Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

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Fig. 2. Procedure for two-step immunopanning method.

PROCEDURE

Coating the Coverslips

Materials

1)12-mm coverslips (Matsunami Glass, Osaka, Japan) in 95% EtOH

2)Sterile forceps in 95% EtOH

3)10-mm Petri dish

4)24-well culture plate

5)Poly-l-lysine (P2636; 1 mg/ml, store at –20°C)

6)Sterile water

7)EHS Laminin (GIBCO BRL 23017-015; 1 mg/ml, store at –20°C)

8)EAGLE-MEM (Earle’s Salts): EBSS or HBSS

Method

1)Put 12-mm coverslips on top of the 10-mm Petri dish with sterile forceps

2)Put coverslips on the bottom of the 24-well plate (culture plate)

3)Add sterile water (9.5 ml) + poly-l-lysine (0.5 ml); final concentration: 50 μg/ml

4)Put 400 μl of diluted poly-l-lysine into each well (let stand overnight at 4°C)

5)Wash each well twice with 0.5 ml of EBSS

6)Add EBSS (10 ml) + dissolved laminin (10 μl); final concentration: 1 μg/ml)

7)Put 400 μl of diluted laminin into each well for 2 h at room temperature (RT)

8)Wash each well twice with 0.5 ml of EBSS

Dissecting the Retinal Tissue

Materials

1)Neurobasal (Gibco 21103-049; store at 4°C) with B-27 supplement (Gibco 17504– 044 × 50; store at –20°C). This is known as the NB mix medium.

a.Gentamicin (10 μg/ml)

b.l-Glutamine (G-7513; 1 mM) 50 ng/ml each of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) and 10 μM of forskolin (F-6886)

c.Warm the mixture to 37°C

2)Enzyme solution in EBSS (10 ml)

a.0.02% BSA (A-7888) (2 mg)

b.0.2 mg/ml DL-Cysteine (C-4022) (2 mg)

c.70 U/ml Collagenase (C-0130) (1 ml) with stock solution (700 U/ml collagenase in EBSS with Ca2+, Mg2+)

d.15 Units/ml papain (Worthington, Lakewood, NJ) (130 μl) (1170 Units/ml)

e.9 ml of EBSS with Ca2+, Mg2+, and 1 ml of collagenase stock solution (700 Units/ml); add 2 mg BSA (store at 4°C) and 2 mg of DL-Cysteine*1 (store at RT) (Solution A)

f.Combine 3 ml of solution A with 40 μl of papain*2 (final concentration of 15 Units/ml, store at 4°C) (Solution B)

g.Filter the enzyme solution through Millipore and warm it to 37°C

h.*1and *2 to be digested, *2should be mixed in last.

3)Ovomucoid (T-2011)/BSA in EBSS

a.Ovo1: BSA (5 mg) + Ovomucoid (10 mg) in 5 ml of EBSS

b.Ovo2: BSA (30 mg) + Ovomucoid (30 mg) in 3 ml of EBSS

c.Control pH with 0.4N NaOH and 1N HCl, the best color is light pink

d.0.1% BSA in PBS: BSA 15 mg + PBS 15 ml

Method

1)Decapitate 6- to 8-day-old rats (4 retinas are needed for each experiment)

2)Cut the skin around the lid

3)Remove the eyeball, taking care not to perforate it

4)Puncture the area around the limbus with a disposable knife

5)Remove the anterior segment with micro-scissors

6)Remove the vitreous from the retina and store the retina in EBSS with Ca2+ and Mg2+

7)Aspirate EBSS, add 0.5 ml/eye of the enzyme solution in EBSS, and incubate for 30 min at 37°C

8)Triturate the tissue sequentially with a narrow-bore Pasteur pipette in a solution containing Ovo1 solution in EBSS to yield a suspension of single cells

9)After centrifugation at 800 × g for 5 min, wash the cells again with Ovo2 solution in EBSS

10)After centrifugation at 800 × g for 5 min, resuspend the cells in 4.5 ml of 0.1% BSA in PBS and then filter them through 20-μm mesh

Purification of RGCs

Materials

1)24-well plate with sterile 12-mm coverslips

2)Primary antibodies: anti-mouse macrophage antiserum (Mouse anti-rat SIRP monoclonal antibody for rat retina, MAB1407P, MRC OX-41, Chemicon, Temecula, CA); mouse anti-rat and mouse Thy 1.1 monoclonal antibody, MAB1406, MRC OX-7, Chemicon)

3)25-mm2 flask (Nunc, Wiesbaden, Germany) for anti-macrophage antibody

4)50-ml Falcon or Corning tubes for anti-Thy 1.1 antibody

5)Sterile PBS, 0.1% BSA in PBS

Method

(Two-step immunopanning procedure)

