Héctor Coirini

Changes of NADPH-diaphorase reactivity in lumbar spinal cord of short-term streptozotocin induced diabetic rats.

Diabetes is an endocrine and metabolic disorder often associated with erectile dysfunction and peripheral neuropathy. Among other factors, penile erection is induced by activation of nitric oxide synthase (NOS). Hypothalamic paraventricular nuclei neurons produce NO and project to spinal cord areas implicated in penile reflexes. These nuclei have shown an increase of NOS in streptozotocin-induced diabetic rats. NOS-containing neurons are identical to the populations of neurons selectively stained for NADPH-diaphorase activity. Using this technique, we have evaluated changes of NOS in the lumbar spinal cord of diabetic rats with or without insulin treatment. Positive staining was found in motoneurons, dorsal horn neurons (layer II), neurons surrounding the ependimus (layer X) and neurons at the intermediolateral cell column (ILCC). Diabetic animals showed significant decrease in reactive area and increase of the histochemical reaction in motoneurons from the sexual dimorphic nuclei and in neurons of the ILCC. A marked decrease of the number of reactive neurons was also observed in layer II. Morphologic alterations were observed in neurons of layer X as an increase in the percentage of multipolar neurons and a decrease in the number and length of secondary processes. The alterations observed in these animals were absent in the insulin treated diabetic animals. These results show the plasticity of lumbar spinal cord neurons, suggesting a direct participation of NO synthesis in the physiopathology of the erection dysfunction in diabetes.

Alterations of LXRα and LXRβ expression in the hypothalamus of glucose-intolerant rats

Liver X receptor (LXR) α and β are nuclear receptors that are crucial for the regulation of carbohydrate and lipid metabolism. Activation of LXRs in the brain facilitates cholesterol clearance and improves cognitive deficits, thus they are considered as promising drug targets to treat diseases such as atherosclerosis and Alzheimers disease. Nevertheless, little is known about the function and localization of LXRs in the brain. Here, we studied the expression of LXR in the brains of rats that received free access to 10% (w/v) fructose group (FG) in their beverages or water control drinks (control group (CG)). After 6 weeks rats in the FG presented with hypertriglyceridemia, hyperinsulinemia, and became glucose intolerant, suggesting a progression toward type 2 diabetes. We found that hypothalamic LXR expression was altered in fructose-fed rats. Rats in the FG presented with a decrease in LXRβ levels while showing an increase in LXRα expression in the hypothalamus but not in the hippocampus, cerebellum, or neocortex. Moreover, both LXRα and β expression correlated negatively with insulin and triglyceride levels. Interestingly, LXRβ showed a negative correlation with the area under the curve during the glucose tolerance test in the CG and a positive correlation in the FG. Immunocytochemistry revealed that the paraventricular and ventromedial nuclei express mainly LXRα whereas the arcuate nucleus expresses LXRβ. Both LXR immunosignals were found in the median preoptic area. This is the first study showing a relationship between glucose and lipid homeostasis and the expression of LXRs in the hypothalamus, suggesting that LXRs may trigger neurochemical and neurophysiological responses for the control of food intake and energy expenditure through these receptors.

NEOSTRIATAL CYTOSKELETON CHANGES FOLLOWING PERINATAL ASPHYXIA: EFFECT OF HYPOTHERMIA TREATMENT

Long-term changes of different types of neurofilaments (NF) and glial fibrilar acid protein (GFAP) were studied in neostriatal rat subjected to perinatal asphyxia (PA) under normothermic and hypothermic (15°C) conditions, using immunohistochemistry for light and electron microscopy. Neostriatal neurons of 6-month-old rats that were subjected to 19 and 20 min of PA, showed an increase of NF 200 kDa immunostaining mainly in the axon fascicles in comparison with the control and hypothermia groups. In contrast, no alterations were seen with NF68 and NF160 neurofilament antibodies. Furthermore, the same PA groups showed astroglial cells with enhanced GFAP immunoreactivity, evidencing a typical astroglial reaction with a clear hypertrophy of these cells. A quantitative image analysis confirmed these observations. Hypothermic treated animals did show neither astroglial nor neuronal cytoskeletal changes in comparison to the control group. These findings showed that PA produces chronic cytoskeletal alterations in the neostriatum cells that can be prevented by hypothermia.

