Three experimental glaucoma models in rats: Comparison of the effects of intraocular pressure elevation on retinal ganglion cell size and death

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Abstract

Glaucoma is a chronic and progressive optic nerve neuropathy involving the death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is considered to be the major risk factor associated with the development of this neuropathy. The objective of the present study was to compare the effects on RGC survival of three different experimental methods to induce chronic elevation of IOP in rats. These methods were: (i) injections of latex microspheres into the eye anterior chamber; (ii) injections into the anterior chamber of a mixture of microspheres plus hydroxypropylmethylcellulose (HPM) and (iii) cauterization of three episcleral veins. The IOP of right (control) and left (glaucomatous) eyes was measured with an applanation tonometer in awake animals. Thirteen to 30 weeks later, RGCs were retrogradely labeled with 3% fluorogold. Subsequently, we analyzed the density of RGCs, as well as the major axis length and area of RGC soma resulting from the application of each method. A significant increase in IOP was found following application of each of the three methods. Cell death was evident in the glaucomatous eyes as compared to controls. However, no statistical differences were found between the extent of cell death associated with each of the three methods. IOP increase also induced a significant increase in the size of the soma of the remaining RGCs. In conclusion, the three methods used to increase IOP induce a similar degree of RGC death. Moreover, the extent of cell death was similar when the retinas were maintained under conditions of elevated IOP for 24 weeks in comparison to 13 weeks.

Introduction

Elevated intraocular pressure (IOP) is one of the most important risk factors for developing glaucoma. In order to understand in detail the mechanisms which take place in glaucoma and lead to retinal damage, fast, inexpensive and reproducible animal models need to be developed (Chew, 1996).

The currently available models of experimental glaucoma involve the induction of a chronic increase in IOP. This increase can be achieved by reducing aqueous humor outflow through the eye. Thus, aqueous humor drainage can be interrupted by cauterizing two or three episcleral veins (Shareef et al., 1995) or by injecting hypertonic saline into the episcleral veins in rats (Morrison et al., 1997). Moreover, laser energy has been employed as a tool to perform burns directed at the trabecular meshwork (TM) (Ueda et al., 1998) and at both TM and episcleral veins (Levkovitch-Verbin et al., 2002). Other methods are based on the blockage of aqueous humor drainage at the level of the trabecular meshwork, avoiding manipulation of the eye vascular system. Thus, injection of different substances such as ghost red blood cells (Quigley and Addicks, 1980) or latex microspheres into the eye anterior chamber (Weber and Zelenak, 2001) leads to TM channel blockade. Finally, injection of viscoelastic agents into the eye anterior chamber has been reported to induce IOP spikes by a mechanical obstruction of the trabecular meshwork in rabbits (Benson et al., 1983, Manni et al., 1996, Törngren et al., 2000).

It has been widely demonstrated that retinal ganglion cells (RGCs) are selectively affected during glaucoma. However, few works have analyzed the pattern of RGC death by means of quantification of specifically labeled RGCs (Ko et al., 2001, Morgan et al., 2000, Naskar et al., 2002, Shareef et al., 1995). In this sense, during the first 10 weeks after IOP increase in rats, the rate of RGC death has been estimated to be uniform and almost linear at about 3–4% per week, but the degree of cell death seems to depend on the retinal region analyzed (Laquis et al., 1998).

Apart from RGC death, changes in RGC soma size have been reported in experimental glaucoma. Thus, an overall hypertrophy of all RGC types has been reported in rats after IOP elevation by episcleral vein cauterization (Ahmed et al., 2001). This increase in RGC soma size is also observed after optic nerve crush and axotomy (Moore and Thanos, 1996, Rousseau and Sabel, 2001). The increase in RGC soma size is likely to be in part a response to the space made available by the culling of RGC (Ahmed et al., 2001).

Glaucoma is a chronic and progressive disease and consequently, it is vital to develop experimental models which analyze RGC death following different periods of sustained elevation of IOP. The effect of experimental IOP elevation on RGC death has been analyzed during periods no longer than 12–16 weeks, due to the transitory effect of surgery. The main aim of the present study was to compare the effect of three different glaucoma models on RGC death and soma size. Moreover, we have also compared the effect of different periods of elevation of IOP in the same experimental glaucoma model to determine if a correlation exists between the duration of IOP elevation and the extent of RGC death.

Section snippets

Animals

Twenty-one female Sprague–Dawley rats weighing 250–300 g were used in the present work. Animals were housed in a standard animal room with food and water provided ad libitum, a constant temperature of 21 °C and a 12-h light/dark cycle. All animal experimentation adhered to the ARVO Statement for the use of Animals in Ophthalmic and Vision Research.

Experiments were designed and carried out in such a way as to minimize animal suffering in accordance with European Communities Council Directive of 24

IOP increase

In control animals, the mean IOP was similar in both the right (23.06 ± 0.8 mmHg) and left eye (23.59 ± 0.7 mmHg) throughout all the experimental period (p = 0.61) (Fig. 1A).

Injections of latex microspheres into the eye anterior chamber led to a significant increase in IOP. Thus, the mean IOP in injected eyes was 28.1 ± 0.7 mmHg during the experimental period and this increase reached maximum values during the 27th week when the average IOP during this period was 1.69 times that of the control (37.6 ± 2.6 

Discussion

IOP measurements were made using a tonopen XL applanation tonometer, which permits non-invasive determinations of IOP (Moore et al., 1993). Following the methodology used by others (Benozzi et al., 2002, Cohan and Bohr, 2001, Jia et al., 2000a), IOP measurements were performed using only topical anesthesia, thus avoiding any hypotensive (Jia et al., 2000b) or even neuroprotective effects (Fujikawa, 1995, Ozden and Isenmann, 2004) associated with general anesthesia. IOP determinations were

Acknowledgements

Grants from The Glaucoma Foundation (TGF 2004), ONCE (III Convocatoria I+D), First Price FUNDALUCE 2005, Spanish Ministry of Science and Technology (BFI 2003-07177) and the University of the Basque Country (E15350/2003).

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