1)Incubate 25-mm2 flasks and 50-ml tubes with anti-macrophage antiserum and antiThy 1.1 antiserum in PBS, respectively, and leave overnight at 4°C (2 retinas/25mm2 flask or 50-ml tube; anti-macrophage antiserum PBS: 5 ml PBS + 100 μl macrophages (× 50) anti-Thy 1.1 antiserum PBS: 6 ml PBS + Thy 1.1: 20 μl

(× 300)

2) To remove free Thy 1.1 and macrophages, rinse the flasks and tubes with 3 ml of PBS once for macrophage-coated flasks, twice for Thy 1.1-coated tubes

3)Incubate the retinal suspension in macrophage-coated 25-mm2 flasks for 30 min at RT in a dark environment (the flasks should be swirled every 10 min)

4)Collect non-adherent cells in Thy 1.1-coated 50-ml tubes and incubate for 30 min at RT in a dark environment (the tubes should be swirled every 10 min)

5)Aspirate the non-adherent cells and rinse the tubes five times with 3 ml of PBS and put 1.2 ml of NB mix medium into each tube

6)After centrifugation at 800 × g for 5 min, seed the cells on coverslips coated with poly-l-lysine (50 μg/ml) and laminin (1 μg/ml) (NB—mix medium: 400 μl/well)

7)Incubate cultures at 37°C in 5% CO2/95% O2

The purified RGCs were plated at a low density of approximately 500–2000 cells/coverslip of growth substrate. On the first day in vitro (1 DIV), most cultured cells were round, and some cells had begun to grow short processes (see Fig. 3A). On 3 DIV, the cells displayed a neuron-like morphology and extended processes (see Fig. 3B), and on 7 DIV, almost all cells had numerous processes, some with a diameter of more than five cells (see Fig. 3C).

Recombinant human BDNF: PeproTech. EC Ltd., London, UK; Cat. No. 450-02 (10 μg), Diaclone, Besançon, France; Cat. No. IM-36 (10 μg)

Recombinant rat CNTF: Diaclone; Cat. No. IM-202 (25 μg)

Unless noted, all other reagents were obtained from Sigma (St. Louis, MO). 100,000 RGCs/postnatal day 7 retina

Panning procedure: more than 85% purity, 2–4% yields (7) The entire procedure takes about 4 h

CONTROVERSY CONCERNING NMDA EXCITOTOXICITY IN RGCS

Excitotoxic neuronal death is believed to be an important contributor to neuronal death caused by brain and spinal cord injuries as well as by many neurological diseases. The ionotropic glutamate receptors are ligand-gated ion channels that are grouped into three pharmacologically defined classes: N-metyl-D-aspartate (NMDA), -amino- 3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA), and kainate (KA) receptors. NMDA receptor-mediated excitotoxicity is presently thought to be an important contributor to RGC death in glaucoma and retinal ischemia (9,10). However, recent

Fig. 3. Morphology of purified retinal ganglion cells (RGCs) in culture. Phase-contrast images of rat RGCs after 1 (A), 3 (B), and 7 (C) days of culture. Bar = 20 μm (13).

reports show that RGCs are not vulnerable to NMDA excitotoxicity in vitro (7,11,12). A previous report of ours dealt with AMPA-KA receptor-mediated excitotoxicity in postnatal RGCs (7). Ullian et al. (11) reported that, compared with amacrine cells, RGCs in vitro and in situ are relatively unaffected by glutamate and NMDA excitotoxicity. Luo et al. (12) found that adult mouse RGCs were less sensitive than postnatal mouse RGCs to glutamate, NMDA and KA excitotoxicity. These findings do not support the notion of an important contribution by NMDA excitotoxicity to glaucoma, which is characterized by selective loss of RGCs.

CONCLUSIONS

The procedure described above constitutes a simplified purification of rat RGCs. To detect the neuroprotective effects of some compounds in cultured RGCs, the RGCs need to be healthy. Purified RGCs can be cultured at present, but these cells are regenerated, so that it is necessary to confirm data obtained from in vitro experiments by using experimental glaucoma or ischemic models to examine whether the compounds can

prevent RGC death. The procedure described here can produce healthy RGCs in a relatively short time.

REFERENCES

1.Meyer-Franke, A., Kaplan, M.R., Pfrieger, F.W., Barres, B.A. (1995). Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 15:805–819.

2.Meyer-Franke, A., Wilkinson, G.A., Kruttgen, A., Hu, M., Munro, E., et al. (1998) Depolarization and c AMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons. Neuron 21:681–693.

