Effects of neonatal exposure to cocaine in the development of the neurotransmitters retinal systems: an immunocytochemical and neurochemical study.

The visual system of rodents is affected if exposure to drugs, e.g., cocaine, occurs during prenatal or early postnatal development. This study aims to evaluate, in an experimental model of neonatal exposure to cocaine in the rat, the immunocytochemical expression of tyrosine hydroxylase in the retina and the levels of different neurotransmitters and its metabolites. Male Wistar rats were given 15 mg cocaine hydrochloride/kg body weight/day, subcutaneously, in two daily doses, from the day after birth (PND1) to PND6, 13, and 29. Controls were given 0.9% saline. Groups of rats were perfused at PND7, 14, and 30 with fixative, and the retinas were processed as wholemounts, and immunostained with the antibody anti-TH. Other groups were decapitated, and the retinas were dissected and processed by high performance liquid chromatography with electrochemical detection (HPLC-EC) for determination of dopamine and metabolites (DOPAC and HVA). A reduction in the retinal surface area was detected in the PND30 cocaine group, and a decrease in the density of the small TH-IR cells was found in the PND14 cocaine group although not reaching significant levels. The other quantitative parameters did not differ between the control and cocaine groups. The levels of neurotransmitters did not significantly differ between the groups at any age. These results show a differential vulnerability of the dopaminergic system of rats exposed neonatally to cocaine when compared with the effects found after prenatal exposure to the same drug.

Development of the eye after gestational exposure to cocaine. Vascular disruption in the retina of rats and humans.

Clinical and basic research in the area of drugs of abuse are of utmost importance since they provide the necessary background for health programs in one of the main problems of contemporary society. The available data in this field demonstrate that acute, subacute and/or chronic abuse of illicit drugs, e.g., cocaine, alters the neurochemistry and functioning of the neural circuitries. Although recent works demonstrated that the visual system is lesioned after exposure to cocaine during the active periods of development, no studies have provided detailed information on the effects of these substances on the development of this sensory system. The present paper will report: 1) the vulnerability of the developing visual system to gestational exposure to cocaine; 2) the effects of cocaine in the visual system during the more active periods of development in humans and, as far as possible, the establishment of homologies with animal models where exposure is made in corresponding periods of human gestation, and 3) the characterization of the vascular disruption caused by ischemic/hypoxic mechanisms. The clinical study focused the ophthalmologic evaluation of newborns exposed in utero to illicit drugs. Newborns exposed to cocaine in utero showed marked vascular disruption in the retina: superficial and deep hemorrhages that, although morphologically similar to neonatal retinal hemorrhages, presented a longer reabsorption time when compared with the neonatal hemorrhagic lesions due to birth trauma in the general population. Prolonged eyelid edema was also a prominent finding. The animal study was conducted in Wistar rats exposed prenatally (gestational days 8 to 22) and postnatally (postnatal days 1-6, 1-13 and 1-29) to 60 mg/kg body weight/day and 15 mg/kg body weight/day, respectively, to cocaine hydrochloride administered subcutaneously; control groups included pair-feeding during the same experimental periods. Similar alterations to those observed in the newborns where exposure to cocaine was affirmative, were found: intraretinal hemorrhages allied to signs of chronic ischemia both in the outer retina-photoreceptor rosettes and in the inner retina-epiretinal glial membranes. Taking into consideration that the visual system is one of the more important sensory systems, the identification and characterization of these alterations, the similarity between animal and human findings, and their relation with cocaine per se, can provide a sound data base for illicit drug prevention programs.

Effects of prenatal exposure to amphetamine in the medial prefrontal cortex of the rat.

