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Jul 2019

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Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration
视神经挤压对小鼠视网膜神经节细胞存活和再生的影响   

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Abstract

In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to assay RGC survival and regeneration in mice following AAV2-mediated transduction and ONC injury including 1) intravitreal injection of AAV2 viral vectors, 2) optic nerve crush, 3) cholera-toxin B (CTB) labeling of regenerating axons, 4) optic nerve clearing, 5) flat mount retina immunostaining, and 6) quantification of RGC survival and regeneration. In addition to providing all the materials and procedural details necessary to execute this protocol, we highlight its advantages over other similar published approaches and include useful tips to ensure its faithful reproduction in any modern laboratory.

Keywords: Glaucoma (青光眼), Retinal ganglion cells (RGCs) (视网膜神经节细胞), Degeneration (退化), Neuroprotection (神经保护), Regeneration (再生), AAV2 (AAV2)

Background

Glaucoma is the leading cause of irreversible blindness worldwide characterized by the progressive degeneration and loss of retinal ganglion cells (RGCs), the central projecting neurons that form the optic nerve connecting the retina to the brain (Quigley, 2011; Tham et al., 2014). Glaucomatous RGC cell death is thought to be induced, in part, by an increase in intraocular pressure (IOP) and concomitant compression of RGC axons as they exit the retina through the optic nerve head (Quigley, 2011; Chang and Goldberg, 2012). Several models of glaucoma in rodents have been developed to study the cellular and molecular mechanisms that underlie RGC degeneration including optic nerve crush (ONC), intracameral injection of microbeads, and intravitreal injection of silicon oil (Sappington et al., 2010; Tang et al., 2011; Templeton and Geisert, 2012; Ito et al., 2016; Zhang et al., 2019). While both the microbead and silicon injection models recapitulate the increase in IOP and induce progressive RGC cell death associated with glaucoma, they are not conducive to study axon regeneration due to variability of the insult and incomplete degeneration of RGC axons. Alternatively, ONC has served as a useful preclinical model to study both neuronal survival and regeneration as it induces significant RGC death with little variability and severs all axons allowing confidence that any fibers found past the site of injury are regenerating, rather than spared. Adeno-associated virus serotype 2 (AAV2) specifically and efficiently transduces RGCs following intravitreal injection, making it the principal means to deliver genes (recombinant DNA, shRNA, etc.) into RGCs (Martin et al., 2002; Nickells et al., 2017). Indeed, we’ve reported the use of AAV2 to deliver therapeutic peptides and shRNA to promote RGC survival and axon regeneration following ONC injury (Moore et al., 2009; Apara et al., 2017; Galvao et al., 2018; Boczek et al., 2019). Here, we describe a comprehensive protocol detailing the use of AAV2 in combination with ONC as a means to study the mechanisms that promote neuroprotection and regeneration, and to identify and characterize candidate molecules with therapeutic potential for the treatment of glaucoma and other optic neuropathies.

Materials and Reagents

  1. Falcon 3 ml transfer pipette (Corning, catalog number: 357524 )
  2. 6-inch Cotton Tipped Applicators (VWR, catalog number: 89031-270 )
  3. VWR® Microcentrifuge Tubes (VWR, catalog number: 87003-294 )
  4. VWR® Superfrost® Plus Micro Slide (VWR, catalog number: 48311-703 )
  5. Polycin® Ophthalmic Ointment, USP (Perrigo, catalog number: NDC 0574-4021-35 )
  6. Absorbent Bench Underpads (VWR, catalog number: 82020-845 )
  7. Kimwipes (Fisher Scientific, catalog number: 06-666 )
  8. Falcon® 48-well clear flat bottom plate (Corning, catalog number: 353078 )
  9. 35 x 10 mm tissue culture dish (Corning, catalog number: 353001 )
  10. SecureSealTM imaging spacer (Grace Bio-Labs, catalog number: 654006 )
  11. 22 x 22 mm (No. 1.5) micro coverslip (VWR, catalog number: 48366-227 )
  12. Adult (> P21) male and female C57BL/6J mice (JAX, catalog number: 000664 )
  13. FlurisoTM Isofluorane, USP (VetOne, catalog number: NDC 13985-528-60 )
  14. Ketamine (or similar as approved by your local regulations)
  15. Xylazine (or similar as approved by your local regulations)
  16. Buprenorphine HCl (or similar as approved by your local regulations)
  17. Refresh Tears® Lubricant Eye Drops (Allergan, catalog number: NDC 0023-0798-01 , or similar)
  18. Proparacaine Hydrochloride Ophthalmic Solution, USP (Sandoz, catalog number: NDC 61314-016-01 )
  19. Cholera Toxin Subunit B (CTB), Alexa FluorTM 555 Conjugate (Thermo Fisher, catalog number: C34776 )
  20. 10x Phosphate Buffered Saline (Thermo Fisher, catalog number: AM9624 )
  21. Paraformaldehyde 16% Solution (Fisher Scientific, catalog number: 50-980-488 )
  22. FocusClearTM (CelExplorer, catalog number: FC-101 )
  23. MountClearTM (CelExplorer, catalog number: MC-301 )
  24. TritonTM X-100 (Sigma, catalog number: X100 )
  25. Non-specific Goat serum (Thermo Fisher, catalog number: 16-210-064 )
  26. Anti-RBPMS, Guinea Pig (Phospho Solutions, catalog number: 1832-RBPMS )
  27. Anti-Guinea Pig-Alexa Fluor 647 (Thermo Fisher, catalog number: A21450 )
  28. ProLong® Gold Antifade Mountant (Thermo Fisher, catalog number: P36934 )
  29. Flat mount blocking buffer (see Recipes)

Equipment

  1. Surgical Microscope (WPI, catalog number: PSMB5N )
  2. Laboratory Stereo Microscope (VWR, catalog number: 10836-004 )
  3. Hot Bead Sterilizer (FST, catalog number: 1800050 )
  4. Mouse Heating Pad (Stryker, catalog number: TP600/700 )
  5. Tabletop Isoflurane Anesthesia System with Induction Box (Harvard Apparatus, catalog number: 72-6468 )
  6. Dumont #5 Straight Forceps (FST, catalog number: 11251-10 )
  7. Dumont #7 Curved Forceps (FST, catalog number: 11271-30 )
  8. Dumont #N5 Forceps Cross Action Inox Thin Tips Size .05 x .01 Biologie Tips (Roboz Surgical, catalog number: RS-5020 )
  9. Hamilton Syringe (Hamilton Company, 5 μl, 700 series, catalog number: 7634-01 )
  10. Hamilton 33 gauge needle (Hamilton Company, catalog number: 7803-05 , 33 gauge, point 4, 0.375 inches)
  11. Vannas Spring Scissors-2.5 mm Cutting Edge (FST, catalog number: 15000-08 )
  12. Surgical Scissors (FST, catalog number: 91402-14 )
  13. Zeiss 880 LSM Confocal Microscope (Zeiss)

