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Method Article

Examination of Anatomical Features of Retinal Ganglion Cells Under N-methyl-D-aspartic Acid (NMDA)-induced Excitotoxicity

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DOI:

10.3791/68537

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September 19th, 2025

 ,  , 
1Ruiz Department of Ophthalmology and Visual Science, McGovern Medical School, University of Texas Health Science Center at Houston (UTHealth)

In This Article

Summary

Here, we describe the use of a genetically directed alkaline phosphatase (AP) labeling method to investigate the anatomical features and phenotypes of mouse retinas under NMDA-induced excitotoxicity. In conjunction with several retinal ganglion cell (RGC) subtype-specific reporter lines, we uncover novel phenotypes in dying RGCs throughout the degenerative process.

Abstract

Retinal ganglion cells (RGCs) are the output neurons of the retina, responsible for transmitting visual information to the brain. In the mature mouse retina, over 40 RGC types have been identified based on genomic, morphological, and functional characteristics. RGC degeneration is a hallmark of various debilitating retinal diseases, including glaucoma and traumatic optic neuropathy. Gaining deeper insight into the cellular and molecular mechanisms underlying RGC degeneration, survival, and resilience across different disease conditions is crucial for developing effective therapeutic interventions. To investigate RGC degeneration, we adopted a simple yet powerful technique previously used extensively in retinal development studies for RGC morphologies and central projection. Here, we apply this method to assess the anatomical phenotypes of RGCs under NMDA-induced excitotoxicity. This approach enables the visualization of large numbers of degenerating RGCs over time, allowing us to map the sequence of degenerative events, identify hallmark phenotypes of dying RGCs, and distinguish RGCs that are resistant to NMDA-induced damage.

Introduction

Traumatic brain injury (TBI) is a sudden injury that causes neuronal damage to the brain. In severe forms of TBI, such as those caused by car accidents or sports collisions and blast injuries due to explosions, mechanical forces may disrupt cell architecture, leading to inflammation, oxidative stress, excitotoxicity, and eventually neuronal death1,2.

Retina, the most accessible part of the central nervous system (CNS), has served as a model TBI system to study the molecular mechanisms underlying excitotoxicity and to develop strategies for therapeutic intervention3,4,5. The most pertinent retina models for TBI studies are the optic nerve crush model and N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity of retinas. In the NMDA-induced excitotoxicity retina model, retinal ganglion cells (RGCs) were identified as the most susceptible type of retinal neurons because RGCs utilize glutamate as a major excitatory neurotransmitter and various types of glutamate-receptors, including AMPA receptors (AMPARs), KAPA receptors (KAPARs), and NMDA receptors, to trigger axon potential6. In addition to RGCs, other retinal cells and neural circuitries could also be affected by NMDA treatment7,8,9. Intra-vitreous application of NMDA elicits prolonged opening of the NMDA-receptor, which leads to an excess influx of calcium into cells, causing over-excitatory neuronal activity and imbalanced calcium homeostasis10,11,12,13.

The NMDA receptor is a heteromeric complex composed of three subunits: GluN1, GluN2, and GluN314,15,16,17,18. GluN1, encoded by Grin1, contains eight isoforms in mice due to alternative splicing of Grin1. There are four different GluN2 subunits (a-d), which are encoded by four separate genes: Grin2a-2d. There are two different GluN3 subunits (a and b), which are encoded by Grin3a and 3b. According to scRNA-seq data19,20, Grin1 is highly expressed in most RGC subtypes (Figure 1A) as well as a few amacrine cell (AC) subtypes with weak to moderate expression levels (Figure 1B). Grin2a is enriched in fewer RGC and AC subtypes, whereas Grin2b is expressed moderately in most RGC and many AC subtypes (Figure 1A,B). Expression levels of Grin2c, Grin2d, and Grin3b in RGCs and ACs are extremely low (Figure 1A,B). Grin3a expression can be seen in a few RGC subtypes and weakly in a few ACs (Figure 1A,B). In addition to the role of NMDA receptors, calcium influx-triggered excitotoxicity through calcium-permeable AMPARs has been described in multiple disease models21. The expression of NMDA receptor subunits may provide clues as to why NMDA-induced excitotoxicity targets mainly RGCs as well as ACs9,10,14,22,23.