This study was designed to investigate the effects of prenatal exposure to amphetamine in the organization of the medial prefrontal cortex of the rat, by an evaluation of growth, morphometric and neurochemical parameters. Pregnant Wistar rats were given 10 mg/kg body weight/day of D-amphetamine sulfate, subcutaneously, from gestational days 8 to 22. Control groups of pregnant rats were injected with saline, pair-fed or non-manipulated; litters were culled to eight pups (four males and four females), weighed every other day until postnatal day 30 and every week until day 90. The Gompertz model was used to study body weight evolution and the estimated growth parameters were not significantly different in the experimental groups. At postnatal days 14 and 30, the volumes of the prefrontal cortex, the fraction of neuropile occupied by neurons and the number of neurons per unit surface are were determined. The number of neurons per unit volume of reference area was calculated using the stereological technique of the dissector. For neurochemical analysis, the medial prefrontal cortex was dissected to measure the concentration of dopamine, serotonin and their metabolites. The allometric relationship of forebrain/body growth pointed to a mechanism of sparing and compensatory growth in the amphetamine exposed group. The changes found in the number of neurons per unit volume at postnatal day 14 show a catch-up at postnatal day 30. A decrease in serotonin levels was found in the amphetamine group compared with the pair-fed control, which was reflected in the ratio of serotonin to its metabolite, 5-hydroxyindolacetic acid. These changes, whether permanent or transitory, raise the possibility that some of the effects of prenatal exposure to amphetamine may be due to modifications in the neurotransmitter levels of serotonin.

The effects of prenatal exposure to cocaine on the dopaminergic cells in the rat retina. An immunocytochemical and neurochemical study.

There is a growing consensus that the development of the eye is affected by prenatal exposure to cocaine. Considering that the retina is affected by prenatal cocaine exposure, that this drug affects the dopaminergic systems, that the dopaminergic cells in the retina show a well-defined pattern of development and that they can be specifically stained in wholemounts by the antibody anti-tyrosine hydroxylase (TH), this study was undertaken to evaluate the effects of in utero cocaine exposure on the dopaminergic cells of the rat retina. Pregnant Wistar rats were given 60 mg (kg body weight)-1 day-1 of cocaine hydrochloride, subcutaneously, from gestational days 8 to 22. Control groups of pregnant rats were pair-fed. At PND14, 30 and 90, male offspring from different litters were perfused with fixative and the retinas processed as wholemounts and immunostained with the antibody anti-TH. Rats from other groups were decapitated at the same post-natal ages, the retinas dissected and processed by neurochemical techniques to measure the concentrations of dopamine, its metabolites and the turnover of dopamine. There was a significant increase of the retina surface area between PND14-30 in the control group, which was not found in the cocaine group. The density of the immunostained small TH cells was lower in the cocaine groups. No drug-effects were detected in the density of the large TH cells. The densities of the total large and small cells in the superior, inferior and nasal hemiretinas were similar to those found in the whole retinas; however, in the temporal hemiretinas of the cocaine groups, the density of the large TH cells was higher and of the small TH cells was lower than in controls, resulting in an absence of effects on the total density of TH-cells in this hemiretina. A transient increase in the level of dopamine metabolite (DOPAC) and of the turnover of dopamine at PND14 was detected in the cocaine groups. All quantitative parameters reached normal values, in all groups, at PND90. These results show that, during the critical periods in which catecholamines can influence the development of neurons, cocaine transiently affects the pattern of dopaminergic neurons in the retina. This may have functional importance due to the role of this neurotransmitter as a regulatory and/or trophic factor in developing neuronal circuitries.

Retinal hemorrhages associated with in utero exposure to cocaine. Experimental and clinical findings.

BACKGROUND: The use of drugs of abuse--e.g., cocaine--during pregnancy has been associated with abnormalities of the visual system. The authors studied the effects of prenatal exposure to drugs of abuse, especially cocaine, on the vascular system of the retina in newborn infants and in an experimental model in the rat. METHODS: The animal study was conducted in pregnant Wistar rats injected subcutaneously with cocaine hydrochloride (60 mg/kg body weight/day) from gestation days 8 to 22. Male offspring were killed at postnatal days 7, 14, and 30 and perfused with fixative, and the retinas were dissected and processed for microscopic observation. The ophthalmologic observations were conducted in a population of newborn infants born to women who abused many drugs during pregnancy and in a control group of women with no history of illicit drug use. RESULTS: Vascular disruptive lesions were seen after prenatal exposure to cocaine in the rat: round intraretinal hemorrhages, ischemic and hypoperfused areas located at the temporal part and often extending from the posterior pole to the periphery of the retina. The ophthalmologic observation of the newborns showed a higher incidence of vascular disruptive lesions in infants in whom exposure to drugs of abuse was affirmative during pregnancy. In the cases in which cocaine consumption was reported, they consisted in blot full-thickness hemorrhages with rounded domed contours suggestive of venous occlusion and retinal ischemia, very similar to the lesions seen in the animal model. These hemorrhagic lesions, morphologically similar to neonatal retinal hemorrhages, had a higher incidence than in controls; they also took longer to resolve when compared with the reabsorption time of the neonatal hemorrhages due to birth trauma and the hemorrhagic lesions in newborns of mothers in whom consumption of other drugs--but not cocaine--were reported. CONCLUSION: A topographic and morphologic parallelism can be established between the retinal vascular alterations found in humans consuming cocaine and in the animal model of prenatal exposure to this drug of abuse; although findings from animal studies may be difficult to apply directly to humans, these data strongly support that cocaine can be a causal factor for the occurrence of retinal vascular disruption in newborns.

