A modified chronic ocular hypertension rat model for retinal ganglion cell neuroprotection

Abstract

This study aimed to modify a chronic ocular hypertension (OHT) rat model to screen for potential compounds to protect retinal ganglion cells (RGCs) from responding to increased intraocular pressure (IOP). A total of 266 rats were prepared and randomly grouped according to different time-points, namely, weeks 3, 8, 16, and 24. Rats were sedated and eye examination was performed to score as the corneal damage on a scale of 1 to 4. The OHT rat model was created via the injection of a hypertonic saline solution into the episcleral veins once weekly for two weeks. OHT was identified when the IOP at week 0 was≥6 mmHg than that at week -2 for the same eye. Viable RGCs were labeled by injecting 4% FluoroGold. Rats were sacrificed, and the eyes were enucleated and fixed. The fixed retinas were dissected to prepare flat whole-mounts. The viable RGCs were visualized and imaged. The IOP (meanβ±βSD) was calculated, and data were analyzed by the paired t-test and one-way ANOVA. The OHT model was created in 234 of 266 rats (87.97%), whereas 32 rats (12.03%) were removed from the study because of the absence of IOP elevation (11.28%) and/or corneal damage scores over 4 (0.75%). IOP was elevated by as much as 81.35% for 24 weeks. The average IOP was (16.68β±β0.98)βmmHg in non-OHT eyes (n = 234), but was (27.95±0.97)βmmHg in OHT eyes (n = 234). Viable RGCs in the OHT eyes were significantly decreased in a time-dependent manner by 29.41%, 38.24%, 55.32%, and 59.30% at weeks 3, 8, 16, and 24, respectively, as compared to viable RGCs in the non-OHT eyes (P<β0.05). The OHT model was successfully created in 88% of the rats. The IOP in the OHT eyes was elevated by approximately 81% for 24 weeks. The number of viable RGCs was decreased by 59% of the rats in a time-dependent manner. The modified OHT model may provide an effective and reliable method for screening drugs to protect RGCs from glaucoma.

Keywords

chronic ocular hypertension / intraocular pressure / retinal ganglion cells / neuroprotection / glaucoma

References

[1] Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol2006; 90(3): 262–267

[2] Muñoz B, West SK. Blindness and visual impairment in the Americas and the Caribbean. Br J Ophthalmol2002; 86(5): 498–504

[3] Shields BM, Ritch R, Krupin T. Classifications of the glaucomas. St. Louis: Mosby1996.

[4] Garcia-Valenzuela E, Shareef S, Walsh J, Sharma SC. Programmed cell death of retinal ganglion cells during experimental glaucoma. Exp Eye Res1995; 61(1): 33–44

[5] Reichstein D, Ren L, Filippopoulos T, Mittag T, Danias J. Apoptotic retinal ganglion cell death in the DBA/2 mouse model of glaucoma. Exp Eye Res2007; 84(1): 13–21

[6] Quigley HA, Nickells RW, Kerrigan LA, Pease ME, Thibault DJ, Zack DJ. Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci1995; 36(5): 774–786

[7] Sappington RM, Sidorova T, Long DJ, Calkins DJ. TRPV1: contribution to retinal ganglion cell apoptosis and increased intracellular Ca2+ with exposure to hydrostatic pressure. Invest Ophthalmol Vis Sci2009; 50(2): 717–728

[8] Farkas RH, Grosskreutz CL. Apoptosis, neuroprotection, and retinal ganglion cell death: an overview. Int Ophthalmol Clin2001; 41(1): 111–130

[9] Kuehn MH, Fingert JH, Kwon YH. Retinal ganglion cell death in glaucoma: mechanisms and neuroprotective strategies. Ophthalmol Clin North Am2005; 18(3): 383–395, vi (vi.)

[10] Levin LA. Neuroprotection and regeneration in glaucoma. Ophthalmol Clin North Am2005; 18(4): 585–596, vii (vii.)