Conventional methods of assessing gross NMDA-induced retinal phenotypes include hematoxylin and eosin (H&E) staining to detect histological abnormalities, TUNEL assay for cell death, and immunofluorescent (IF) staining using a cell-type-specific biomarker8,10,24,25,26. At the cellular level, confocal imaging and dye-filling techniques have been used to investigate the sequence of events in dying RGCs in the NMDA-induced retinal model. These methods have provided valuable insights into the subcellular compartments of the dying RGCs and allowed for the identification of the types of resilient RGCs that survive under NMDA insult27,28.

Here, we employ genetically activated alkaline phosphatase (AP) staining to investigate the anatomical features and phenotypes of mouse retinal neurons under NMDA-induced excitotoxicity29,30,31. The rationale for selecting this method is due to its simplicity, cost-effectiveness, and ability to obtain a comprehensive overview of the anatomical features of neuronal subtypes across the entire retina, making it well-suited for gross assessments and large-scale comparative analyses. This approach provides novel insights into how distinct, genetically marked retinal neurons respond to NMDA at both gross and subcellular levels. We anticipate that this strategy will be broadly applicable for assessing morphological changes in various disease models that affect neuronal architecture and synaptic organization4,26,32,33.

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Protocol

All animal procedures followed the US Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at The University of Texas McGovern Medical School at Houston. The compositions of the solutions are listed in the Table of Materials.

1. Injection of tamoxifen

  1. Mix 30 mg of tamoxifen with 30 μL of ethanol. In sub-boiling water, heat 1 mL of sunflower seed oil and the tamoxifen/ethanol mixture.
  2. After the tamoxifen has dissolved in the ethanol, quickly add 1 mL of heated sunflower seed oil to the tamoxifen solution. Mix well by pipetting.
  3. Mice of either sex at various ages between 2 to 6 months were used. Inject the tamoxifen solution at a dose of 100 μg/g body weight intraperitoneally using an insulin syringe. Determine the number of tamoxifen injections based on the mouse line. In this protocol, tamoxifen was administered once daily for four consecutive days.
    NOTE: Since the tamoxifen solution is mixed with dense oil, it is difficult to handle with a syringe that has a detachable needle. Thus, an insulin syringe with an attached needle is preferred for tamoxifen injection.
  4. House the mouse in groups of no more than 5 in individually ventilated cages. until the NMDA injection.

2. Injection of NMDA solution into the mouse eyes

  1. Prepare 20 mM of NMDA solution in sterile 1x PBS. Wash the Nano-injector with a 35G needle attached using 100% ethanol 20x, then wash with sterile ultrapure water an additional 20x. Fill the Nano-injector with 1 μL of NMDA solution.
  2. Anesthetize the mouse by intraperitoneal injection of a ketamine/xylazine in 1X PBS (80/10 mg per kg body weight) solution mixture. Assess unconsciousness by reflex withdrawal response to tail and toe pinching.
  3. Place the anesthetized mouse on a stereomicroscope. Position the mouse with its right eye facing upward and its nose pointing toward the right side. Apply a drop of 0.5% proparacaine hydrochloride solution to the right eyeball for local anesthesia.
  4. Use a sterile 30G injection needle to puncture the limbus at the 7 o'clock position, creating a small hole. Monitor the needle's location and direction under the stereomicroscope. Ensure the size of the injection needle is slightly larger than the nano-injection needle to allow easy insertion.
  5. Carefully remove the injection needle from the eyeball and dispose of it properly. Use the stereomicroscope for guidance, insert the nano-injection needle into the vitreous through the limbus at a 45° angle.
  6. Slowly inject 1 μL of NMDA solution into the vitreous. After the injection, keep the nano-injection needle in the vitreous for approximately 10 s to prevent the outflow of the injected solution.
  7. Carefully and slowly withdraw the nano-injection needle from the eye. Apply 0.5% erythromycin ophthalmic ointment to the cornea of the injected eye.
  8. Place the mouse in a cage on a heating pad. Monitor the mouse every 15 min until it has fully recovered from anesthesia. House the mouse for one to two weeks until tissue collection.