Effects of prenatal cocaine exposure in the photoreceptor cells of the rat retina.

Despite the increasing evidence of eye abnormalities, the effects of prenatal exposure to cocaine on the visual system are still poorly understood. This study was aimed at analyzing the qualitative and quantitative organization of the retinal photoreceptor cells (PR) and outer nuclear layer (ONL) after prenatal exposure to cocaine in the rat. Pregnant Wistar rats were given sc injections of cocaine hydrochloride (60 mg/kg body wt/d) or saline or were not manipulated; analyses were performed in the 14- and 30-d-old male offspring. Radial semithin and ultrathin sections of epon-embedded flat mounts of the retina showed displaced PR-like cells in the inner nuclear layer (INL), picnotic PR nuclei in INL, and ONL, and retinal PR rosettes and outer-segment debris in the subretinal space. The quantitative study showed an increased density of PR-like nuclei in the INL in PND14 cocaine-treated rats that were within normal values at PND30; no changes were detected in the PR mean nuclear diameter and in the packing density of PR nuclei in the ONL. These data constitute the first morphological demonstration of photoreceptor damage after prenatal cocaine-exposure probably owing to a direct action of the drug and/or to the cocaine-induced ischemia/hypoxia.

Effects of prenatal cocaine exposure in the prefrontal cortex of the rat. A morphometric evaluation.

This work was undertaken in order to assess the organization of the prelimbic area of the medial prefrontal cortex of rats exposed prenatally to cocaine. Pregnant Wistar rats were assigned to the following groups: 1. Cocaine--60 mg/kg body wt/d sc, from gestational days 8-22; 2. Saline; 3. Pair-fed; and 4. Nonmanipulated. Male offspring were perfused on postnatal days 14 and 30. Six brains per group and per age were embedded in celloidin to calculate the volumes of the prelimbic area; sections from the other six brains were embedded in resin and processed for electron microscopy. Using semithin sections (2 microns) of layers II-III and V-VI, the following parameters were calculated: 1. The fraction of the neuropil occupied by neurons (VV); 2. The packing (NA) density; and 3. The numerical (NV) density. Qualitative alterations consisted of dispersed profiles of degenerated neurons and dendrites in the medial prefrontal cortex. No significant differences were found in the gross morphometric parameters when the cocaine group was compared with the other groups. A high interanimal variation was shown in the prelimbic volumes of postnatal day (PND) 14 cocaine-treated rats, and a a decrease in volumes was detected at PND30. Although there are some alterations in the main afferent cortical target area for dopaminergic input, its gross morphometric parameters do not seem to be sufficiently affected to account for the behavioral alterations referred to as being dependent on this brain region.

Effects of prenatal cocaine exposure in the retinal ganglion cell layer of the rat. A morphometric analysis.

To study the effects of prenatal cocaine-exposure on the developing retinal ganglion cell layer of the rat, female Wistar rats were administered subcutaneously (sc) cocaine hydrochloride (60 mg/kg body wt/d) or saline, or were not manipulated from gestational d 8-22. Male offspring were sacrificed at postnatal day 14 and 30. Radial semithin sections of epon-embedded flat mounts of the retinal quadrants were used to evaluate the following parameters along the centroperipheral axis: 1. Thickness of ganglion cells plus nerve fiber layer; 2. Nuclear size of ganglion cell layer neurons; and 3. Linear density (number per unit length) of ganglion cell layer neurons. To study the effects of cocaine and age on the retinal areas (temporal/nasal, dorsal/ventral), a repeated measures analysis of variance was used for each of the parameters mentioned above. All parameters were affected by prenatal exposure to cocaine. The thickness of the ganglion cell plus nerve fiber layer was reduced in cocaine-exposed rats in comparison with the saline group. Nuclear diameters were smaller in the cocaine than in the saline and control groups. The linear density was higher in the cocaine-exposed group than in the control and saline groups. The age-dependent decrease in the linear density from postnatal day 14-30 was higher in the cocaine-exposed rats than in the saline group; the decrease in the linear density along the centroperipheral axis found in both the control and saline groups was not significant in the cocaine-treated group. These morphometric findings strongly support the view that prenatal cocaine-exposure induces marked changes in the organization of the developing retina.