Software

  1. Fiji ImageJ (https://imagej.net/Fiji)
  2. Adobe Photoshop (https://www.adobe.com/)

Procedure

  1. Intravitreal AAV2 injection
    1. Anesthetize mice in accordance with your lab’s and your Institutional Animal Care and Use Committee (IACUC)-approved protocols or country-specific regulations. For the purpose of intravitreal injections, isoflurane administered using a tabletop induction box and nose cone is sufficient.
    2. While animals are under anesthesia, make sure to keep their eyes moist at all times by applying Refresh Tears® lubricating eye drops as needed (one drop per eye every 5 min is sufficient).
      Note: To prevent the cornea from drying out during intravitreal injections and ONC surgeries liberally apply Refresh Tears® lubricating eye drops as required.
    3. Once anesthetized, transfer a mouse to the surgical scope and attach nose cone (make sure isoflurane is flowing).
    4. Instill 1 drop of proparacaine (or similar topical anesthetic) to each eye to block local reflexes. Wait ~1-2 min and confirm the depth of anesthesia by gently touching the conjunctiva.
    5. Tilt the mouse’s head such that the targeted eye is facing the scope (Figure 1A). Use a cotton tipped applicator to wipe away any excess liquid.
    6. Using #5 forceps, slide the tips between the upper and lower eyelids such that the eyeball protrudes out of the socket and lightly pinch the conjunctiva ~1 mm below the limbus (Figure 1B).
    7. While holding the eyeball steady with forceps, pick up a preloaded 5 μl Hamilton syringe with your free hand and at a 60° angle with respect to the optic nerve, carefully insert the needle ~1 mm (bevel facing towards the lens) through the sclera ~1 mm below the limbus into the vitreous cavity (Figure 1C). Since the lens occupies the majority of the vitreous cavity, care should be taken to avoid piercing the lens.
      Note: Purified in vivo grade AAV2 with a titer ≥ 1 x 1012 GC/ml should be used to efficiently transduce RGCs.
    8. Using your index finger, gently push on the plunger to inject 1 μl of virus.
      Note: Check the lens before optic nerve crush. If there is cataract, the eye should be excluded.
    9. After injecting, leave the needle in the vitreous cavity for ~15-20 sec to prevent leakage, then slowly remove.
      Note: If liquid starts to flow back out of the site of injection you may have injected too much virus and the eye may be excluded from future analysis.
    10. Remove forceps and gently place the eye back into the socket by closing the eyelids.
    11. Apply a generous amount of Polycin® Ophthalmic Ointment (or similar antibiotic ointment) over the cornea and injection area.
    12. Flip mouse and repeat the injection in the contralateral eye if desired.
    13. Place animals on a heating pad to fully recover (~2-3 min) before transferring back to their home cages.
    14. House animals for 2 weeks, or optimized length, to allow for maximum virus expression then proceed to the optic nerve crush.


      Figure 1. Intravitreal injection of AAV2. A. Transfer an anesthetized mouse to the surgical scope and tilt the head such that the targeted eye is facing the scope. Instill one drop of proparacaine on each eye to block local reflexes and clean away any excess liquid with a cotton tipped applicator. B. Pinch the conjunctiva just below the limbus with forceps and hold the eye steady. C. With your free hand, insert preloaded Hamilton syringe at a 60° angle just below the limbus ~1 mm into the vitreous cavity and gently inject 1 μl of virus by depressing the plunger with your index finger. Hold needle in place for ~15-20 s before removing to prevent leakage and to allow virus to distribute evenly.

  2. Optic nerve crush surgery
    1. Deeply anesthetize mice by isoflurane induction and intraperitoneal (IP) or subcutaneous (SC) injection of ketamine (100 mg/kg)/xylazine (20 mg/kg) (or similar approved cocktail) in accordance with your lab’s and institutional IACUC protocols or local regulations
      Note: To prevent the cornea from drying out during intravitreal injections and ONC surgeries liberally apply Refresh Tears® lubricating eye drops as required.
    2. Transfer a mouse to the surgical scope and instill 1 drop of proparacaine (or similar topical anesthetic) to each eye. Wait ~1-2 min and confirm the depth of anesthesia by hindlimb pinch.
    3. After ensuring the depth of anesthesia, tilt the mouse’s head a little such that the targeted eye is facing the surgeon.
    4. Brush any hair and excess liquid away from the eye with a wet cotton tipped applicator.
    5. Before starting the surgical procedure, surgical tools should be sterilized using a hot bead sterilizer. Wait a few seconds after sterilization to allow tools to cold down.
    6. Slide the tips of #5 forceps between the upper and lower eyelids such that the eye protrudes out of the socket.
    7. With the opposite hand pinch the conjunctiva along the superior-temporal border of the globe with #7 curved forceps making a small fold in the tissue (this will prevent cutting too deep and damaging the sclera) (Figure 2A).
    8. Retract #5 forceps and pick up spring scissors maintaining your hold of the eyeball with curved forceps.
    9. With spring scissors, cut a 1 mm hole in the superior-temporal conjunctiva using the fold as a guide (Figure 2A) and then blunt dissect the conjunctiva to make the hole bigger (Figure 2B).
    10. Gently separate the tissue with forceps till you have a clear view of the superior muscle (this can be challenging to identify but is important to prevent damage of the orbital sinus) (Figures 2C-2D).
    11. With your dominant hand, insert #5 forceps through the newly formed opening past the superior muscle and carefully push away any fat that is obstructing the optic nerve (Figure 2E). Take care not to disrupt any blood vessels, particularly the orbital sinus, or the cavity will fill with blood and completely obstruct your view of the optic nerve.
      Note: There can be a little bit of bleeding during the tissue separation; if so, use cotton tips to wipe away blood.
    12. Once the optic nerve is visualized and cleared, insert the Cross Action self-closing forceps in the open position around the optic nerve 2 mm behind the eyeball, loosen grip and hold steady for 3 secs to crush (Figure 2F). This should be adequate to induce complete degeneration of optic nerve axons up to the crush site and significant RGC cell death.
      Note: The central retinal artery (CRA) of mice branches off the ophthalmic artery (OA), where is very close to the globe (May and Lütjen-Drecoll, 2002). If you clear the tissue around the ON and crush 2 mm behind the globe, the crush won’t obstruct the blood supply of retina.
    13. Following crush, open and retract Cross Action self-closing forceps.
    14. Gently coax the eye back to its natural position with forceps and close the eyelids.
    15. Apply a generous amount of Polycin® Ophthalmic Ointment (or similar antibiotic ointment) over the incision and cornea.
    16. Turn the mouse’s head and repeat crush on the contralateral optic nerve experimental plan.
    17. Administer Buprenorphine HCl (or similar analgesic) after surgery in accordance with your lab’s and institutional IACUC protocols or local regulations.
    18. Place mice on a heating pad to fully recover (~60 min) before transferring back to their home cages.
    19. Check for infection every 12-24 h as dictated by your lab’s and institutional IACUC protocols or local regulations.
    20. House mice for 12 days then proceed to the CTB injections.


