Ignacio Pérez de Vargas

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.

Quantitative age-related changes in dorsal lateral geniculate nucleus relay neurons of the rat

An ultrastructural and quantitative study of the age-related changes occurring in the relay neurons of the dorsal lateral geniculate nucleus (dLGN) was carried out using male Wistar rats aged 3, 18, 24, and 28 months. Morphometric techniques were used to obtain data regarding cellular activity including soma, nuclear, and nucleolar size. Volume fractions for rough endoplasmic reticulum (RER), mitochondria, and lipofuscin, as well as numbers and sizes of mitochondria and dense bodies (DB) was also calculated. Among the few alterations found in the perikaryon, we can highlight the redistribution and fragmentation of RER and an increase and progressive aggregation of lipofuscin. Quantitative data show a significant decrease in the volume of the soma (-42.77%) and the nucleus (-33.66%), and in the volume fraction of the RER (-18.81%) and mitochondria (-10.16%). A significant increase in lipofuscin (+213.29%), and variations in size and number of mitochondria and dense bodies were also found. Some histophysiological considerations about the findings are discussed. The findings lead to the conclusion that a relative degree of morphological stability is exhibited by relay neurons, although the quantitative data show evident intracellular changes, especially from 24 to 28 months. These changes suggest that accompanying physiological alterations may occur, with putative effects on visual function during ageing.

Stereological Age-Related Changes in Neurons of the Rat Dorsal Lateral Geniculate Nucleus

Quantitative methods were used to compare the changes taking place in the volume of the dorsal lateral geniculate nucleus (dLGN) and corresponding neurons of young, adult and old rats. The study was carried out on male albino rats aged 3, 18, 24 and 28 months. In order to estimate the volume of the dLGN, neuronal volume density, numerical density and total number of neurons, we used serial sections stained according to the Klüver‐Barrera technique and stereological methods. We found that dorsal lateral geniculate nucleus volume increases between 3 and 28 months, with a larger increase between 24 and 28 months. Neuronal volume density and numerical density of neurons are greater at 3 months and undergo a significant decrease between 24 and 28 months. Finally, the total number of neurons is shown to be smaller in adult and old animals than in younger ones, even though no significant variations are found between 18 and 28 months. Furthermore, this study confirms the need to analyze the total number of neurons and not just neuronal density if we want to correctly evaluate some of the microscopic changes occurring during senescence. Anat Rec 255:396–400, 1999.

Quantitative morphological changes in neurons from the dorsal lateral geniculate nucleus of young and old rats

We studied the morphological changes occurring in neurons from the dorsal lateral geniculate nucleus (dLGN) during aging by analysing the size and shape of cell bodies and nuclei.

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.

DNA Ploidy Changes in Rhino Mouse Skin Induced by All-Trans Retinoic Acid and Retinol

OBJECTIVE In order to assess the proliferative changes induced by all-trans retinoic acid (RA) and retinol (ROL), we have carried out a study of the DNA content of basal and suprabasal keratinocytes after epicutaneous application on the rhino mouse. STUDY DESIGN Skin sections were analyzed stereologically and cytophotometrically using the Feulgen technique. The diploid DNA value (2C) was obtained from hepatocyte nuclei of control animals. Whereas cells in phase G0-G1 will show a 2C content, cells during phase S and in phase G2-M will show DNA values ranging from 2C to 4C and 4C, respectively. RESULTS Although epidermal thickness (ET) increased significantly in all treated animals, surface density only increased in animals treated with all-trans RA. Quantification of DNA content of basal keratinocytes showed reduction of 2C and 2C-4C populations with a commensurate increase in proportions of cells with 4C and > 4C in the animals treated with 0.025% all-trans RA and ROL. Suprabasal keratinocytes of mice treated with 0.025% all-trans showed a decrease of the 2C population and an increased proportion of cells with 4C. Whereas 0.025% all-trans RA induced an increase of both basal and suprabasal DNA indices, ROL enhanced only the basal DNA index significantly. CONCLUSION Animals treated with 0.025% ROL showed a significant increase in the basal proliferative index (PI) while the suprabasal PI remained constant; treatment with 0.025% all-trans RA produced a significant increase of both basal and suprabasal PIs and parakeratotic hyperkeratosis probably due to incomplete differentiation.

Study of certain clinical variables in patients with psoriasis and their relation to DNA content of keratinocytes

BACKGROUND In a previous study of 26 patients with psoriasis we analyzed cytophotometrically the nuclear DNA content of the germinative compartment of involved and uninvolved skin by means of the Feulgen technique. These subjects were classified into three groups according to their DNA profile. Group 1 had a monomodal diploid profile, group 2 showed a significantly increased 2C-4C population, and group 3 demonstrated high proportions of 4C and hyperdiploid keratinocytes. OBJECTIVE Our purpose was to analyze clinical variables implicated in the development of psoriasis in reference to the three groups. METHODS Nuclear DNA content of each group by quantitative histochemical studies was analyzed and correlated with variables such as chronologic age, sex, age at onset, duration of flare during the study, stress, and the Koebner phenomenon. RESULTS No significant differences in DNA profile were observed in the involved epidermis among the clinical variables. The only differences in the uninvolved skin pertained to the duration of the flare, where a statistically significant difference was observed between groups 1 and 3 in the basal (p < or = 0.0459) and suprabasal keratinocytes (p < or = 0.06), and in the Koebner phenomenon, which was induced in all subjects (100%) in groups 2 and 3 and in only 44% of subjects in group 1. CONCLUSION Uninvolved skin of patients with psoriasis should be included in analysis of the clinical behavior of the disease. Furthermore, the Koebner phenomenon is a good clinical indicator of the DNA profile of these subjects.