[11] Baltmr A, Duggan J, Nizari S, Salt TE, Cordeiro MF. Neuroprotection in glaucoma- Is there a future role? Exp Eye Res2010; 91(5): 554–566

[12] Danesh-Meyer HV. Neuroprotection in glaucoma: recent and future directions. Curr Opin Ophthalmol2011; 22(2): 78–86

[13] Link BA, Gray MP, Smith RS, John SW. Intraocular pressure in zebrafish: comparison of inbred strains and identification of a reduced melanin mutant with raised IOP. Invest Ophthalmol Vis Sci2004; 45(12): 4415–4422

[14] Sherpa T, Hunter SS, Frey RA, Robison BD, Stenkamp DL. Retinal proliferation response in the buphthalmic zebrafish, bugeye. Exp Eye Res2011; 93(4): 424–436

[15] Quigley HA, Addicks EM. Chronic experimental glaucoma in primates. I. Production of elevated intraocular pressure by anterior chamber injection of autologous ghost red blood cells. Invest Ophthalmol Vis Sci1980; 19(2): 126–136

[16] Quigley HA, Addicks EM. Chronic experimental glaucoma in primates. II. Effect of extended intraocular pressure elevation on optic nerve head and axonal transport. Invest Ophthalmol Vis Sci1980; 19(2): 137–152

[17] Levkovitch-Verbin H, Quigley HA, Martin KR, Valenta D, Baumrind LA, Pease ME. Translimbal laser photocoagulation to the trabecular meshwork as a model of glaucoma in rats. Invest Ophthalmol Vis Sci2002; 43(2): 402–410

[18] Johnson MA, Drum BA, Quigley HA, Sanchez RM, Dunkelberger GR. Pattern-evoked potentials and optic nerve fiber loss in monocular laser-induced glaucoma. Invest Ophthalmol Vis Sci1989; 30(5): 897–907

[19] Quigley HA, Hohman RM. Laser energy levels for trabecular meshwork damage in the primate eye. Invest Ophthalmol Vis Sci1983; 24(9): 1305–1307

[20] Morrison JC, Moore CG, Deppmeier LM, Gold BG, Meshul CK, Johnson EC. A rat model of chronic pressure-induced optic nerve damage. Exp Eye Res1997; 64(1): 85–96

[21] Danias J, Shen F, Kavalarakis M, Chen B, Goldblum D, Lee K, Zamora MF, Su Y, Brodie SE, Podos SM, Mittag T. Characterization of retinal damage in the episcleral vein cauterization rat glaucoma model. Exp Eye Res2006; 82(2): 219–228

[22] Mittag TW, Danias J, Pohorenec G, Yuan HM, Burakgazi E, Chalmers-Redman R, Podos SM, Tatton WG. Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model. Invest Ophthalmol Vis Sci2000; 41(11): 3451–3459

[23] Nissirios N, Chanis R, Johnson E, Morrison J, Cepurna WO, Jia L, Mittag T, Danias J. Comparison of anterior segment structures in two rat glaucoma models: an ultrasound biomicroscopic study. Invest Ophthalmol Vis Sci2008; 49(6): 2478–2482

[24] Morrison JC, Fraunfelder FW, Milne ST, Moore CG. Limbal microvasculature of the rat eye. Invest Ophthalmol Vis Sci1995; 36(3): 751–756

[25] Cohan BE, Bohr DF. Goldmann applanation tonometry in the conscious rat. Invest Ophthalmol Vis Sci2001; 42(2): 340–342

[26] Jia L, Cepurna WO, Johnson EC, Morrison JC. Patterns of intraocular pressure elevation after aqueous humor outflow obstruction in rats. Invest Ophthalmol Vis Sci2000; 41(6): 1380–1385

[27] Mermoud A, Baerveldt G, Minckler DS, Lee MB, Rao NA. Intraocular pressure in Lewis rats. Invest Ophthalmol Vis Sci1994; 35(5): 2455–2460

[28] Morrison JC, Magann EF, Paulson KA, Chauhan SP, Berghella V, Siddiqui D. Evidence-based medicine guidelines in obstetrics/gynecology and trauma surgery. J Miss State Med Assoc2009; 50(9): 302–305

[29] Grozdanic SD, Betts DM, Sakaguchi DS, Kwon YH, Kardon RH, Sonea IM. Temporary elevation of the intraocular pressure by cauterization of vortex and episcleral veins in rats causes functional deficits in the retina and optic nerve. Exp Eye Res2003; 77(1): 27–33