      Figure 2. Optic nerve crush surgery. A. Pinch the conjunctiva along the superior-temporal border of the globe with forceps making a small fold in the tissue. Using spring scissors incise a 1 mm hole cutting posteriorly using the folded conjunctiva as a guide. B. Bluntly but gently separate the tissue with forceps to enlarge the hole ~2-3 mm. C-D. Carefully push away any fat obstructing the optic nerve taking care not to disrupt the orbital sinus. E. Once the optic nerve is cleared and visualized. F. use Cross Action self-closing forceps to crush the optic nerve 2 mm behind the globe for 3 s.

  3. CTB labeling of regenerating retinal ganglion cell axons
    1. Resuspend CTB-555 (or desired fluorophore, 500 µg) in 50 µl of sterile PBS to a final concentration of 10 µg/µl.
    2. 12 days post-crush, anesthetize mice accordingly and inject 1 µl of CTB-555 into each eye intravitreally as per the AAV2 injection protocol described above.
      Notes:
      1. To prevent the cornea from drying out during intravitreal injections and ONC surgeries liberally apply Refresh Tears® lubricating eye drops as required.
      2. CTB-555 should be used with AAV2-GFP viral vectors. Alternatively, CTB-488 or -647 can be used depending on the AAV2 reporter.
      3. Regenerating neurons can alternatively be visualized by GAP43 staining as described in Leon et al. (2000).

  4. Tissue dissection and optic nerve clearing
    1. Sacrifice mice 14 days post-crush by transcardial perfusion in accordance with your lab’s and institutional IACUC protocols or local regulations.
    2. Decapitate fixed mice and remove all the skin and hair around the eyes and top of the head with surgical scissors (Figure 3A).
    3. Using #5 forceps and spring scissors, carefully dissect away the conjunctiva connecting the globe to the socket such that it can freely move within the orbit.
    4. Gently pull the globe nasally to access the orbital cavity and remove the lacrimal glands as well as any fat from behind the eye to expose the optic nerve.
    5. Enucleate eyes by cutting the optic nerve as close to the globe as possible with spring scissors and transfer to 48-well dish containing ~500 μl 4% PFA. Post-fix for 1 h at room temperature (Figures 3A-3B).
    6. After 1 h, aspirate the PFA and replace with 1x PBS. Eyes can be stored in sterile 1x PBS for 12+ hours at 4 °C.
    7. Once all the eyes have been post-fixed proceed to Flat mount dissection.
    8. To isolate optic nerves, first cut away the hindbrain with large surgical scissors.
    9. Then carefully cut along the sagittal (dorsal) and squamous (temporal) sutures with surgical scissors and peel the skull nasally to expose the cortex (Figures 3B-3C).
    10. Bluntly separate the olfactory bulbs from the cortex with #5 forceps, then slide forceps under the forebrain, and gently lift ~45° from front to back to expose the optic nerves at the base of the skull (Figure 3D).
    11. Make sure the nerves can freely slide through the meningeal sheath and flip the brain out of the skull (Figure 3E).
      Note: If the nerves get stuck dissect away the meninges before removing the brain.
    12. With the underside of the brain exposed, use spring scissors to cut the nerves just beyond the optic chiasm (Figures 3F-3G).
    13. If desired, separate the right and left optic nerves at the chiasm using spring scissors.
    14. Transfer nerves to 48-well dish containing ~300 μl 4% PFA/well and post-fix for 1 h at room temperature.
    15. After fixation, remove PFA with a transfer pipette and replace with ~100 μl FocusClearTM for ≥ 3 h or until optic nerve is totally transparent (Diekmann et al., 2015; Wang et al., 2019) 
    16. Place a SecureSealTM imaging spacer on a clean slide to build a small chamber to prevent squashing the nerve while mounting.
    17. Place optic nerve in chamber, cover with MountClearTM and coverslip with a 22 x 22 mm micro coverslip.
      Note: Make sure to warm MountClearTM in a 55 °C water bath for about 30 min prior to the mounting.
    18. Image whole mounted cleared optic nerves from the crush site to the chiasm at 20x by tile scanning and Z-stacking on a confocal microscope. Stacked images should be acquired at 8 µm intervals.
    19. Maximum project z-stack images and proceed to Quantification of cleared optic nerve regeneration.


      Figure 3. Eye and optic nerve dissection. A. Remove skin and hair around the eyes and top of the head with large surgical scissors, then using forceps and spring scissors carefully dissect away the conjunctiva connecting the globe to the socket and cut the optic nerve as it enters the globe. Transfer eyes to 48-well dish containing 4% PFA. B. Using large surgical scissors cut away the hindbrain (solid line), then superficially cut along the sagittal and squamous sutures (dashed lines). C. Gently peel the skull (black asterisks) nasally to expose the cortex (red asterisk) and olfactory bulbs (yellow asterisk). D. Bluntly separate the olfactory bulbs from the cortex, then gently slide forceps under the forebrain and lift ~45° to expose the optic nerves (green arrow). E. To isolate nerves, carefully flip the brain out of the skull making sure the nerves can freely slide through the meningeal sheath before removal (if necessary, dissect away the meninges to prevent optic nerves from catching and tearing). F. With the underside of the brain exposed, G. cut the nerves just beyond the chiasm and transfer to a 48-well dish containing 4% PFA.

  5. Flat mount dissection
    1. Under a dissection scope, transfer fixed eyes to 35 x 10 mm tissue culture dish filled with sterile 1x PBS (Figure 4A).
    2. Hold the eyeball steady by pinching the cornea with #5 forceps and make a small hole just above the limbus with a needle (Figure 4B).
    3. Pinch the cornea with forceps and dissect away with spring scissors using the newly formed hole as an access point (Figure 4C).
    4. Carefully remove the lens and any remaining vitreous from the surface of the retina using #5 forceps taking care not to touch the retinal surface (Figure 4D).
    5. Slide #5 forceps behind the retina into the subretinal space and hold the globe steady by pinching the sclera.
    6. Slide closed spring scissors into the subretinal space and move them around the circumference of the eye to completely detach the retina from the RPE/sclera.
    7. Once detached, use spring scissors to partition the retina into equally sized quadrants by making 4 evenly spaced 2-3 mm cuts towards the center of the retina (Figure 4E).
    8. Gently slide retina out of the eye cup and transfer into a separate 35 x 10 mm tissue culture dish filled with sterile 1x PBS (Figure 4F).
    9. Inspect the surface of the retina and remove any remaining vitreous.
      Note: It is critical that all the vitreous is removed from the surface of the retina otherwise it will interfere with antibody staining.
    10. Transfer dissected retina to a 48-well tissue culture dish containing 1x PBS using a cut 3 ml transfer pipette (Figures 4G-4H) and proceed to Flat mount immunostaining.


      Figure 4. Retinal flat mount dissection and staining. A. Transfer fixed eyes to a small tissue culture dish containing fresh 1x PBS. B. Hold eyeball steady with forceps and poke a small hole in the cornea with a needle just above the limbus. C. Using the newly formed hole as an access point, dissect away the cornea with spring scissors. Cut just below the limbus to ensure the ciliary body and iris are separated from the retina. D. Gently remove the lens and vitreous with forceps. E. Slide forceps into the subretinal space and use spring scissors to detach the retina from the RPE/sclera. Partition retina into quadrants by making 4 evenly spaced ~2-3 mm cuts towards the optic nerve head. F. Carefully slide the retina out of the eye cup and remove any remaining vitreous on the retinal surface with forceps. G. Using a cut 3 ml transfer pipette, H. transfer dissected retina (yellow arrow) to a 48-well dish containing 1x PBS and proceed with retinal staining as described below. I. Following staining, transfer retinas ganglion cell side up to a clean slide and unfurl petals. Remove any excess liquid with a twisted KimWipe taking care not to touch the retina. J. Add 1-2 drops ProLong® Gold Antifade Mountant on the surface of each retina and coverslip. Let dry overnight on a flat surface before imaging.