Effects of prenatal cocaine exposure on postnatal growth patterns of male Wistar rats.

The purpose of this study was to investigate basic parameters regarding the postnatal effects of prenatal exposure to cocaine. Timed-pregnant Wistar rats were injected SC with 60 mg/kg body weight/day of cocaine from gestational day 8 to 22. Control females were nonmanipulated and given food and lib; saline females received saline injections and pair-fed received saline and were nutritionally controlled to the cocaine-treated rats. Litters were restricted to 8 pups, weighed every other day until postnatal day (PND) 30 and every week from PND 30 to PND 90. The rats were perfused at PND 14, 30, and 90. The adequacy of adjustment of the logistic and Gompertz models to the body weights of the offspring was tested for the whole experimental period. The results from the Gompertz curve showed a higher growth rate and less time to reach 37% of expected mature body mass for the offspring of cocaine and pair-fed dams as compared with that of control and saline dams. No significant differences in the estimated adult weight were found among the experimental groups. The allometric relationship between forebrain and body weight is described by two postnatal growth phases with a first phase of rapid growth between PND 14 and 30 and a decelerating phase between PND 30 and 90. This relationship was not different among the experimental groups; however, the cocaine and pair-fed offspring showed a constant deficit in the forebrain weight as compared with the control and saline offspring.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological and biochemical assessment of the cornea in a Gaucher disease carrier with keratoconus.

Ocular abnormalities such as corneal opacities and some specific alterations in ocular movements have been described in the neuropathic forms of Gaucher disease. This study was designed to correlate the clinical, morphological and biochemical findings in the corneal button obtained after keratoplasty in a Gaucher disease carrier with keratoconus. Morphologically, the cornea showed keratocytes with marked dilatations of the rough endoplasmic reticulum and intracytoplasmic "dark inclusions"; the acidic lipid profiles presented alterations in the cornea of the Gaucher disease carrier when compared with healthy controls and a clear deficiency in beta-glucosidase activity was detected as well. Our data suggest that the cornea may serve as a good marker of an early target organ in lipid metabolism disorders such as Gaucher's disease.

Expression of glial fibrillary acidic protein in the rat retina after exposure to psychostimulants.

PURPOSE: Neonatal exposure to psychostimulants is known to produce morphologic alterations in the developing visual system of rats. This study was designed to evaluate the extent of retinal astrocytic response by using the astrocyte localized protein, glial fibrillary acidic protein, after neonatal exposure to cocaine and amphetamine. METHODS: Male Wistar rats were given 15 mg/kg body weight/day of cocaine hydrochloride, subcutaneously, from postnatal days 0 to 6, 13, and 29 and were killed at postnatal days 7, 14, and 30; other rats received 10 mg/kg body weight/day of d-amphetamine sulfate following the same protocols. Control rats were given saline or were not manipulated. After transcardiac perfusion with 4% paraformaldehyde whole mounts of flat retinas were processed for glial fibrillary acidic protein immunohistochemistry. Retinas displaying lesions from the amphetamine groups were processed for electron microscopy; vertical semithin and ultrathin sections were observed. RESULTS: In cocaine-treated rats extensive sheets of proliferating astrocytes presenting a preferential peripheral localization could be identified. In the amphetamine-exposed rats an enhanced expression of glial fibrillary acidic protein was detected in widespread "loci" of astrocytes observed throughout the whole retinal surface; at postnatal day 30 sheets of proliferating astrocytes could also be identified. CONCLUSION: Exposure to psychostimulants during active development induced different types of astrocytic responses in the retina, which, as an end result, can lead to functional changes due to the disruption of the retina structural organization.

