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

doi:10.1016/j.exer.2015.03.006

Experimental Eye Research

Volume 141, December 2015, Pages 3-8

https://doi.org/10.1016/j.exer.2015.03.006 Get rights and content

Highlights

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We describe a model of acute intraocular pressure elevation in the mouse eye.
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Intraocular pressure is elevated by anterior chamber cannulation.
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Injury induces inner retinal dysfunction, oxidative stress and glial cell activation.
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Injury does not result in significant cell loss or retinal ischemia.

Abstract

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.

Introduction

We adapted an acute intraocular pressure (IOP) elevation injury model to investigate how retinal ganglion cells (RGCs) respond to a titratable and highly reproducible injury. Our goal in generating this “optic nerve stress test” was to identify levels of injury that, in a healthy young mouse retina, induce selective but reversible loss of RGC function while maintaining normal outer retinal function. Additionally, we sought an injury that does not lead to manifest ischemia of the inner retina with minimal RGC death. Our interest lies in determining the parameters that influence RGC recovery over time.

Glaucoma is a chronic, progressive optic neuropathy characterized by the selective death of RGCs and their axons. How then does our acute IOP injury model inform us of processes that are relevant to this disease? In the very early stages of glaucoma, the optic nerve head is repeatedly exposed to cycles of relatively minor injury (Downs et al., 2011). We propose that healthy RGCs are initially able to withstand and recover from such minor injuries. However, over time, repeated insults or an intrinsic impairment in the capacity of RGCs to recover, will overwhelm their ability to recover resulting in induction of a cell death program. Measuring the capacity for RGC recovery following an acute IOP injury, we believe, will shed light on the very early stages of glaucoma. Furthermore, this test will enable us to investigate factors such as aging, diet and exercise that can improve or impair RGC recovery. By incrementally increasing the IOP level or duration of injury we will also be able to investigate the critical switch point at which full functional recovery is not possible.

Section snippets

Overview

Our injury model involves acute elevation of IOP through insertion of a fine needle attached to either a column of fluid or a motorized syringe pump. RGC function is measured using the positive scotopic threshold response (pSTR) or the photopic negative response (PhNR) components of the full-field electroretinogram (ERG). A single animal can be monitored over a number of timepoints post-injury but to mitigate potential effects of repeat anesthesia, we leave 7 days between injury and functional

Pros and Cons of this injury model

An advantage of an IOP challenge is that it can be easily titrated by altering the magnitude or duration of IOP elevation (Table 1). It provides a precise and reproducible injury within and between experiments. This level of control contrasts with many of the more chronic IOP elevation models where the degree and duration of IOP elevation are much more unpredictable and often vary substantially between animals exposed within the same experiment. The discrete temporal nature of a single acute

Funding acknowledgment

The Centre for Eye Research Australia receives Operational Infrastructure Support from the Victorian Government.