  6. Flat mount immunostaining
    1. Aspirate PBS from each well and replace with 500 μl of Flat Mount Blocking Buffer. Incubate for 1-2 h at room temperature on a rocker.
    2. While blocking, dilute the primary antibody (Guinea Pig-Anti-RBPMS) 1:250 in Flat Mount Blocking Buffer.
    3. After 1-2 h, aspirate the blocking buffer and add ~200 μl of the diluted primary antibody to each well. Incubate for 24-48 h at 4 °C on a rocker.
    4. Aspirate primary antibody and wash retinas 3 times with 1x PBS for 15 min each.
    5. Dilute the secondary antibody (Anti-Guinea Pig-Alexa Fluor 647) 1:500 in Flat Mount Blocking Buffer.
    6. Aspirate final wash and add ~200 ul of the diluted secondary to each well. Incubate for 2-4 h at room temperature on a rocker.
      Note: Alternatively, retinas can be incubated for 24 h at 4 °C on a rocker.
    7. Aspirate secondary antibody and wash retinas 3 times with 1x PBS for 15 min each.
    8. Following the final wash, transfer the retinas to a clean slide using a cut 3 ml transfer pipette.
    9. Place retinas ganglion cell side facing up and unfurl the petals such that they are as flat as possible (Figure 4I). Remove any excess PBS using a Kimwipe taking care to not touch the retina.
    10. Place 1-2 drops of ProLong® Gold Antifade Mountant on the surface of each retina and coverslip with a 22 x 22 mm micro coverslip (Figure 4J).
    11. Allow slides to set overnight on a flat surface protected from light.
    12. Image the retinas at 10x on a confocal microscope acquiring a tile-scan of the whole flat-mounted retinas Z-stacked through the RGC layer (Figures 5A-5B).
    13. Max intensity project the Z-stacks and proceed to Quantification of RGC survival.

Data analysis

  1. Quantification of RGC survival
    1. Before counting, set scale for each exported TIFF images accordingly.
    2. Manually count RGCs in the peripheral (~150-200 μm away from the edge of the retina), middle (~700-800 μm away from the edge) and central (~1,500-1,600 μm away from the edge) regions of each quadrant using the “cell counter” function in FIJI ImageJ (Figure 5C).
    3. Calculate average RGC cell density (D) per region (central, middle, periphery) using the formula: D = [(R1 + R2 + R3 + R4)/4]/A where R equals the number of RGCs for a given image and A equals area (mm2).
    4. Calculate whole retinal RGC density using the formula: (DC + DM + DP)/3 where DC, DM, and DP represent the average densities of the central, middle, and peripheral regions, respectively (Figure 5D).


      Figure 5. Quantification of RGC survival. 10x tile scanned/Z-projected confocal images of A. AAV2-mCherry and B. AAV2-4D3(E)-mCherry treated retinal flat mounts C. Count the number of RBPMS+ cells in the central (C), middle (M), and peripheral (P) regions of each quadrant and calculate RGC density as describe above. D. Comparison of RGC survival of mCherry and 4D3(E) treated retinas adapted from Boczek et al., 2019.

  2. Quantification of cleared optic nerve regeneration
    1. Export and convert confocal images to TIFF format with 500 µm scale bar.
    2. Using Photoshop, make a metric scale ruler with red lines evenly spaced 250 µms apart and save as PSD format (Figure 6A).
    3. Open both the optic nerve TIFF and PSD ruler images. Select the ruler and copy/paste to the optic nerve images. Use “Free Transform” under “Edit” to change the length of the ruler so that the distance between the first and third red lines equals 500 µm (Figure 6B). Apply 2-3 rulers if the optic nerve is not straight (Figure 6C).
    4. Manually count the number of CTB positive puncta that cross each red line starting 0.5 mm from the crush site and continuing all the way to the chiasm or to the end of longest regenerating axons.


      Figure 6. Quantification of regenerating axons. A. Create a ruler in Photoshop where the distance between two red lines is equal to 250 µm. B. Copy ruler to image layer and choose Free Transform to set the scale such that the distance between the first and third red line is 500 µm. C. Rotate the ruler and place the first red line over the crush site (red asterisks). If the optic nerve is not straight, copy another ruler and rotate according to the actual shape of optic nerve. White asterisk shows the connection of the first and second ruler.

Recipes

  1. Flat Mount Blocking Buffer
    1.2 ml (3%) Triton X-100
    0.2 ml (0.5%) Tween-20
    0.4g (1%) BSA
    1 ml (0.1%) 4% sodium azide
    Fill to 40 ml 2x PBS

Acknowledgments

This work was supported in part by National Institutes of Health Grants EY026766 (M.S.K. and J.L.G.), EY031167 (M.S.K.), P30-EY026877 to Stanford University, EY025915 (E.G.C.), and an unrestricted grant from Research to Prevent Blindness, Inc.
  E.G.C., X.X., J.G., and M.A. participated in the design, data acquisition, and writing of the manuscript. J.L.G. and M.S.K. funded and supervised the work. All authors read and edited the manuscript. This protocol was adapted from Boczek et al. (2019) and Apara et al. (2017).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments were approved and conducted in accordance with the guidelines of the Administrative Panel on Laboratory Animal Care (Protocol #: 30550, exp.06/19/2020) at Stanford University and in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

References

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  10. May, C. A. and Lütjen-Drecoll, E. (2002). Morphology of the murine optic nerve. Invest Ophthalmol Vis Sci 43(7): 2206-2212.
  11. Moore, D. L., Blackmore, M. G., Hu, Y., Kaestner, K. H., Bixby, J. L., Lemmon, V. P. and Goldberg, J. L. (2009). KLF family members regulate intrinsic axon regeneration ability. Science 326(5950): 298-301.
  12. Nickells, R. W., Schmitt, H. M., Maes, M. E. and Schlamp, C. L. (2017). AAV2-mediated transduction of the mouse retina after optic nerve injury. Invest Ophthalmol Vis Sci 58(14): 6091-6104.
  13. Quigley, H. A. (2011). Glaucoma. Lancet 377(9774): 1367-1377.
  14. Sappington, R. M., Carlson, B. J., Crish, S. D. and Calkins., D. J. (2010). The microbead occlusion model: A paradigm for induced ocular hypertension in rats and mice. Invest Ophthalmol Vis Sci 51 (1): 207-216.
  15. Tang, Z., Zhang, S., Lee, C., Kumar, A., Arjunan, P., Li, Y., Zhang, F. and Li, X. (2011). An optic nerve crush injury murine model to study retinal ganglion cell survival. J Vis Exp (50).
  16. Tham, Y. C., Li, X., Wong, T. Y., Quigley, H. A., Aung, T. and Cheng, C. Y. (2014). Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology 121(11): 2081-2090.
  17. Wang, J., Geisert, E. E. and Struebing, F. L. (2019). RNA sequencing profiling of the retina in C57BL/6J and DBA/2J mice: Enhancing the retinal microarray data sets from GeneNetwork. Mol Vis 25: 345-358.
  18. Zhang, J., Li, L., Huang, H., Fang, F., Webber, H. C., Zhuang, P., Liu, L., Dalal, R., Tang, P. H., Mahajan, V. B., Sun, Y., Li, S., Zhang, M., Goldberg, J. L. and Hu, Y. (2019). Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse. Elife 8: e45881.

