Mercedes Moreno

Reaction of Müller cells in an experimental rat model of increased intraocular pressure following timolol, latanoprost and brimonidine

The aim of this study was to evaluate the reaction of Müller cells in an experimental rat model of intraocular pressure (IOP) and their response to treatment with ocular hypotensive drugs. Episcleral vein cauterization in unilateral eyes of Wistar rats was performed to produce elevated IOP. The animals were divided into five groups: control, experimental, and experimental treated with timolol, latanoprost or brimonidine. Histological sections of retina were studied by immunochemistry with antibodies to glial fibrillary acidic protein (GFAP), and the percentage of labeled area was measured to evaluate the degree of reactive gliosis. In the experimental group, the Müller cells showed hypertrophy and a significant increase in GFAP (4.39+/-0.32%) in relation to retinas of the control group (2.05+/-0.14%). Gliosis was detected in all three treated groups, with a varying increase in GFAP intensity. The timolol-treated group showed the most intense and persistent glial reactivity after 3 months of treatment (13.89+/-0.63%). Treatment with brimonidine, however, resulted in a decrease in the level of GFAP immunoreactivity (8.37+/-0.4%). The group treated with latanoprost showed the lowest glial reactivity (4.8+/-0.36%). Given that all three drugs are effective hypotensive agents, their neuroprotective effect could be related with other factors, such as gliosis, which, over long periods may have noxious effects on the neurons. Thus, hypotensives like brimonidine, and specially latanoprost, may afford greater neuroprotection to the ganglion cells by attenuating the retinal glial reaction.

Nitric oxide synthase in retina and optic nerve head of rat with increased intraocular pressure and effect of timolol

We investigated the expression of nitric oxide synthase (NOS) isoforms -1, -2 and -3 in the retina and optic nerve head (ONH) in an experimental rat model of elevated intraocular pressure (IOP) before and after treatment with timolol, to assess whether its neuroprotective action is associated with the activity of these enzymes. Episcleral vein cauterization in unilateral eyes of Wistar rats was performed to produce elevated IOP. Histological sections of retina and ONH from animals with normal IOP, with elevated IOP, and elevated IOP treated with timolol, were studied by immunohistochemistry with antibodies to NOS-1, NOS-2, and NOS-3. In the control rats, NOS-1 was localized to photoreceptor inner segments, amacrine cells and bipolar cells in the retina, and in astrocytes, pericytes and vascular nitrergic terminals in the ONH. NOS-3 immunostaining localized to the endothelial cells. The rats with elevated IOP showed increased expression of NOS-1 in the plexiform layers of the retina and reactive astrocytes in the ONH. These cells also showed NOS-2 positivity. The rats treated with timolol showed reduced expression of NOS-1 in the retina and ONH. NOS-2 was only detected in a few groups of astrocytes in the ONH. NOS-3 was unchanged in both elevated IOP and timolol-treated groups. These results show that excessive levels of NO synthesized by the NOS-1 and -2 isoforms, considered neurotoxic, might contribute to the progressive lesions of retinal ganglion cell axons. Their reduction after treatment suggests a possible neuroprotective effect of timolol in neurons exposed to excessive amounts of NO.

Impression cytology of the conjunctival epithelium after antiglaucomatous treatment with latanoprost.

Purpose To study nongoblet and goblet epithelial conjunctival cells after several treatment periods with latanoprost, a prostaglandin analogue. Methods Twelve patients (20 eyes) were studied before the onset of treatment and after 1, 3, and 6 months of latanoprost use. Impression cytology was carried out to analyze cellular density and morphologic parameters such as minimum and maximum diameter and area. Results Nongoblet epithelium cell density did not change over the treatment period. The density of goblet cells increased after 1 month of use, but returned to initial cell density after longer treatment periods. Nongoblet epithelial cells underwent a significant reduction in size after 1, 3, and 6 months of treatment. In addition, the minimum/maximum diameter ratio suggested that after 1 month there were some changes in shape (a slight elongation) when compared to cells of untreated patients. Nevertheless, after longer treatment periods, the cells regained their original shape. No changes in size were observed in goblet cells, except for a slight decrease in maximum diameter after 6 months of treatment, which suggests that the cells became more rounded. Conclusions The density of nongoblet epithelial cells does not change after different treatment periods with latanoprost. However, their size decreases and after short treatment periods their shape also undergoes changes. The density of goblet cells increases after 1 month of treatment, but decreases again after longer periods. Their size does not undergo any modification, although there is a variation in shape after 6 months of treatment.