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The photopic negative response of the mouse electroretinogram: reduction by acute elevation of intraocular pressure
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24-Hour IOP telemetry in the non-human primate: implant system performance and initial characterization of IOP at multiple timescales
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Effect of anterior chamber cannulation and acute IOP elevation on retinal macrophages in the adult mouse
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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.
Inhibition of p66Shc attenuates retinal ischemia-reperfusion injury-induced damage by activating the akt pathway
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This makes it a valuable and widely used model to study disease changes and evaluate neuroprotective methods (Osborne et al., 1999). In this model, the degree and time of IOP elevation can be precisely controlled, and surgical trauma to the anterior segment of the eye is small, which allows to better evaluate the changes in the structure and function of the eye (Crowston et al., 2015). However, this model also has its disadvantages and limitations.
Retinal ganglion cell (RGC) death is the direct cause of several optic neuropathies. Several studies have reported that the loss of p66Shc ameliorates neuronal injury and vascular abnormalities in ischemia-reperfusion (I/R) injury. However, whether p66Shc is involved in the loss of RGC remains unclear. Therefore, this study aimed to investigate the function of p66Shc due to retinal ischemia in mice. The retinal I/R model was constructed after an intravitreal injection of recombinant adeno-associated viruses (rAAV-EGFP or rAAV-p66Shc-EGFP) for 4 weeks. The expression of p66Shc was detected by western blotting, quantitative real-time polymerase chain reaction, and immunofluorescence staining. The survival of RGCs was determined using immunofluorescence staining. Retinal function was analyzed based on electroretinogram (ERG) findings. Retinal cell apoptosis was detected by TdT-mediated dUTP nick-end labeling staining. The protein expressions of Akt, phospho-Akt, Bax, and PARP were analyzed by western blotting. After rAAVs were successfully transfected, enhanced green fluorescent protein was expressed in all retinal cell layers, and the level of p66Shc after I/R injury was successfully reduced. We found that inhibition of p66Shc expression remarkably decreased the death of RGCs and prevented the loss of ERG a- and b-wave amplitudes caused by retinal ischemia. Mechanistically, downregulation of p66Shc resulted in reduced Bax, whereas increased phospho-Akt and PARP. Taken together, our study revealed that p66Shc acts through the Akt pathway to protect RGCs from retinal I/R injury-induced apoptosis and retinal dysfunction, making p66Shc a possible therapeutic target for glaucoma treatment.
Restoring the oxidative balance in age-related diseases – An approach in glaucoma
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As human life expectancy increases, age-related health issues including neurodegenerative diseases continue to rise. Regardless of genetic or environmental factors, many neurodegenerative conditions share common pathological mechanisms, such as oxidative stress, a hallmark of many age-related health burdens. In this review, we describe oxidative damage and mitochondrial dysfunction in glaucoma, an age-related neurodegenerative eye disease affecting 80 million people worldwide. We consider therapeutic approaches used to counteract oxidative stress in glaucoma, including untapped treatment options such as novel plant-derived antioxidant compounds that can reduce oxidative stress and prevent neuronal loss. We summarize the current pre-clinical models and clinical work exploring the therapeutic potential of a range of candidate plant-derived antioxidant compounds. Finally, we explore advances in drug delivery systems, particular those employing nanotechnology-based carriers which hold significant promise as a carrier for antioxidants to treat age-related disease, thus reviewing the key current state of all of the aspects required towards translation.
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During this period, if the insult is not severe enough that it will result in the death of the cell, the cell recovers function. This reversal of function after transient IOP insult has been demonstrated in mouse and rat models of glaucoma.14,15,33 Before axonal loss, elevated IOP induces a deficit in anterograde and retrograde transport in the RGC axon, thereby impairing transport of proteins, mitochondria, trophic signals; consequent decrease in retinal function as measured by pattern electroretinogram (PERG) amplitude has been confirmed to occur.12,19,77
Glaucoma is characterized by retinal ganglion cell loss that can lead to permanent visual loss. Current clinical management practice assumes that glaucomatous visual loss is irreversible; however, there is increasing evidence that permanent vision loss and cell death are preceded by reversible functional and structural changes. We propose that these changes should be considered by glaucoma specialists when treating their patients. We discuss the neurobiological basis of this phenomenon and provide clinical evidence of reversibility in both structure and function. Specifically, we review the findings of visual field testing, contrast sensitivity, electroretinography, and imaging of the optic nerve and their correlation with functional changes. We then discuss the clinical value of these observations in helping guide approaches toward the diagnosis and treatment of patients with glaucoma.