简介

[摘要 ] 在青光眼等疾病中,视网膜神经节细胞(RGC)神经元无法存活或无法再生视神经轴突,这是部分视力丧失的原因,在某些情况下,甚至是完全的视力丧失。视神经挤压术(ONC)不仅可以作为创伤性视神经病变的一种有用模型,而且还可以作为青光眼损伤的有用模型,因为它类似地诱导RGC细胞死亡和变性。腺相关病毒血清型2(AAV2)的玻璃体内注射已被证明特别地和有效地转导视网膜神经节细胞在体内和已因而被提出作为基因递送用于治疗青光眼的治疗的有效手段。确实,我们和其他人常规使用AAV2来研究促进ONC 后RGC中神经保护和轴突再生的机制。本文中,我们描述了分步操作的方案,以测定AAV2介导的转导和ONC损伤后小鼠中RGC的存活和再生,包括1)玻璃体内注射AAV2病毒载体,2)视神经挤压,3)霍乱毒素B (CTB)标记再生轴突,4)视神经清除,5)视网膜平面免疫染色和6)定量RGC存活和再生。除了提供执行此协议所需的所有材料和程序详细信息之外,我们还强调了它比其他相似的已发表方法的优势,并提供了有用的技巧以确保其在任何现代实验室中都能如实复制。

[背景 ] 青光眼是世界范围内不可逆失​​明的主要原因,其特征是视网膜神经节细胞(RGCs)逐渐退化和丧失,这是构成连接视网膜与大脑的视神经的中央投射神经元(Quigley ,2011 ; Tham 等。,2014)。青光眼的RGC细胞死亡被认为部分是由于眼内压(IOP)的增加和RGC轴突通过视神经头离开视网膜时伴随的压缩所致(Quigley,2011; Chang和Goldberg,2012)。已经开发了几种啮齿动物青光眼模型来研究RGC变性的基础细胞和分子机制,包括视神经挤压(ONC),前腔内注射微珠和玻璃体内注射硅油(Sappington 等,2010; Tang 等); 2011;Templeton and Geisert,2012; Ito 等,2016; Zhang 等,2019)。虽然微珠注射模型和硅注射模型都重新确定了IOP的增加并诱导了与青光眼相关的进行性RGC细胞死亡,但由于RGC轴突的损伤和不完全变性,它们不利于研究轴突再生。另外,ONC已成为研究神经元存活和再生的有用的临床前模型,因为它诱导了显着的RGC死亡,几乎没有变异,并且切断了所有的轴突,使人有信心发现受伤部位以外的任何纤维都在再生而不是幸免。甲杰诺相关病毒血清型2 (AAV2 )具体地和有效地转导小号的RGC玻璃体内我njection ,使其成为的原理与人手段将基因递送(重组DNA,shRNA的,等等)转换成视网膜神经节细胞(马丁等人,2002; Nickells 等人。,2017) 。确实,我们已经报道了AAV2在ONC损伤后使用治疗性肽和shRNA来促进RGC存活和轴突再生的用途(Moore 等,2009; Apara 等,2017; Galvao 等,2018; Boczek 等)等。,2019)。在这里,我们描述了一个详尽的协议,详细介绍了将AAV2与ONC结合使用的方法,以研究促进神经保护和再生的机制,并鉴定和表征具有治疗青光眼和其他视神经病变治疗潜力的候选分子。

关键字:青光眼, 视网膜神经节细胞, 退化, 神经保护, 再生, AAV2

材料和试剂


 


猎鹰3毫升移液器(Corning,目录号:357524)
6英寸棉头涂抹器(VWR,目录号:89031-270)
VWR ® 微量离心管(VWR,目录号:87003-294)
VWR ® 的Superfrost ® 此外微型滑动(VWR,目录号:48311-703)
Polycin ® Ophth almic软膏,USP (百利高,目录号:NDC 0574-4021-35 )
吸收剂工作台垫(VWR,目录号:82020-845)
Kimwipes (Fisher Sci entific ,目录号:06-666)
猎鹰® 48孔透明平底板(Corning,目录号:353078)
35 x 10 mm组织培养皿(Corning,目录号:353001)
SecureSeal TM 成像垫片(Grace Bio-Labs,目录号:654006)
22 x 22毫米(1.5号)微盖玻片(VWR,目录号: 48366-227)
成年(> P21)雄性和雌性C57BL / 6J小鼠(JAX,目录号:000664)
美国药典Fluriso TM 异氟烷(VetOne ,目录号:NDC 13985-528-60 )
氯胺酮(或当地法规批准的类似物质)
赛拉嗪(或您当地法规批准的类似品)
盐酸丁丙诺啡(或当地法规批准的类似品)
刷新眼泪® 润滑剂眼药水(Allergan,目录号:NDC 0023-0798-01或类似产品)
普罗卡因盐酸盐眼药水,美国药典(Sandoz,目录号:NDC 61314-016-01)
霍乱毒素B亚基(CTB),Alexa的氟TM 555偶联物(热费舍尔,目录号:C34776)
10x磷酸盐缓冲盐水(Thermo Fisher,目录号:AM9624)
16%多聚甲醛溶液(Fisher Scientific ,目录号:50-980-488)
FocusClear TM (CelExplorer ,目录号:FC-101)
MountClear TM (CelExplorer ,目录号:MC-301)
Triton TM X - 100(Sigma,目录号:X100)
非特异性山羊血清(Thermo Fisher,目录号:16-210-064)
抗RBPMS,豚鼠(Phospho Solutions,目录号:1832-RBPMS)
Anti-Guinea Pig-Alexa Fluor 647(Thermo Fisher,目录号:A21450)
的ProLong ® 黄金抗淬灭封固(赛默飞世,目录号:P36934)
平面安装阻塞缓冲器(请参见配方)
 


设备


 


手术显微镜(WPI,目录号:PSMB5N)
实验室立体显微镜(VWR,目录号:10836-004)
热珠灭菌器(FST,目录号:1800050)
鼠标加热垫(Stryker,目录号:TP600 / 700)
带感应箱的台式异氟烷麻醉系统(哈佛仪器,目录号:72-6468)
杜蒙#5直钳(FST,目录号:11251-10)
杜蒙#7弯曲钳(FST,目录号:11271-30)
Dumont#N5镊子交叉作用Inox细尖尺寸.05 x .01 生物学尖(Roboz Surgical,目录号:RS-5020)
汉密尔顿注射器(Hamilton Company,5μl ,700 系列,目录号:7634-01)
汉密尔顿33号针头(汉密尔顿公司,目录号:7803-05,33规格,点4 0.375英寸)
Vannas 弹簧剪刀-2.5毫米切削刃(FST,目录号:15000-08)
手术剪刀(FST,目录号:91402-14)
蔡司880 LSM共焦显微镜(蔡司)
 