Study of the effects of ocular hypotensive drugs on number of neurons in the retinal ganglion layer in a rat experimental glaucoma

Purpose We investigated the effects of antiglaucomatous drugs on neurons in the retinal ganglion layer (RGL) in an experimental model of elevated intraocular pressure (IOP). Methods Three episcleral veins of rats with normal IOP were cauterized. Three months later, we examined the effects on the number of neurons in the RGL as well as in rats submitted to treatment with timolol, latanoprost, or brimonidine. The IOP was measured using a calibrated Tono-Pen XL tonometer before and immediately after cauterization and every 2 weeks for the following 3 months as well as immediately before perfusion. Results The IOP was 14.85±0.65 mmHg in the control group, whereas it was 1.25-fold higher (33.5±1.06 mmHg) in the experimental group. After treatment, the IOP returned to baseline levels. The mean number of neurons per mm2 in the RGL was 33% lower in the experimental group (283±10 cells/mm2) compared with the control group (423±11 cells/mm2). In the groups treated with timolol, latanoprost, or brimonidine, the neuronal loss was less (331±10, 360±15, and 333±3 cells/mm2, respectively), although values did not return to baseline levels. Conclusions This experimental model provokes an immediate, constant, and prolonged increase in IOP and the application of hypotensive agents affords a certain degree of protection to neurons in the RGL.

Quantitative age-related changes in NADPH-diaphorase-positive neurons in the ventral lateral geniculate nucleus

Age-related changes in nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) were examined in the rat ventral lateral geniculate nucleus (vLGN) using histochemical methods. Eighteen rats aged 3, 24, and 26 months were studied using quantitative methods to investigate the number of neurons per mm(2), the cross-sectional area, and the orientation of dendritic processes of NADPH-d-positive neurons. We have described three types of neurons: types A and B are both located in the lateral and medial vLGN (vLGN-l and vLGN-m, respectively), and type C neurons over the optic tract. The number of NADPH-d-positive neurons was significantly reduced in the old rats (-39%) when compared with controls (3-month-old rats). The quantitative analysis of cell areas revealed a significant decrease of somatic size in type B neurons, both in the lateral and medial vLGN, and in C neurons; however, type A neurons did not show significant changes. By quantifying the orientation of dendritic processes, we observed a predominant dorsolateral orientation in type A and B neurons. During aging, there are no changes in the dendritic orientation of neurons located in the vLGN-m; however, vLGN-l neurons show an increase in dendritic processes with dorsoventral orientation. In type C neurons, our results show that 87.4% of dendritic processes are lateromedially oriented at 26 months old. Therefore, the types A and B neurons behave differently during senescence. Type A neurons do not change in size, but those located in the vLGN-l modify the orientation of their dendritic processes; however, type B neurons, reduce their size and those located in the vLGN-l also modify their dendritic process orientation. Finally, the type C neurons modify their size and dendritic process.

Experimental model of ocular hypertension in the rat: study of the optic nerve capillaries and action of hypotensive drugs.

PURPOSE To investigate quantitatively the effect of elevated intraocular pressure (IOP) on the microvasculature of the optic nerve with and without topical treatment with two hypotensive drugs, timolol and latanoprost. METHODS Three groups of rats underwent cauterization of three episcleral veins to produce elevated IOP in the right eye. Two of these groups were treated with timolol or latanoprost for 3 months. Eyeballs were incubated with anti-GLUT-1 polyclonal antibody. GLUT-1-positive capillaries of the optic nerve head (ONH) and optic nerve exit (ON) were examined and analyzed for their number per square millimeter, volume fraction, length per unit volume, surface area per unit volume, and mean diameter. RESULTS An increase in IOP resulted in a significant decrease in microvessel density in the laminar region (LR) and postlaminar region (PR) and ON compared with the control group. The other parameters fell significantly in all regions of the optic nerve. Topical treatment with timolol or latanoprost did not modify the density of the capillaries, although the other parameters increased significantly compared with the untreated experimental group. Additionally, the mean diameter of the capillaries in the LR and the PR recovered after treatment. CONCLUSIONS The results indicate that the capillaries of the LR and the PR of the ONH are the most susceptible to IOP elevation. The authors suggest that timolol and latanoprost have a certain vascular action by increasing the available blood volume, surface area per unit volume, length per unit volume, and diameter of the capillaries of the ONH in these two regions.

Cambios morfológicos y morfométricos en los capilares del nervio óptico de rata en un modelo experimental de glaucoma

AIM To study the morphological and morphometric changes produced in the capillaries of the optic nerve (ON) head and initial portion after the experimental increase in intraocular pressure (IOP). MATERIAL AND METHODS Wistar rats underwent cauterization of three episcleral veins, which produced an immediate increase in the IOP, and was maintained for 3 months. Sagittal sections of the eyeball were studied with immunohistochemical techniques, using a primary antibody to GLUT-1. The GLUT-1 positive capillaries were counted, and measurements were made of the area, perimeter and mean diameter. RESULTS Microscopic examination of sections of the ON of control rats revealed a lower density and larger caliber of capillaries in the prelaminar region as compared with the other regions of the ON (P<.05). Comparison between the control and the experimental groups showed a reduction in capillary density (except in the prelaminar region) and a smaller size in all the areas of the ON studied, but less evident in the initial portion (P<.05). CONCLUSIONS The increase in IOP was associated with significant qualitative and quantitative changes in the capillaries of the laminar and poslaminar regions of the ON head. These changes appear to return towards parameters compatible with normality in the initial portion of the ON, an area where the vascular collapse was less evident. These findings might explain the significant reduction in ocular blood flow seen in patients with primary open-angle glaucoma.