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Glaucoma, a major cause of irreversible blindness worldwide, is associated with elevated intraocular pressure (IOP) and progressive loss of retinal ganglion cells (RGCs) that undergo apoptosis. A mechanism for RGCs injury involves impairment of neurotrophic support and exogenous supply of neurotrophic factors has been shown to be beneficial. However, neurotrophic factors can have widespread effects on neuronal tissues, thus targeting neurotrophic support to injured neurons may be a better neuroprotective strategy. In this study, we have encapsulated LM22A-4, a small neurotrophic factor mimetic, into Annexin V-conjugated cubosomes (L4-ACs) for targeted delivery to injured RGCs in a model of acute IOP elevation, which is induced by acute IOP elevation. We have tested cubosomes formulations that encapsulate from 9% to 33% LM22A-4. Our data indicated that cubosomes encapsulating 9% and 17% LM22A-4 exhibited a mixture of Pn3m/Im3m cubic phase, whereas 23% and 33% showed a pure Im3m cubic phase. We found that 17% L4-ACs with Pn3m/Im3m symmetries showed better in-situ and in-vitro lipid membrane interactions than the 23% and 33% L4-ACs with Im3m symmetry. In vivo experiments showed that 17% L4-ACs targeted the posterior retina and the optic nerve head, which prevented RGCs loss and improved functional outcomes in a mouse model of acute IOP elevation. These results provide evidence that Annexin V-conjugated cubosomes-based LM22A-4 delivery may be a useful targeted approach to prevent the progression of RGCs loss in glaucoma.
Recent studies suggest that the therapy of effectively delivering neurotrophic factors to the injured retinal ganglion cells (RGCs) could promote the survival of RGCs in glaucoma. Our present work has for the first time used cubosomes as an active targeted delivery system and have successfully delivered a neuroprotective drug to the damaged RGCs in vivo. Our new cubosomal formulation can protect apoptotic cell death in vitro and in vivo, showing that cubosomes are a promising drug carrier system for ocular drug delivery and glaucoma treatment. We have further found that by controlling cubosomes in Pn3m phase we can facilitate delivery of neuroprotective drug through apoptotic membranes. This data, we believe, has important implications for future design and formulation of cubosomes for therapeutic applications.
Fluctuations in Macular Thickness in Patients with Retinal Vein Occlusion Treated with Anti–Vascular Endothelial Growth Factor Agents
2020, Ophthalmology Retina
Citation Excerpt :
Another study used in vitro devices to model mechanical stress on retinal pigment epithelium cells as a model of AMD and similarly determined that stress induces VEGF expression and contributes to the progression of choroidal neovascularization.28 In addition, many studies of glaucoma have related intraocular pressure-induced mechanical strain of retinal neurons and glial cells to complex molecular cascades resulting in impaired function and cell death.29–32 In RVO eyes, another potential explanation is that there is a third, unknown factor that predisposes some eyes to the recurrence of edema after transient resolution with each injection because of suboptimal response or inconsistent therapeutic effects of anti-VEGF.
To evaluate macular thickness fluctuations in patients with retinal vein occlusions (RVOs) treated with anti–vascular endothelial growth factor (VEGF) agents and to assess whether patients with larger fluctuations have poorer visual outcomes.
Retrospective cohort study.
Treatment-naive patients with RVO.
Central subfield thickness (CST), cube volume (CV), and cube average thickness (CAT) were collected from OCT images obtained at baseline and 3, 6, 9, and 12 months, and standard deviations (SDs) across 12 months were calculated. Mixed-effects regression was performed to examine the relationship between macular thickness SD and 12-month visual acuity (VA). Standard multiple regression was performed to identify predictors of macular thickness SD.
Standard deviations across 12 months for CST, CV, and CAT and VA at 12 months.
One hundred thirty-four eyes, including 71 with branch RVO (BRVO) and 63 with central RVO (CRVO), were evaluated. Mean baseline and 12-month CST were 488.6 ± 165.0 μm and 334.3 ± 131.9 μm (change, –154.3 ± 210.2 μm; P < 0.001), with CST SD of 114.1 ± 77.0 μm. Baseline and 12-month VA were 52.8 ± 20.9 letters and 65.9 ± 17.3 letters (change, +13.1 ± 20.3 letters; P < 0.001). Central subfield thickness SD was a significant negative predictor of 12-month VA (–5.21 letters/100 μm; 95% confidence interval [CI], –10.21 to –0.22 letters/100 μm; P = 0.041) when adjusting for baseline factors and injections. Baseline CST and number of injections were not predictive (P ≥ 0.101). Stratification by CST SD demonstrated a 10-letter difference in 12-month VA between the first and fourth quartiles. Baseline CST and RVO diagnosis were the only significant predictors of CST SD (CRVO vs. BRVO: +34.64 μm/100 μm [95% CI, 29.33–39.94 μm/100 μm; P < 0.001] and +22.13 μm/100 μm [95% CI, 4.81–39.44 μm/100 μm; P = 0.013]). Associations using CV and CAT were similar.
Larger macular thickness fluctuations are associated with poorer visual outcomes in patients with RVO treated with anti-VEGF agents. Macular thickness fluctuations, in addition to absolute macular thickness, may be an important prognostic biomarker in these patients.

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View full text

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

Highlights

Abstract

Introduction

Section snippets

Overview

Pros and Cons of this injury model

Funding acknowledgment

Neurobiol. Aging

Ophthalmology

Evaluation of retinal nerve fiber layer thickness and axonal transport 1 and 2 weeks after 8 hours of acute intraocular pressure elevation in rats

Invest. Ophthalmol. Vis. Sci.

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

Invest. Ophthalmol. Vis. Sci.

The status of cones in the rhodopsin mutant P23H-3 retina: light-regulated damage and repair in parallel with rods

Invest. Ophthalmol. Vis. Sci.

24-Hour IOP telemetry in the non-human primate: implant system performance and initial characterization of IOP at multiple timescales

Invest. Ophthalmol. Vis. Sci.

Improvement of visual field indices after surgical reduction of intraocular pressure

Ophthalmic Surg.

Improvement of spatial contrast sensitivity threshold after surgical reduction of intraocular pressure in unilateral high-tension glaucoma

Invest. Ophthalmol. Vis. Sci.

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

Invest. Ophthalmol. Vis. Sci.