软件


 


斐济ImageJ(https://imagej.net/Fiji)
Adobe Photoshop(https://www.adobe.com/)
 


程序


 


玻璃体内AAV2注射
根据您的实验室和我们的机构动物护理和使用委员会(IACUC)批准的规程或特定国家/地区的规定对小鼠进行麻醉。对于玻璃体内注射,使用桌面诱导盒和鼻锥施用异氟烷就足够了。
虽然动物麻醉下,一定要保持自己的眼睛湿润了在任何时候通过应用刷新泪® 润滑滴眼液需要(每只眼睛一滴每隔5分钟就足够了)。
注意:为防止角膜的过程中玻璃体内注射晒出与ONC手术宽松申请刷新泪® 根据需要润滑滴眼液。


麻醉后,将鼠标移至手术范围并连接鼻锥(确保异氟烷在流动)。
向每只眼睛滴入1滴Proparacaine(或类似的局部麻醉药)以阻止局部反射。等待〜1-2分钟,然后轻轻触摸结膜以确认麻醉深度。
倾斜鼠标的头部,使目标眼睛面向示波器(图1A)。使用棉签涂抹器擦去多余的液体。
使用#5镊子,在上下眼睑之间滑动尖端,使眼球突出于眼窝,并轻轻将结膜捏到角膜缘下方约1 mm (图1B)。
在用镊子稳定眼球的同时,用一只手拿起预装的5μlHamilton 注射器,相对于视神经成60 °角,小心地将巩膜针〜1 mm(斜面朝向晶状体)穿过巩膜角膜缘下方约1毫米进入玻璃体腔(图1C)。由于晶状体占据了玻璃体腔的大部分,因此应注意避免刺穿晶状体。
注意:效价≥1 x 10 12 GC / ml的纯化的体内AAV2级抗体应用于有效转导RGC。


用食指轻轻推动柱塞以注入1μl 病毒。
注意:在视神经压伤之前检查镜片。如果有白内障,则应排除眼睛。


注射后,将针头留在玻璃体腔中约15-20秒,以防止泄漏,然后慢慢取出。
注意:如果液体开始从注射部位回流,则可能注射了太多病毒,并且可能无法将眼睛从以后的分析中排除。


取下镊子,然后闭上眼睑,将眼睛轻轻地放回插槽中。
适用的慷慨量Polycin ® 在角膜和喷射区域眼用软膏(或类似抗生素软膏)。
翻转鼠标并根据需要在对侧眼中重复注射。
将动物放到加热垫上以使其完全康复(约2-3分钟),然后再转移回其家中笼子。
饲养动物2周或优化长度,以使病毒最大表达,然后进行视神经挤压。
              D:\ Reformatting \ 2020-1-6 \ 1902757--1314 Michael Kapiloff 799095 \ Figs jpg \ Fig 1.jpg


              图1. AAV2玻璃体内注射。A. 将麻醉的小鼠转移到手术镜上,并倾斜头部,使目标眼面向镜。在每只眼睛上滴入一滴Proparacaine,以阻止局部反射,并用棉签涂抹器清除多余的液体。B. 用镊子捏住角膜缘正下方的结膜,并保持眼睛稳定。C. 用一只手将预装的汉密尔顿注射器以60 °角插入角膜缘下方约1 mm处,进入玻璃体腔,然后用食指压下柱塞轻轻注入1μl 病毒。取下针头之前,请将其保持在适当的位置约15-20 s,以防止泄漏和使病毒均匀分布。


 


视神经挤压手术
深麻醉话筒通过异氟烷诱导和腹膜内(IP)或皮下(SC)注射氯胺酮(100 E 毫克/ ķ 克)/ 符合您实验室和机构IACUC协议或当地法规的甲苯噻嗪(20 mg / k g)(或类似的获批混合物)
注意:为防止干燥角膜过程中玻璃体内注射了荷兰国际集团和ONC手术宽松申请刷新泪® 根据需要润滑滴眼液。


将鼠标移至手术范围,并向每只眼睛滴入1滴普罗卡因(或类似的局部麻醉药)。等待〜1-2分钟,然后通过后肢捏捏确认麻醉深度。
确保麻醉深度后,稍微倾斜鼠标头,使目标眼睛面向外科医生。
用湿的棉签涂抹器将所有头发和多余的液体从眼睛刷去。
在开始外科手术之前,应使用热珠灭菌器对外科工具进行灭菌。灭菌后等待几秒钟,以使工具冷却。
在上下眼睑之间滑动#5镊子的尖端,使眼睛从插座中突出。
用另一只手捏住#7弯曲镊子沿球体的上颞边界结膜,在组织中形成小折叠(这将防止切得太深并损害巩膜)(图2A)。
缩回#5镊子并拿起弹簧剪刀,用弯曲的镊子保持眼球的抓握。
用弹簧剪刀,以褶皱为指导在颞上结膜上切一个1 mm的孔(图2A),然后用钝器解剖结膜,使孔变大(图2B)。
直到你有优越的肌肉的清晰视图(这可以是具有挑战性的,以确定,但重要的是防止眶窦的损伤)轻轻分开用钳子组织(图小号2C- 2 d) 。
用您的优势手,将#5镊子穿过新形成的开口,越过上肢肌肉,并小心地推开阻碍视神经的脂肪(图2E)。注意不要破坏任何血管,尤其是眼眶窦,否则腔中会充满血液并完全阻塞视神经的视野。
注意:在组织分离过程中可能会有一点点出血;如果是这样,请使用棉签擦拭血液。


视神经可视化并清除后,将Cross Action自闭合钳在眼球后2 mm的视神经周围打开位置插入,松开握持并稳定3秒钟以压碎(图2F)。这应该足以引起视神经轴突完全变性直至挤压部位,并导致RGC细胞大量死亡。
注意:小鼠的视网膜中央动脉(CRA)从眼球(OA)分支,而眼球非常靠近地球(May和Lütjen-Drecoll ,2002年)。如果您清除ON周围的组织并在球体后方2毫米处挤压,那么挤压不会阻碍视网膜的血液供应。


挤压后,打开和收回Cross Action自动关闭钳。
用镊子轻轻地使眼睛回到自然位置,然后闭上眼睑。
适用的慷慨量Polycin ® 在切口和角膜眼用软膏(或类似抗生素软膏)。
转动鼠标的头部,并重复对侧视神经实验计划。
手术后应按照实验室和机构的IACUC规程或当地法规管理盐酸丁丙诺啡(或类似的镇痛药)。
将小鼠放在加热垫上以使其完全康复(〜60分钟),然后再转移回其家中笼子。
按照实验室和机构的IACUC协议或当地法规的规定,每12-24 小时检查一次感染。
家鼠12天,然后进行CTB注射。
 