3. Tissue collection and Alkaline Phosphatase (AP) staining

  1. Euthanize the mouse with CO2 asphyxiation, followed by cervical dislocation. Carefully cut the muscles connecting to the eyeball and the optic nerve with Vanna scissors.
  2. The eyeball was then picked up with forceps and transferred into separate labeled 2.0 mL tubes.
  3. Fix the eyeballs in 10% neutral buffered formalin for 10 min at room temperature. Flat mount the retinas onto a cellulose nitrate membrane filter as described below.
    1. Transfer the eyeballs to 10 cm Petri dishes containing 1x PBS. Hold any muscle tissue attached to the eyeball using forceps. Create a hole at the limbus using a 30G injection needle. Insert the tip of Vanna scissors into the hole and cut around the ora serrata. Remove the lens from the eyecup using forceps.
    2. Make 3-4 cuts on the eyecup and use forceps to hold the RPE and gently remove the RPE from the retina.
    3. Cut a small piece of cellulose nitrate membrane filter and soak it in 1x PBS. Place the isolated retina onto the filter. Gently push a peripheral area of the retina with the tip of a fine paintbrush to attach the edge of the retina to the filter.
    4. Hold the edge of the filter and slowly pull it out of the 1x PBS, ensuring the retina remains attached and flattens onto the filter. Place the retina/filter on a paper towel for 1 min to help secure the attached retina to the filter.
    5. Transfer the retina/filter back to a petri dish with 1x PBS. Carefully remove any debris, hair, and vitreous from the retina using forceps and/or a fine paintbrush, as much as possible. To distinguish the injected and uninjected retinas, trim filters in different shapes.
  4. Transfer the retina/filter back to a Petri dish. Fix the retina in 10% neutral buffered formalin for 5 min at room temperature with gentle agitation.
  5. Prepare a 65 °C water bath. Preheat 30 mL of 1x PBS in a 50 mL glass beaker. Transfer the retina/filters directly into the preheated 1x PBS. Incubate retinas at 65°C for 30 min.
    NOTE: The heat treatment inactivates the endogenous AP activity, which causes background staining in the retina. It is crucial to treat the retina in the heated solution to effectively eliminate this background.
  6. After heat treatment, transfer the retinas to a 30 mm Petri dish containing AP buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2).
  7. Incubate the retinas in the AP buffer at room temperature for 5 min with gentle agitation.
    NOTE: This step exchanges the buffer in the retina from 1x PBS to AP buffer, preventing chemical precipitation during color development.
  8. Replace the AP buffer with staining solution (30 μL of 10 mg/mL BCIP and 60 μL of 10 mg/mL NBP in 10 mL of AP buffer).
  9. Place the Petri dish in a light-excluding box. Develop the AP reaction at room temperature for 1 to 2 days with gentle agitation until the intensity of the AP staining is satisfactory for imaging.
  10. Stop the reaction by washing the retinas with 1x PBS for 5 min, 2x, at room temperature with gentle agitation.
  11. Postfix the retinas with 10% neutral buffered formalin for 10 min at room temperature with gentle agitation.
  12. Wash the retinas with 1x PBS for 5 min, 3x, at room temperature with gentle agitation. Dehydrate the retinas by incubating them in 30% ethanol, followed by 50% ethanol for 30 min each at room temperature with gentle agitation.
  13. Use a fine paintbrush to gently remove the retinas from the filter paper. Dehydrate the filter-free retinas in 70% ethanol for 30 min, followed by two 30-min incubations in 100% ethanol at room temperature with gentle agitation.
  14. Clear the retinas in a glass Petri dish with a benzyl benzate/benzyl alcohol (2:1, v/v) clearing solution for 30 min to 2 h at room temperature with gentle agitation.
    NOTE: This clearing procedure should not be performed overnight, as prolonged incubation can often result in the loss of AP staining. Clearing solution can melt plastic, so the procedure should be performed in a glass container.
  15. To mount the retinas onto a glass slide, apply double-sided tape to the slide to create a platform for the coverslip and retina. Place the retinas on the slide, fill the space with clearing solution, apply the coverslip, and seal the slide with nail polish to secure the retina and coverslip.
  16. Image the AP-stained retinas using a Zeiss Apotome microscope.