[30] Bui BV, Edmunds B, Cioffi GA, Fortune B. The gradient of retinal functional changes during acute intraocular pressure elevation. Invest Ophthalmol Vis Sci2005; 46(1): 202–213

[31] Fortune B, Bui BV, Morrison JC, Johnson EC, Dong J, Cepurna WO, Jia L, Barber S, Cioffi GA. Selective ganglion cell functional loss in rats with experimental glaucoma. Invest Ophthalmol Vis Sci2004; 45(6): 1854–1862

[32] Nguyen CT, Bui BV, Sinclair AJ, Vingrys AJ. Dietary omega 3 fatty acids decrease intraocular pressure with age by increasing aqueous outflow. Invest Ophthalmol Vis Sci2007; 48(2): 756–762

[33] Wu Q, Zhang M, Song BW, Lu B, Hu P. Expression of ciliary neurotrophic factor after induction of ocular hypertension in the retina of rats. Chin Med J (Engl)2007; 120(20): 1825–1829

[34] Martin KR, Quigley HA, Zack DJ, Levkovitch-Verbin H, Kielczewski J, Valenta D, Baumrind L, Pease ME, Klein RL, Hauswirth WW. Gene therapy with brain-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model. Invest Ophthalmol Vis Sci2003; 44(10): 4357–4365

[35] WoldeMussie E, Ruiz G, Wijono M, Wheeler LA. Neuroprotection of retinal ganglion cells by brimonidine in rats with laser-induced chronic ocular hypertension. Invest Ophthalmol Vis Sci2001; 42(12): 2849–2855

[36] Hains BC, Waxman SG. Neuroprotection by sodium channel blockade with phenytoin in an experimental model of glaucoma. Invest Ophthalmol Vis Sci2005; 46(11): 4164–4169

[37] Robertson JV, Golesic E, Gauldie J, West-Mays JA. Ocular gene transfer of active TGF-beta induces changes in anterior segment morphology and elevated IOP in rats. Invest Ophthalmol Vis Sci2010; 51(1): 308–318

[38] Díaz F, Villena A, Vidal L, Moreno M, García-Campos J, Pérez de Vargas I. Experimental model of ocular hypertension in the rat: study of the optic nerve capillaries and action of hypotensive drugs. Invest Ophthalmol Vis Sci2010; 51(2): 946–951

[39] Wang WH, Millar JC, Pang IH, Wax MB, Clark AF. Noninvasive measurement of rodent intraocular pressure with a rebound tonometer. Invest Ophthalmol Vis Sci2005; 46(12): 4617–4621

[40] Ko ML, Hu DN, Ritch R, Sharma SC. The combined effect of brain-derived neurotrophic factor and a free radical scavenger in experimental glaucoma. Invest Ophthalmol Vis Sci2000; 41(10): 2967–2971

[41] Roh M, Zhang Y, Murakami Y, Thanos A, Lee SC, Vavvas DG, Benowitz LI, Miller JW. Etanercept, a widely used inhibitor of tumor necrosis factor-α (TNF-α), prevents retinal ganglion cell loss in a rat model of glaucoma. PLoS ONE2012; 7(7): e40065

[42] Husain S, Abdul Y, Crosson CE. Preservation of retina ganglion cell function by morphine in a chronic ocular-hypertensive rat model. Invest Ophthalmol Vis Sci2012; 53(7): 4289–4298

[43] Park HY, Kim JH, Park CK. Activation of autophagy induces retinal ganglion cell death in a chronic hypertensive glaucoma model. Cell Death Dis2012; 3(4): e290

[44] Schallenberg M, Prokosch V, Thanos S. Regulation of retinal proteome by topical antiglaucomatous eye drops in an inherited glaucoma rat model. PLoS ONE2012; 7(7): e33593

[45] Cordeiro MF, Guo L, Luong V, Harding G, Wang W, Jones HE, Moss SE, Sillito AM, Fitzke FW. Real-time imaging of single nerve cell apoptosis in retinal neurodegeneration. Proc Natl Acad Sci USA2004; 101(36): 13352–13356

[46] Guo LX, Zhang M, Teo EC. Influences of denucleation on contact force of facet joints under whole body vibration. Ergonomics2007; 50(7): 967–978

