULTS: The density of hyalocytes and subretinal macrophages was significantly increased 1 week after IOP elevation and AC cannulation compared with naïve eyes. CD68 and MHC Class II expression was upregulated in both cannulated eyes and eyes with elevated IOP. Electroretinographic signals derived from retinal ganglion cells were significantly reduced in response to acute IOP elevation, but not in response to cannulation alone. CONCLUSIONS: Cannulation of the AC causes an increase in hyalocyte density, microglial activation, and accumulation of macrophages in the subretinal space. These macrophage changes are similar to those observed in eyes subject to IOP elevation. Additional IOP elevation led to significant Müller cell activation, which was not evident after cannulation alone. These data highlight the importance of using appropriate controls in models of acute retinal injury.