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Results

NMDA application in the vitreous induces RGC death, which has conventionally been detected using histological staining or immunofluorescence (IF) staining with RGC markers. In our experimental setting, by using the conventional IF strategy with a pan-RGC marker RBPMS, we confirmed that the number of RGCs decreased by approximately 65%, 1 week after NMDA injection (Figure 2E-G). Previous studies have discovered that Tbr2-expressing RGCs are indeed ipRGCs34

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Discussion

In this study, we utilized a genetically directed sparse AP labeling system in conjunction with a number of genetically manipulated mouse lines to examine NMDA-induced degenerative events in various RGC and AC subtypes and uncovered several hallmark phenotypes29,31,39. The most noticeable phenotype is the significant reduction of RGCs and ACs in the NMDA-injected retinas (Figure 2A-D

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Disclosures

The authors declare no competing interests.

Acknowledgements

This work was supported by grants from the National Institutes of Health-National Eye Institute to C.A.M. (EY024376) and in part by National Eye Institute Vision Core Grants P30EY028102 (UTHealth). We also acknowledge the financial support from the Stephen Lasher Endowment (UTHealth) and the Raye White Endowment (UTHealth).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.5% erythromycin ophthalmic ointment Bausch+Lomb24208-910-55
0.5% proparacaine hydrochloride Bausch+Lomb24208-730-06
10% neutral buffered formalin VWR89370-094
10 cm petridishCorning430167
10x PBSFisher ScientificBP399Dilute in water to create 1x PBS and autoclave. 
2.0 ml tubeVWR525-1136
30 gauge injection needleEXELINT International26437
30 mm petridishFalcon35208
Benzyl alchohol Acros Organics447000010
Benzyl benzateAcros Organics105860010
Cellulose nitrate membrane filterCytiva7188-002
Cover slipElectron Microscopy Sciences72204-01
Double sided tapeScotch597020
EthanolDecon Labs2716Avoid Fisher Bioreagents (Cat#: BP2818) for dehydtration after AP staining. This reagent distorts retinas.
ForcepsTED PELLA505
Heating padTheratherm710298-001
Insulin syringeBD329424
KetamineDechra17033010110
MicroscopeZeissAxio Imager 2
Nail polishElectron Microscopy Sciences72180
Nanofil injectorWorld Precision InstrumentsNANOFIL
Nanofil needleWorld Precision InstrumentsNF25BL
NBT/BICP reagent kitInvitrogenN6547
NMDASigma-AldrichM3262
Paint brushKingart6650
Slide glassFisher Scientific12-550-15
Stereo microscopeMoticDM143
Sunflower seed oilSigma-AldrichS5007
TamoxifenSigma-AldrichT5648Store tamoxifen solution at 4 °C in a light-tight box. Use tamoxifen solution within one week.
Vanna scissors Fine Science Tools15000-00
WaterbathLab-Line18052
XylazineDechra17033-0099-05Product name: Rompun

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NMDA ExcitotoxicityRGC DegenerationMouse RetinaCell MorphologyCell SurvivalCell ResilienceRetinal DiseasesAnatomical PhenotypesCell Labeling
Examination of Anatomical Features of Retinal Ganglion Cells Under N-methyl-D-aspartic Acid (NMDA)-induced Excitotoxicity
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