[47] Hackett RB. MeDonald TO. Ophthalmic Toxicology. Washington, D.C.: Taylor & Francis1996

[48] Zhong L, Bradley J, Schubert W, Ahmed E, Adamis AP, Shima DT, Robinson GS, Ng YS. Erythropoietin promotes survival of retinal ganglion cells in DBA/2J glaucoma mice. Invest Ophthalmol Vis Sci2007; 48(3): 1212–1218

[49] Nishijima K, Ng YS, Zhong L, Bradley J, Schubert W, Jo N, Akita J, Samuelsson SJ, Robinson GS, Adamis AP, Shima DT. Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury. Am J Pathol2007; 171(1): 53–67

[50] Morrison JC, Jia L, Cepurna W, Guo Y, Johnson E. Reliability and sensitivity of the TonoLab rebound tonometer in awake Brown Norway rats. Invest Ophthalmol Vis Sci2009; 50(6): 2802–2808

[51] Minton AZ, Phatak NR, Stankowska DL, He S, Ma HY, Mueller BH, Jiang M, Luedtke R, Yang S, Brownlee C, Krishnamoorthy RR. Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma. PLoS ONE2012; 7(8): e43199

[52] Guo L, Normando EM, Nizari S, Lara D, Cordeiro MF. Tracking longitudinal retinal changes in experimental ocular hypertension using the cSLO and spectral domain-OCT. Invest Ophthalmol Vis Sci2010; 51(12): 6504–6513

[53] Linden R. Dendritic competition in the developing retina: ganglion cell density gradients and laterally displaced dendrites. Vis Neurosci1993; 10(2): 313–324

[54] Fileta JB, Huang W, Kwon GP, Filippopoulos T, Ben Y, Dobberfuhl A, Grosskreutz CL. Efficient estimation of retinal ganglion cell number: a stereological approach. J Neurosci Methods2008; 170(1): 1–8

[55] Chang ZY, Lu DW, Yeh MK, Chiang CH. A novel high-content flow cytometric method for assessing the viability and damage of rat retinal ganglion cells. PLoS ONE2012; 7(3): e33983

[56] Nadal-Nicolás FM, Jiménez-López M, Salinas-Navarro M, Sobrado-Calvo P, Alburquerque-Béjar JJ, Vidal-Sanz M, Agudo-Barriuso M. Whole number, distribution and co-expression of brn3 transcription factors in retinal ganglion cells of adult albino and pigmented rats. PLoS ONE2012; 7(11): e49830

[57] Glovinsky Y, Quigley HA, Dunkelberger GR. Retinal ganglion cell loss is size dependent in experimental glaucoma. Invest Ophthalmol Vis Sci1991; 32(3): 484–491

[58] Pease ME, Zack DJ, Berlinicke C, Bloom K, Cone F, Wang Y, Klein RL, Hauswirth WW, Quigley HA. Effect of CNTF on retinal ganglion cell survival in experimental glaucoma. Invest Ophthalmol Vis Sci2009; 50(5): 2194–2200

[59] Wang X, Li Y, He Y, Liang HS, Liu EZ. A novel animal model of partial optic nerve transection established using an optic nerve quantitative amputator. PLoS ONE2012; 7(9): e44360

[60] Coassin M, Lambiase A, Sposato V, Micera A, Bonini S, Aloe L. Retinal p75 and bax overexpression is associated with retinal ganglion cells apoptosis in a rat model of glaucoma. Graefes Arch Clin Exp Ophthalmol2008; 246(12): 1743–1749

[61] Guo L, Moss SE, Alexander RA, Ali RR, Fitzke FW, Cordeiro MF. Retinal ganglion cell apoptosis in glaucoma is related to intraocular pressure and IOP-induced effects on extracellular matrix. Invest Ophthalmol Vis Sci2005; 46(1): 175–182

[62] Ishii Y, Kwong JM, Caprioli J. Retinal ganglion cell protection with geranylgeranylacetone, a heat shock protein inducer, in a rat glaucoma model. Invest Ophthalmol Vis Sci2003; 44(5): 1982–1992

RIGHTS & PERMISSIONS

2014 Higher Education Press and Springer-Verlag Berlin Heidelberg