D:\ Reformatting \ 2020-1-6 \ 1902757--1314 Michael Kapiloff 799095 \ Figs jpg \ Fig 2.jpg


图2.视神经挤压手术。A. 用镊子在地球的上颞边界捏结膜,在组织中形成小折叠。使用弹簧剪刀在后部以折叠的结膜为向导切开一个1毫米的孔。B. 用镊子直截了当地轻轻分离组织,以将孔扩大到2-3 mm。光盘。小心地推开任何阻碍视神经的脂肪,注意不要破坏眼眶窦。E. 一旦视神经被清除并可视化。F. 使用Cross Action自闭合镊子将视神经压在球体后2 mm处3秒钟。


 


再生视网膜神经节细胞轴突的CTB标记
将CTB-555(或所需的荧光团,500 µg)重悬于50 µl无菌PBS中,使其终浓度为10 µg / µl。
后12天挤压,麻醉小鼠,并相应地注入1 μ 上述CTB-555的升到每只眼睛玻璃体内地具体根据AAV2注射方案。
笔记:


为了防止从角膜中玻璃体内注射干燥和ONC手术宽松申请刷新泪® 根据需要润滑滴眼剂。
CTB-555应该与AAV2-GFP病毒载体一起使用。或者,根据AAV2报告基因,可以使用CTB-488或-647。
如Leon等人所述,可通过GAP43染色可视化再生神经元。(2000 )。
 


组织解剖和视神经清除
根据您的实验室和机构的IACUC规程或当地法规,经心灌注灌注后14天将小鼠牺牲。
脱去固定的小鼠,并用手术剪刀去除眼睛和头部顶部周围的所有皮肤和头发(图3A)。
使用#5镊子和弹簧剪刀,仔细解剖掉将球体连接到球窝的结膜,使其可以在轨道内自由移动。
轻轻地将球体拉向鼻腔以进入眼眶,并去除泪腺以及眼后部的任何脂肪,以露出视神经。
通过用弹簧剪刀将视神经尽可能靠近球体切开,使眼睛充满魅力,并转移至包含约500个孔的48孔培养皿中 μ升4%PFA。后修复1个小时在室温下(图小号3A- 3 B) 。
1小时后,吸出PFA,并用1x PBS代替。眼睛可在4 °C的无菌1x PBS中保存12小时以上。
固定好所有眼睛后,继续进行平口解剖。
要隔离视神经,请先用大号手术剪刀切除后脑。
然后沿矢状(背)和鳞状(时间的)缝线手术剪小心切开和剥离经鼻颅骨以暴露皮质(F igure 小号3B- 3 C) 。
用#5镊子将嗅球从皮层中直截了当分离,然后将镊子滑到前脑下方,并从前向后轻轻抬起〜45 ° ,露出颅底的视神经(图3D)。
确保神经可以自由地滑过脑膜鞘并将大脑翻转出颅骨(图3E)。
注意:如果神经被卡住,请在切除大脑之前解剖掉脑膜。


在露出大脑下方的情况下,使用弹簧剪刀将神经切断到正好位于视交叉的上方(图s 3F- 3 G)。
如果需要,可以用弹簧剪刀将左,右视神经分开在as骨处。
神经传递到48孔培养皿的含〜300 μ 升4%PFA /孔和后固定1 ħ 在室温下。
固定后,除去PFA与移液管,并用〜100替换μ 升FocusClear TM 为≥3小时或直到视神经是完全透明的(狄克曼等人,2015;王等人,2019)
将SecureSeal TM 成像垫片放在干净的幻灯片上,以建立一个小腔室,以防止在安装时挤压神经。
置于腔室视神经,盖与MountClear TM 和盖玻片用22×22毫米的微型盖玻片。
注意:安装前,请确保在55 °C的水浴中加热MountClear TM 约30分钟。


通过平铺扫描和共聚焦显微镜在Z轴上进行堆叠,从粉碎部位到胫骨的完整清晰的视神经图像以20倍成像。堆叠的图像应以8 µm的间隔获取。   
最大投影z-stack图像,并进行清晰的视神经再生的量化。
 


D:\ Reformatting \ 2020-1-6 \ 1902757--1314 Michael Kapiloff 799095 \ Figs jpg \ Fig 3.jpg


图3.眼睛和视神经解剖。A. 用大号外科手术剪刀除去眼睛和头顶周围的皮肤和头发,然后用镊子和弹簧剪刀仔细解剖结膜,将球体连接到承窝,并在视神经进入球体时切断视神经。将眼睛转移到含有4%PFA的48孔培养皿中。B. 用大号外科剪刀剪掉后脑(实线),然后沿矢状和鳞状缝合线(虚线)浅切。C. 轻轻地将头骨(黑色星号)鼻翼剥离,露出皮质(红色星号)和嗅球(黄色星号)。D. 直截了当地将嗅球从皮层中分离出来,然后将镊子轻轻滑到前脑下方并抬起〜45 ° ,露出视神经(绿色箭头)。E. 要分离神经,请小心地将大脑从颅骨中翻转出来,以确保在移除之前神经可以自由地滑过脑膜鞘(如有必要,请解剖脑膜以防止视神经被抓住和撕裂)。F. 在暴露出大脑下方的情况下,G。切断了神经,使其恰好超出了神经干,并转移到含有4%PFA的48孔培养皿中。


 


平装解剖
在解剖范围内,将固定的眼睛转移到装有无菌1x PBS的35 x 10 mm组织培养皿中(图4A)。
用#5镊子捏住角膜,保持眼球稳定,并用针在角膜缘上方钻一个小孔(图4B)。
用镊子捏住角膜,并用弹簧剪刀将新形成的孔作为接入点解剖(图4C)。
使用#5镊子小心地从视网膜表面取下晶状体和所有残留的玻璃体,注意不要触摸视网膜表面(图4D)。
将#5镊子滑入视网膜后方的视网膜下空间,并通过捏紧巩膜使球保持稳定。
将闭合的弹簧剪刀滑入视网膜下间隙,并在眼周周围移动它们,以使视网膜与RPE /巩膜完全脱离。
分离后,使用弹簧剪刀通过向视网膜中心进行4个均匀间隔的2-3 mm切口,将视网膜划分为相等大小的象限(图4E)。
轻轻地将视网膜滑出眼罩,然后转移到另一个装有无菌1x PBS的35 x 10 mm组织培养皿中(图4F)。
检查视网膜表面并清除所有残留的玻璃体。
注意:至关重要的是,必须从视网膜表面去除所有玻璃体,否则会干扰抗体染色。


使用切开的3 ml移液管将解剖的视网膜转移至包含1x PBS的48孔组织培养皿中(图s 4G- 4 H),然后进行平装免疫染色。
 


D:\ Reformatting \ 2020-1-6 \ 1902757--1314 Michael Kapiloff 799095 \ Figs jpg \ Fig 4.jpg


