The role of Ketamine in Optic Nerve Injury

1. Overview

(A) Scientific Rationale and Importance:

Ketamine has been identified as a novel treatment for mental health diseases in humans [1], and there is conflicting evidence of it as either neuroprotective [2] or neurotoxic [3] in different conditions and environments. In the eye, a single injection of ketamine into the vitreous (intravitreal) prior to induction of ischemia has shown to be protective of neuronal function and health [4] although with somewhat limited evidence. It is unclear how ketamine is causing its neuroprotective effect, and why in some situations it is toxic. Calcium is an ion that is integral for most cell processes, including neuronal activity, where higher baseline levels have recently been shown to be associated with retinal ganglion cell survival to optic nerve degeneration [5]. As ketamine almost fundamentally changes neuronal activity, it is unclear what this does to calcium levels acutely or chronically, and how this is altered in a degenerative condition. This study will address this mystery using in-vivo two photon imaging of homeostatic calcium levels in Retinal Ganglion Cells (RGCs) after optic nerve crush (ONC) axon injury induced neurodegeneration. Ketamine will be evaluated for its neuroprotective (or neurotoxic) effects after acute axon injury, and its potential mechanism through altering of calcium homeostasis.

(B) Confirmation of funding/facilities/ethics approvals: All experimental procedures are approved and validated at Washington University in St. Louis and are conducted under the appropriate IACUC protocols for animal testing.

(C) Estimated Timeline:

Months 1-3: Perform Experiments (Injections+imaging, ONC injury, injections post injury, perfusions,
Months 4-6: Begin analysis, collect more necessary data, perform controls, and complete experimentation/processing.
Months 7-9: Finalize data analysis, prepare figures, and write some version of a report/manuscript (to publish on RH)
Total Time: ~9 months

(D) Data Sharing Commitment:

This study will be pre-registered on ResearchHub. All data will be shared in an open-access repository and made available upon request. A preprint of the findings will be posted on ResearchHub, and regular progress updates will be provided every 3months.

2. Introduction

(A) Summarize theoretical background/key literature:

Ketamine has become a focus of intense research since its 2019 FDA approval for treating depression [6]. Its ability to induce neuroplasticity and alter neuronal activity is well-established [7], but the specific mechanisms, and whether these effects are protective in the context of neuronal injury, are elusive. RGCs, the excitatory output neurons of the retina, offer an ideal model system for studying neurodegeneration due to their accessibility for in vivo imaging [8] and well-characterized degeneration models [9]. I will use two-photon calcium imaging to directly observe how ketamine alters RGC calcium homeostasis and whether this translates to neuroprotection following axon injury.

(B) Research Questions:

Q1) Does intravitreal injection of Ketamine alter RCG calcium homeostasis?
Q2) How does chronic ketamine injection after axon injury alter RGC survival?

(C) Hypotheses:

H1) Ketamine dysregulates calcium homeostasis acutely, potentially a reduction in calcium if its reducing RGC firing and activity through NMDA receptor activation.
H2) Acute Calcium reduction leads to a chronic increase in RGC calcium homeostasis long term.
H3) Longitudinal Ketamine treatment will protect RGCs after ONC injury.

(D) Impact Statement:

This study will investigate the role of Ketamine in RGC specific neuroprotection (or neurotoxicity). Calcium levels will also be evaluated and tested as a mechanism for Ketamine induced RGC effects. This study will provide fundamental mechanistic insight into if and how Ketamine is protective for a well studied and important neuron and nervous system—RGCs and the visual system, and could open doors for future research for ketamine in the eye and beyond to the brain to treat neurodegeneration.

3. Methods

Animal Models

All experimental procedures will adhere to protocols approved by the International Animal Care and Use Committee at Washington University. We will use VGlut2-IRES-Cre mice for Cre-dependent expression of viral vectors. Mice will be housed in a barrier facility with controlled light-dark cycles.

Materials and Procedures:

To measure cytoplasmic calcium levels, I will use a Cre-dependent AAV expressing the Twitch2b FRET-based calcium biosensor. The virus will be injected intravitreally into anesthetized mice. Optic nerve crush (ONC) will be performed approximately two weeks after injection, as previously described [5]. Mice will receive either ketamine or a saline control, administered via intravitreal injections, also as previously described [5]

In vivo Imaging and Analysis:

A Scientifica Hyperscope will be used for in vivo two-photon imaging of RGCs. Anesthetized mice will be placed in a head holder for stable imaging. The Twitch2b sensor will be imaged and evaluated to measure calcium levels. For pharmacological experiments, we will obtain pre-injection images, then administer ketamine or controls, followed by post-injection images to evaluate acute changes.

Analysis will be performed using FIJI and ImageJ. Regions of Interest (ROIs) will be drawn around individual cells, and calcium levels will be determined by analyzing the ratio of the two fluorophores. Cells will be matched between pre- and post-images to quantify changes in calcium homeostasis.

Injury and RGC survival Assessment

Ketamine injections will be performed immediately prior to Optic Nerve Crush (ONC) axon injury. Then longitudinal injections will be performed every 2 days after injury up to 14 days post injury. Mice will be perfused and retinas will be processed for immunohistochemistry (IHC). IHC will be performed on retinal wholemounts with RBPMS staining to label RGCs. Retinas will be imaged on confocal and representative regions will be selected. RGC density will be determined at 14dpi.

Ethics and Data Management

All data will be made avaliable at request. All images and movies will be saved and recorded and shared. Updates will be made every 6 months on ResearchHub.

5. Budget

I am requesting $5,000 in ResearchCoin (RSC) to support this project. The funds will be allocated as follows:

Viral Purchase: $1,500
Mouse Maintenance: $1,500
Ketamine and Other Reagents: $500
Postdoctoral Salary: $1,500

These funds will be instrumental in generating the initial data required to support a larger, career-transition grant application.

The role of Ketamine in Optic Nerve Injury

Proposal

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The role of Ketamine in Optic Nerve Injury

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Funding Progress

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Peer Reviews

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Funding Progress

Nonprofit Recipient

Peer Reviews

Topics

DOI