**2. A model of experimental glaucoma in rat**

Several experimental animal models exist to investigate the ocular pathologies. In our laboratory we have developed a rat model of hypertension that mimics and reproduces the situation found in human glaucoma. This animal model will be briefly reviewed in the following sections [2].

and/or production of neurotoxic and proapoptotic compounds such as nitric oxide synthase (NOS). The nitric oxide thus produced is a reactive free radical present in cells as a response to increased intracellular concentrations of Ca2+. It is known that NOS increases in cerebral ischemia and the over-expression of this enzyme causes relevant damage: the overall result is a detrimental action on the cell membrane. Recent studies demonstrated that an excess of NO is toxic and this compound increases as a consequence of ocular hypertension. In glaucoma, the involvement of inducible NOS (iNOS) has also been suggested. The oxidative stress and the increase of IOP also cause up-regulation of ubiquitin (Ub) and stimulation of the Ubproteasome pathway: this possibly derives from the activation of the apoptotic program. In any case it should be pointed out that we also demonstrated that a well-known natural substance, carnitine, endowed of antioxidant properties and improvement of muscle perform‐ ance, can ameliorate the glaucomatous pathology in the rat model system developed in our

Experimental Glaucoma After Oxidative Stress and Modulation of the Consequent Apoptotic Events in a Rat Model

http://dx.doi.org/10.5772/54628

21

In conclusion, literature data imply that the RGCs are one of the main targets of the oxidative stress in the neural tissue. As shown in our studies, the injection of methylcellulose into the anterior chamber of the eye activates diverse signals of stress at the level of RGCs. Mainly, the up-regulation of the GFAP and DNA damage become evident. Methylcellulose hinders the efflux of fluids from the canals of Schlemm thus increasing the IOP. The consequent oxidative stress is shown by the overexpression of iNOS, which is an enzyme primarily involved in the mitochondrial lipid peroxidation, with consequent damage of the cell membrane. This is validated by the accumulation of intracellular malonal-dihaldehyde: a hallmark of lipoperox‐ idation. The ubiquitin-mediated proteasome pathway is also activated and this is directly related to the execution of the apoptotic death. The antiapoptotic role of carnitine plays a key role in the stabilization and function of the cell membrane, the mitochondrial one in particular. The contemporary treatment with methylcellulose and carnitine reduces the level of typical markers of cell sufferance and apoptotis, this enhances the mitochondrial performance, improves the overall homeostatic response to the hypertensive insult, and limits the apoptotic

Department of Biology and Biotechnology "Charles Darwin"- Sapienza University of Rome,

laboratory [2, 16].

**3. Conclusions**

phenomena.

Rome, Italy

**Author details**

Nicola Calandrella, Simona Giorgini and Gianfranco Risuleo\*

\*Address all correspondence to: gianfranco.risuleo@uniroma1.it

#### **2.1. Induction of the intra-ocular hypertension**

To induce ocular hypertension *in vivo* [50] causing a condition of acute glaucoma in rat we injected in the anterior chamber of the right eye methylcellulose (MTC) suspended in physio‐ logical solution (the contra-lateral eye served as control). The IOP was monitored by tonom‐ etry. The hypertension induced by MTC was also performed in the presence of the antioxidant trolox [50]. The degree of animal sufferance was evaluated by the behavioral Irwin test and by the recovery of bodyweight. Ocular inflammation was assessed by the Drize test adapted to the rat, both approaches were described in detail in [49]. Intra-ocular pressure was monitored on 20 different animals that were finally sacrificed by hemorrhagic shock (decapitation). The eyes were removed and the cornea eliminated at limbus level; vitreous humor, and crystalline lens were discarded. The remaining samples of retina and optic nerve were fixed in paraformaldehyde, quickly washed in PBS finally included in freezing resin and cryostat-cut. Chromatin morphology and structure as well as DNA fragmentation was evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction and validat‐ ed by the formation of the apoptotic ladder after agarose gel electrophoresis. The apoptotic ladder is generated by nucleolytic inter-nucleosomal DNA cleavage since during the late stages of apoptosis the enzyme DNase I is activated. This causes the formation of multiple nucleo‐ somal DNA fragments which can be easily visualized, by gel electrophoresis, by fluorescence after ethidium bromide staining.

#### **2.2. Lipoperoxidative damage of the membrane and apoptosis after induction of cell stress**

The data obtained in our laboratory support the idea that ocular hypertension causes apoptotic death of retinal ganglion cells and over-expression of molecular markers typical of oxidative cell stress response and apoptosis. Glial cells may have a neuroprotective role in a pathological situation; in any case they may contribute protection from neuron damage. In particular, during progression of glaucoma, astrocytes are involved in the re-modeling of the *lamina cribrosa* and they could act as initiators of the pathology. With respect to this see the role of PHB and MMP mentioned in preceding section. Studies on experimental models of ocular hypertension and human glaucoma evidenced an astrocyte hypertrophy and a loss of organ‐ ization both at retina and optic nerve level. The up-regulation of the GFAP was also observed, as mentioned in a previous section of this work, in cultured astrocytes grown at high hydro‐ static pressure. The GFAP is considered a very important stress marker in diverse retinal pathologies. Activation of the glial cells may also have noxious consequences on neurons, as they may cause mechanical damages and alterations of the micro-enviroment also, they may fail to provide the structural/nutritionl support to the neural cells. This could trigger the release and/or production of neurotoxic and proapoptotic compounds such as nitric oxide synthase (NOS). The nitric oxide thus produced is a reactive free radical present in cells as a response to increased intracellular concentrations of Ca2+. It is known that NOS increases in cerebral ischemia and the over-expression of this enzyme causes relevant damage: the overall result is a detrimental action on the cell membrane. Recent studies demonstrated that an excess of NO is toxic and this compound increases as a consequence of ocular hypertension. In glaucoma, the involvement of inducible NOS (iNOS) has also been suggested. The oxidative stress and the increase of IOP also cause up-regulation of ubiquitin (Ub) and stimulation of the Ubproteasome pathway: this possibly derives from the activation of the apoptotic program. In any case it should be pointed out that we also demonstrated that a well-known natural substance, carnitine, endowed of antioxidant properties and improvement of muscle perform‐ ance, can ameliorate the glaucomatous pathology in the rat model system developed in our laboratory [2, 16].