                            图4.视网膜平面安装解剖和染色。A. 将固定的眼睛转移到含有新鲜1x PBS 的小型组织培养皿中。B. 用镊子稳定眼球,并在角膜缘上方用针在角膜上戳一个小孔。C. 使用新形成的孔作为接入点,用弹簧剪刀解剖角膜。在角膜缘下方切开,以确保睫状体和虹膜与视网膜分离。D. 用镊子轻轻取下晶状体和玻璃体。E.将镊子滑入视网膜下腔,并使用弹簧剪刀将视网膜与RPE /巩膜分离。通过在视神经头上形成4个均匀间隔的〜2-3 mm的切口,将视网膜分成四部分。F. 小心地将视网膜滑出眼罩,并用镊子去除视网膜表面上残留的所有玻璃体。G. 使用切割的3ml移液管,H。将解剖的视网膜(黄色箭头)转移到含有1x PBS的48孔培养皿中,并如下所述进行视网膜染色。I. 染色后,将视网膜神经节细胞的一面朝上移至干净的玻片上,并展开花瓣。用扭曲的KimWipe 清除多余的液体,注意不要触摸视网膜。J. 添加1-2滴的ProLong ® 金抗荧光淬灭封固每个视网膜和盖玻片的表面上。在成像之前,让其在平坦的表面上干燥过夜。


 


平装免疫染色
吸PBS从每个孔和替换500 μ 升平坦的载置的封闭缓冲液。在摇杆上于室温下孵育1-2小时。
封闭时,在Flat Mounting封闭缓冲液中以1:250稀释一抗(Guinea Pig-Anti-RBPMS)。
1-2小时后,吸出封闭缓冲液并添加〜200 μ 升稀释的初级抗体的各孔中。在摇杆上于4 °C 孵育24-48小时。
吸出一抗并用1x PBS冲洗视网膜3次,每次15分钟。
在Flat Mounting Blocking Buffer中以1:500稀释二抗(Anti-Guinea Pig-Alexa Fluor 647)。
吸出最后的洗液,并在每个孔中加入〜200 ul稀释的次要溶液。在摇杆上于室温下孵育2-4小时。
注意:或者,视网膜可以在摇杆上于4 °C 孵育24小时。


吸出二抗并用1x PBS冲洗视网膜3次,每次15分钟。
最后一次洗涤后,使用切开的3 ml移液管将视网膜移至干净的玻片上。
将视网膜神经节细胞一侧朝上放置,然后使花瓣去卷曲,使它们尽可能平坦(图4I)。使用Kimwipe除去多余的PBS,注意不要触摸视网膜。
地方1-2滴的的ProLong ® 金抗荧光淬灭封固每个视网膜和盖玻片用22×22毫米的微型盖玻片的表面上(图4J) 。
让幻灯片在避光的平面上放置一整夜。
图像上的共焦显微镜的视网膜在10倍获取瓷砖扫描的整个平坦安装通过RGC层视网膜Z-堆叠(图小号5A- 5 B) 。
最大强度投影Z堆栈并进行RGC生存的量化。
 


数据分析


 


定量RGC生存
在计数之前,为每个导出的TIFF图像设置比例。
人工计数在外围视网膜神经节细胞(〜150-200 μ 米来自视网膜的边缘的距离),中间(〜700-800 μ 米远离边缘)和中央(〜1 ,500-1 ,600 μ 中号远离使用FIJI ImageJ中的“单元格计数器”功能(图5C )显示每个象限的边缘区域。
使用以下公式计算每个区域(中央,中间,外围)的平均RGC单元密度(D):D = [(R1 + R2 + R3 + R4)/ 4] / A其中R等于给定图像的RGC数量,并且等于面积(mm 2 )。
使用以下公式计算整个视网膜RGC密度:(D C + D M + D P )/ 3 其中D C ,D M 和D P分别代表中央,中间和外围区域的平均密度(图5D)。
 


D:\ Reformatting \ 2020-1-6 \ 1902757--1314 Michael Kapiloff 799095 \ Figs jpg \ Fig 5.jpg


图5. RGC生存的量化。10 X 瓦扫描/的Z-投影共聚焦图像A. AAV2-mCherry的和B. AAV2-4D3(E)处理过的-mCherry视网膜铺片C. 计数的RBPMS +细胞的数目在中央(C),中(M)以及每个象限的外围(P)区域,并如上所述计算RGC密度。D. 从Boczek 等人改编的mCherry和4D3(E)处理的视网膜的RGC存活率比较,2019年。


 


清晰的视神经再生的定量
使用500 µm比例尺将共聚焦图像导出并转换为TIFF格式。
使用Photoshop,制作带有红线的度量尺标尺,其均匀间隔250 µms,并另存为PSD格式(图6A)。
打开视神经TIFF和PSD标尺图像。选择标尺并复制/粘贴到视神经图像。使用“编辑”下的“自由变换”更改标尺的长度,以使第一和第三红线之间的距离等于500 µm (图6B)。如果视神经不直,则使用2-3个尺子(图6C)。
手动计算穿过每条红线的CTB阳性点的数量,这些点从挤压部位开始0.5毫米,一直延伸到正畸或最长的再生轴突末端。
 


D:\ Reformatting \ 2020-1-6 \ 1902757--1314 Michael Kapiloff 799095 \ Figs jpg \ Fig 6.jpg


图6.再生轴突的定量。A. 在Photoshop中创建一个标尺,其中两个红色线之间的距离等于250微米。B. 将标尺复制到图像层,然后选择“自由变换”以设置比例,以使第一条和第三条红线之间的距离为500 µm。C. 旋转标尺,并将第一条红线放置在挤压部位上(红色星号)。如果视神经不直,请复制另一把尺子,然后根据视神经的实际形状旋转。白色星号表示第一和第二标尺的连接。


 


菜谱


 


平装阻塞缓冲器
1.2毫升(3%)Triton X-100


0.2毫升(0.5%)吐温20


0.4克(1%)BSA


1毫升(0.1%)4%叠氮化钠


填充至40 ml 2 x PBS


 


致谢


 


这项工作得到了美国国立卫生研究院EY026766(MSK和JLG),EY031167(MSK),斯坦福大学P30-EY026877,EY025915(EGC)的部分支持,以及Research to Prevention Blindness,Inc.的无限制拨款。


EGC,XX,JG和MA参与了手稿的设计,数据采集和编写。JLG和MSK资助并监督了这项工作。所有作者都阅读并编辑了手稿。该协议改编自Boczek 等。(2019 )和Apara 等。(2017年)。


 


利益争夺


 


作者宣称没有利益冲突。


 


伦理


 


所有动物实验均已根据斯坦福大学实验动物管理行政小组(协议编号:30550,exp.06 / 19/2020)的指导原则进行批准并进行,并符合《 ARVO动物使用声明》眼科和视觉研究。


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cameron, E. G., Xia, X., Galvao, J., Ashouri, M., Kapiloff, M. S. and Goldberg, J. L. (2020). Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration. Bio-protocol 10(6): e3559. DOI: 10.21769/BioProtoc.3559.
  2. Boczek, T., Cameron, E. G., Yu, W., Xia, X., Shah, S. H., Castillo Chabeco, B., Galvao, J., Nahmou, M., Li, J., Thakur, H., Goldberg, J. L. and Kapiloff, M. S. (2019). Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment. J Neurosci 39(28): 5466-5480.
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