br Methods br Results br Discussion The measurement of intra

Methods
Results
Discussion The measurement of intraocular pressure was made with a commercially available rebound tonometer, the Tonovet®. In our studies, Tonovet® underestimated the intraocular pressure in both animal models, being this effect more pronounced in the rat. To study the effect of the ghrelin-GHSR-1a system in the modulation of intraocular pressure, we modified a model described in the rabbit [15]. This model has been used previously to study the intraocular hypotensive effect of different compounds [4], [7], [12]. By increasing the concentration of the saline solution and decreasing the volume injected, we were able to extend the ocular PP242 receptor plateau from 90 to 150min. After this period, the pressure started to decrease and remained above normal levels until 240min, reaching baseline values around 300min. The same model was applied to the rat. When compared to the rabbit, the rise in intraocular pressure in the rat was more pronounced and evident earlier (5min post-injection). However, ocular hypertension remained constant until minute 30 post-injection, reaching baseline levels after 120min. The mechanism that explains the elevation of intraocular pressure in this model is the osmolarity gradient created by the NaCl concentration [15]. Ghrelin's aqueous humor levels are reduced in patients with different types of glaucoma, while the plasmatic levels remain normal [8], [16]. Moreover, ghrelin has been shown to relax both the iris’ sphincter and dilator muscles and ghrelin's mRNA has been identified in the eye [17], [18]. In the rat, both ghrelin and des-acyl-ghrelin significantly reduced intra-ocular pressure, but in the rabbit, only ghrelin showed this effect. Ghrelin and des-acyl ghrelin are similar peptides except that des-acyl ghrelin's is unable to bind to GHSR-1a [5]. Moreover, studies have attributed similar biological effects to both des-acyl ghrelin and ghrelin in some organs [2], [13]. These similar effects are independent of the GHSR-1a. Our results suggest that ghrelin's hypotensive effect in the rabbit may be mediated by the GHSR-1a, while in the rat other receptors may be involved. There are considerable differences between the drainage pathways of aqueous humor of the studied animals. In the rabbit, trabecular outflow is the principal drainage route accounting for 92–97% of the total drainage [1]. In the rat, both the trabecular and uveoscleral routes contribute significantly to aqueous humor drainage. Therefore, the differences encountered in our studies regarding ghrelin and des-acyl ghrelin's effects in the different animal models could be related to the relative role of the uveoscleral and trabecular drainage in these species. Nevertheless, further studies are needed to confirm this hypothesis. In the eye, ghrelin is able to relax the iris sphincter muscle. This effect depends on prostaglandins synthesis but is independent from nitric oxide release [18]. Because ghrelin showed a hypotensive effect, we investigated the influence of nitric oxide and prostaglandins on this phenomenon. In the rabbit, the inhibition of NOS or COX, with l-NAME or KETOROLAC respectively, completely blunted ghrelin's effect. Our results suggest the involvement of these two mediators in ghrelin's hypotensive effect in the rabbit ocular hypertension model. Nitric oxide has been implicated in the reduction of intraocular pressure [10], [11]. Nitric oxide promotes an intracellular increase in cGMP that induces the relaxation of the ciliary muscle and increases the uveoscleral pathway promoting a decrease in intraocular pressure. Nitric oxide can also relax the intrinsic contractile elements of the trabecular meshwork, further decreasing intraocular pressure by increasing the trabecular outflow [21], [25]. Prostaglandins are produced by cells related with the aqueous humor outflow pathway and have been suggested as having a role in the regulation of aqueous humor drainage [23], [24]. PGF analogs are currently the most effective therapeutic option in the treatment of glaucoma [22]. In our studies, KETOROLAC-induced COX inhibition abolished ghrelin's hypotensive effect. The release of prostaglandins promoted by ghrelin is known and has already been verified in the ocular tissue [18]. The mechanism responsible for prostaglandin-induced reduction in intraocular pressure is still unclear. However, the increase in uveoscleral outflow is currently considered the main mechanism of action for prostaglandins analogs.