Retinal changes induced by neonatal cocaine exposure in the rat.

Retinal abnormalities have been described in both animals and humans exposed to cocaine during development. The present study was designed to examine the morphological repercussions of neonatal exposure to cocaine on the developing retina of the rat. Male Wistar rats were given 15 mg/kg body weight per day of cocaine hydrochloride subcutaneously on postnatal days (PND) 0-6, 13 and 29 and sacrificed at PND 7, 14 or 30; controls were given saline. The retinas were processed for electron microscopy. Retinal quadrants were embedded flat and vertical semithin and ultrathin sections obtained. PND 7 sections showed discrete intraretinal hemorrhages, PND 14 sections showed massive intraretinal hemorrhages and images of ischemic necrosis in the nerve fiber layer and PND 30 sections showed cavity lesions in the hemorrhagic areas, gliosis and pigmented macrophage-rich epiretinal membranes; photoreceptor rosettes were also found. These results are the first morphological demonstration of retinal hemorrhages and associated epiretinal membranes following neonatal exposure to cocaine in the rat. These changes are probably related to the ischemia/hypoxia induced by cocaine.

Aqueous outflow system in familial amyloidotic polyneuropathy, Portuguese type.

Familial amyloidotic polyneuropathy (FAP) is a hereditary disease which may present a wide range of ocular manifestations. Glaucoma is amongst the most serious complications of FAP. We report the results of ultrastructural study of the trabecular meshwork in a glaucomatous patient with the Portuguese form of FAP. This study showed that there was marked anatomical disruption of the uveoscleral, cornoscleral meshworks and juxtacanalicular tissue characterized by (a) accumulation of amyloid fibrils in the intertrabecular spaces; (b) degeneration of the endothelial cells; (c) homogeneous and/or multilayered plaques of basement membrane-like material loading the intertrabecular spaces or protruding to the lumen of the Schlemm's canal; and (d) degeneration of unmyelinated nerve fibres. These morphological changes and an analysis of the literature suggest that mechanical and neuropathic events can be co-existing factors which enhance the resistance to aqueous humour outflow, leading to increased intraocular pressure and glaucoma in the Portuguese form of FAP.

Changes in the retinal ganglion cell layer and optic nerve of rats exposed neonatally to cocaine.

The effects of neonatal exposure to cocaine upon the structure of the visual system are poorly understood despite the evidence of eye abnormalities in infants exposed in utero to cocaine. We previously demonstrated alterations in the optic nerve of rats exposed neonatally to cocaine, although no changes were detected in the number of its axons. This study was undertaken to investigate the retinal ganglion cell layer and the size distribution of the optic axons, in an attempt to assess further changes in the visual pathways. Groups of rats (Wistar strain) were given subcutaneous injections of cocaine hydrochloride (15 mg kg-1 body weight day-1) divided into two daily doses, from the day following birth until postnatal day 30 Controls were given subcutaneous saline throughout the same experimental period. Per group, five animals from three different litters were evaluated morphometrically. Following perfusion with aldehydes, samples from the median ventral and dorsal parts of the retina and the optic nerves were processed for electron microscopy. Morphometric techniques at light and electron microscopic levels allowed us to determine the following. (a) In the optic nerve: (1) frequency size-distribution of myelinated nerve fibres; and (2) number of myelin sheaths per fibre. (b) In the retina: (1) thickness of the layers; (2) frequency size-distribution of ganglion layer neurons; (3) mean cell nuclear size; and (4) packing density of ganglion cell layer neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological changes in the optic nerve after chronic exposure of neonatal rats to cocaine and amphetamine.

We determined the effects of cocaine hydrochloride (15 mg/kg body weight/day) or amphetamine sulfate (25 mg/kg body weight/day) on the optic nerve in groups of rats exposed from postnatal day 1 to 30. Qualitative and quantitative studies of cross-sections of the optic nerves showed different patterns of organization, namely the presence of degenerative features in drug-treated animals and significant differences in the proportion of the nerve occupied by glial cells and their processes and nerve fiber bundles. No significant differences of the total number of fibers were found. Taken together, these data indicate that the optic nerve is vulnerable to early exposure to cocaine and amphetamine which cause developmental changes in this link of the visual pathways.