3.Beale, R., Osborne, N.N. (1982) Localization of the Thy-1 antigen to the surfaces of rat retinal ganglion cells in culture. Neurochem. Int. 4:587–595.

4.Barnstable, C.J., Dräger, U.C. (1984) Thy-1 antigen: a ganglion cell specific marker in rodent retina. Neuroscience 11:847–855.

5.Barres, B.A., Silverstein, B.E., Corey, D.P., Chun, L.L.Y. (1988) Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1:791–803.

6.Linsey, J.D., Weinreb, R.N. (1994) Survival and differentiation of purified retinal ganglion cells in a chemically defined microenvironment. Invest. Ophthalmol. Vis. Sci. 35: 3640–3648.

7.Otori, Y., Wei, J.-Y., Barnstable, C.J. (1998) Neurotoxic effects of low doses of glutamate on purified rat retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 39:972–981.

8.Otori, Y., Kusaka, S., Kawasaki, A., Morimura, H., Miki, A., Tano, Y. (2003) Protective effect of nilvadipine against glutamate neurotoxicity in purified retinal ganglion cells. Brain Res. 961:213–219.

9.Ritch, R. (2000) Neuroprotection: Is it already applicable to glaucoma therapy? Curr. Opin. Ophthalmol. 11:78–84.

10.Wax, M.B., Tezel, G. (2002) Neurobiology of glaucomatous optic neuropathy: diverse cellular events in neurodegeneration and neuroprotection. Mol. Neurobiol. 26:45–55.

11.Ullian, E.M., Barkis, W.B., Chen, S., Diamond, J.S., Barres, B.A. (2004) Invulnerability of retinal ganglion cells to NMDA excitotoxicity. Mol. Cell. Neurosci. 26:544–557.

12.Luo, X., Baba, A., Matsuda, T., Romano, C. (2004) Susceptibilities to and mechanisms of excitotoxic cell death of adult mouse inner retinal neurons in dissociated culture. Invest. Ophthalmol. Vis. Sci. 45:4576–4582.

13.Miki, A., Otori, Y., Okada, M., Tano, Y. (2006) Expression of AMPA receptor subunit proteins in purified retinal ganglion cells. Jpn. J. Ophthalmol. 50:217–223.

VII

THERAPEUTIC MODALITIES

INTRODUCTION

The treatment of glaucoma entered the current age in the mid-nineteenth century with the introduction of surgical iridectomy by von Graefe although it was not until the 1930s, when Barkan distinguished the open-angle and closed-angle forms of glaucoma, that iridectomy assumed its present role in the management of angle-closure glaucomas. The modern medical therapy of glaucoma began in the 1870s with the application of cholingeric agents, and filtering surgery had its origins in the first decade of the twentieth century.

In two of these areas, iridectomy and medical therapy, we have seen significant advances in the twentieth century. One of the most important advances was the introduction of laser technology and its application to peripheral iridotomy in the 1970s. Today, this is a well accepted and highly effective modality for treating pupillary block angle-closure glaucoma. Laser technology has subsequently been applied to managing other forms of glaucoma, most notably panretinal photocoagulation for neovascular glaucoma, trabeculoplasty for several open-angle forms of glaucoma, and cyclophotocoagulation for some advanced, intractable cases of glaucoma.

Advances in glaucoma pharmacology were slow in the first half of the twentieth century, with the only notable additions to cholinergics being epinephrine and systemic carbonic anhydrase inhibitors. The second half of the century, however, saw a dramatic escalation in new drugs for the chronic management of glaucoma, beginning with beta blockers in the late 1970s, followed by topical carbonic anhydrase inhibitors, alpha-2 adrenergic agonists, and prostaglandin analogs in the 1990s and early twenty-first century. Today, these latter four classes of drugs constitute first-line medical therapy, with a good level of efficacy and safety for a high percentage of glaucoma patients although the need for new and better agents continues.

Unfortunately, the history of glaucoma surgery to drain aqueous humor from the anterior chamber to the subconjunctival space has not been as impressive as that of the iridotomy and medical therapy. The problem remains the unpredictable biological responses of the subconjunctival tissues to excessive fibrosis and surgical failure on the one hand and tissue thinning with leakage and infection on the other. The two major advances during the twentieth century were the introduction of drainage implant devices from the anterior chamber to a plate in the equatorial subconjunctival space and the adjunctive use of anti-fibrotic agents to modulate wound healing. However, both of these modalities are attendant with their own sets of problems, and the need for a better operation for most forms of glaucoma is a priority for glaucoma research in the twenty-first century.