the entire retina have been developed to address this problem (Danias et al., 2002) but are not widely employed.

Experimental methods of producing elevated IOP

The different rat models for simulating optic nerve damage in POAG are defined based on the method used to increase eye pressure. To date, three main approaches have been developed. These include scarring the anterior chamber angle by either injecting hypertonic saline into the aqueous humor outflow pathways or using a laser. The third method, which uses cautery to close off venous outflow from the eye, may not work by the same mechanism, and the reasons for this will be presented.

Hypertonic saline injection of aqueous humor outflow pathways

Aqueous humor outflow in the rat eye has many similarities to that of the primate (Morrison et al., 1995b). These include a trabecular meshwork and a prominent Schlemm’s canal, connected to the episcleral vasculature by trans-scleral collector channels. In the rat, episcleral vessels consist a plexus of aqueous veins that encircles the limbus and drains posteriorly via radial veins into the orbit.

In the hypertonic saline method, the investigator takes advantage of this anatomy by injecting hyperosmolar saline into one of the episcleral radial veins (Morrison et al., 1997). By placing a plastic ring around the equator of the eye, the injection can be directed into Schlemm’s canal and driven across the trabecular meshwork. The hypertonicity of the saline damages cell membranes and produces scarring of the meshwork and the anterior chamber angle, the extent of which parallels the rise in pressure.

Elevation of IOP generally occurs 7–10 days following the injection, depending on the relative time course of the scarring process and resumption of normal aqueous humor production (Morrison et al., 1997). A second injection can be performed if pressure does not rise after 2 weeks, although some investigators have routinely performed a

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second injection in the same eye (Chauhan et al., 2002; Hanninen et al., 2002). Interestingly, we have found that elderly animals are unusually sensitive to this injection, possibly due to a narrower angle owing to enlargement of the lens, and the resulting IOP is often too high (Morrison et al., 2007). Modifying the ring to limit saline to specific portions of the meshwork reduces the extent of IOP elevation in these animals.

The duration of pressure elevation can last several months, although most experiments are limited to a few weeks, so that tissues can be harvested for specific histologic or cell biology studies (Morrison et al., 1997; Johnson et al., 2000; Chauhan et al., 2002; Hanninen et al., 2002; Mckinnon et al., 2002). Injections of greater than 2.0 M saline will produce higher pressures, and concentrations below this produce less severe elevations.

This method generally produces a range of pressures, as high as twofold or more above normal. With this, it is possible to detect responses that might be associated with early nerve injury (Johnson et al., 2007). Other changes will be more linearly related to the extent of injury. In general, optic nerve damage and cellular responses can be related to mean IOP, peak IOP elevation, and cumulative pressure exposure (days of pressure elevation times the mmHg above normal) (Chauhan et al., 2002; Mckinnon et al., 2002; Johnson et al., 2007).

Laser treatment of limbal tissues

This method uses external laser to the limbus, with either an argon or diode laser. While some investigators initially inject India ink into the anterior chamber to improve laser uptake and angle damage (Ueda et al., 1998), others have accomplished a similar result without injecting foreign materials into the anterior chamber (Woldemussie et al., 2001; Levkovitch-Verbin et al., 2002b).

It is possible that this treatment produces elevated IOP by coagulating the limbal vasculature and indirectly obstructing aqueous outflow. However, one study has clearly shown that angle treatment is necessary to achieve chronic IOP

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elevation (Levkovitch-Verbin et al., 2002b). In this manner, the mechanism of pressure rise with this treatment is very similar to hypertonic saline injection. Most likely, pressure increases following laser treatment directed at the limbal vasculature are actually due to collateral damage to the anterior chamber angle (Woldemussie et al., 2001).

This technique is generally used in nonpigmented animals since energy uptake in pigmented eyes can produce a dramatic inflammatory response that can confound interpretation of cellular responses in the retina and the ONH. Because it is difficult to measure IOP in awake albino rats, this restriction will also affect accuracy of pressure monitoring and correlations between pressure and damage.

In most reports, this technique results in a rapid IOP increase, followed by a gradual reduction to normal in a few, often 3, weeks (Levkovitch-Verbin et al., 2002b). In general, additional treatments are used as needed to achieve sustained pressure elevations (Martin et al., 2003). Some groups routinely perform a second treatment during the first 3 weeks (Schori et al., 2001; Bakalash et al., 2002; Ishii et al., 2003; Pease et al., 2006).

Episcleral vein cautery

The third method of creating elevated IOP in rats consists cauterizing large episcleral veins located posterior to the rectus muscle insertions (Shareef et al., 1995; Sawada and Neufeld, 1999). Unlike the limbal laser method, this procedure appears to work in pigmented as well as nonpigmented animals (Neufeld et al., 2002; Grozdanic et al., 2003b; Danias et al., 2006). However, there is little consensus on the effectiveness of this technique. Some groups report that IOP returns to normal after 2–4 weeks (Shareef et al., 1995; Ahmed et al., 2001). Mittag noted that IOP elevations longer than 3 weeks could only be achieved with subconjunctival injections of 5-fluorouracil (Mittag et al., 2000). This suggests that normalization of IOP results from the formation of collateral vessels, and that this is prevented by the antimetabolite. Other investigators have found that pressures will remain elevated longer than this, even up to 7 months, without retreatment (Neufeld et al., 1999, 2002).

The mechanism of pressure rise in this method is also controversial. Some authors feel that the cauterized vessels are simply episcleral veins that drain aqueous humor from the limbal plexus and elevate IOP results from this increased resistance to aqueous outflow (Shareef et al., 1995). Other investigators have determined that these vessels are most likely vortex veins (Grozdanic et al., 2003b; Pang and Clark, 2007). In rats, these veins receive blood from the choroid as well as the anterior uvea and veins at the base of the optic nerve (Morrison et al., 1987, 1999). Given these relationships, cauterization of these veins will instantaneously reduce venous outflow from nearly all parts of the eye and result in ocular congestion and a rise in IOP equivalent to arterial blood pressure (Goldblum and Mittag, 2002).

Several lines of evidence suggest that this model is not equivalent to the other two models. Rats with elevated IOP due to obstruction of aqueous humor outflow following angle closure exhibit a pattern of injury in which the superior region of the optic nerve is damaged first (Morrison et al., 1997). This is also reflected in our injury grading scale in which the focal, grade 2 lesion generally occurs within the superior optic nerve. Within the ONH, early injury is most apparent in the superior portion of the nerve at the level of the sclera (Cepurna et al., 2002) (Fig. 4). Work with the laser model has shown a similar pattern, where the greatest reduction of RGCs occurs in the superior retina (Woldemussie et al., 2001). By contrast, injury following cautery has been described as primarily occurring in peripheral regions of the retina (Sawada and Neufeld, 1999; Ko et al., 2001). Currently, little work on injury within the ONH itself has been reported with this particular model.

A recent high-frequency ultrasound study of the anterior chamber in rats following hypertonic saline treatment has demonstrated a significant deepening of the anterior chamber, suggesting a true aqueous outflow obstruction at the chamber angle (Nissirios et al., 2008) (Fig. 5). On the other hand, vein cautery did not result in any significant alteration in the angle as compared to control eyes. This suggests that elevated IOP in this model does not result from aqueous humor outflow obstruction.