Site-Specific Effects of the Nonsteroidal Anti-Inflammatory Drug Lysine Clonixinate on Rat Brain Opioid Receptors

In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p < 0.05) labeling of mu receptors was observed in thalamic nuclei, gyrus dentate, and layers of the parietal cortex of rats treated for 10 days with lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 × 10–4 mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

Sex differences in LXR expression in normal offspring and in rats born to diabetic dams

Gestational diabetes (GD) alters normal fetal development and is related to a diabetogenic effect in the progeny. Liver X receptors (LXRs) are considered to be potential drug targets for the regulation, treatment, or prevention of diabetes. The aim of this study was to evaluate early and late changes of LXR in the hippocampus and hypothalamus of the male and female offspring of control (CO) and diabetic (DO) mothers. We used an experimental model of streptozotocin-induced GD to assess the protein expression of LXRα (NR1H3) and LXRβ (NR1H2) by western blotting. The tissues were obtained from CO and DO animals at postnatal day 1 (1D), day 10 (10D), and day 35 (35D) and 9 months (9M). In CO, the LXR expression showed significant differences among the groups, which were tissue- and receptor-specific (P<0.05). Sex differences in CO were found only in the hypothalamus for LXRβ expression at 35D and 9M (P<0.05). When CO and DO were compared, differences between them were observed in the majority of the studied groups at 1D (male hippocampus, LXRα 31% and LXRβ 161%; female hippocampus, LXRβ 165%; male hypothalamus, LXRβ 182%; and female hypothalamus, LXRα 85%; P<0.05). However, these differences disappeared later with the exception of LXRβ expression in the male hypothalamus (P<0.05). The area under the curve during the glucose tolerance test correlated negatively with LXRβ in CO but not in DO animals. Moreover, in a male DO subpopulation this correlation was positive as it occurs in intolerant animals. These results indicate that GD affects hypothalamic LXR expression differently in male and female offspring.

Regulation of the expression of LXR in rat hypothalamic and hippocampal explants

Liver X receptors (LXR) are important transcription factors involved in the regulation of carbohydrate and lipid metabolism and are expressed in different brain areas. Recently we described that LXR expression in the hypothalamus is sensitive to serum levels of lipids and carbohydrates. Here, we further characterized the effects of glucose, insulin, cholesterol and cholic acid on the expression of LXRα and LXRβ in hypothalamus and hippocampus explants as in vitro models. The LXR activation products, GLUT2 and ABCA1, were also analyzed by Western blot. Glucose had different effects in the hypothalamus compared to the hippocampus. In the hypothalamus, increases in glucose concentrations decreased LXRβ expression while in the hippocampus increased both receptor subtypes levels. In contrast, insulin treatment decreased LXRβ in the hypothalamus while having no effects on the hippocampus. Cholic acid and cholesterol increased only LXRα expression in the hypothalamus whereas no effects on the hippocampus were detected. The newly expressed LXR receptors may be functional active since the level of the LXR activation product ABCA1 was also increased. Changes in GLUT2 expression was observed only when LXRβ levels were increased. Altogether these data show that LXR are sensitive to glucose, insulin and lipids in vitro, as well as in vivo as we previously showed, suggesting an involvement of LXR in central metabolic pathways and control of energy homeostasis.

Neuroprotective action of synthetic steroids with oxygen bridge. Activity on GABAA receptor

Allopregnanolone (A) and pregnanolone (P) are able to modify neural activities acting through the GABAA receptor complex. This capacity makes them useful as anticonvulsant, anxiolytic, or anti-stress compounds. In this study, the performance of seven synthetic steroids (SS) analogous of A or P containing an intramolecular oxygen bridge was evaluated using different assays. Competition assays showed that compounds 1, 5, 6 and 7 affected the binding of specific ligands for the GABAA receptor in a way similar to that of A and P, whereas compounds 3 and 4 stimulated [(3)H]-flunitrazepam and reduced [(35)S]-TBPS binding. The enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) produces the precursor for A and P, and its activity is regulated by steroids. The action of several SS on 3β-HSD activity was tested in different tissues. All SS analyzed inhibit its activity, but compound 5 was the least effective. Finally, the neuroprotective role of two SS was evaluated in cerebral cortex and hippocampus cultures subjected to hypoxia. Glial fibrillary acidic protein (GFAP) increase was prevented by A, P, and compounds 3 and 5. Only A, P and compound 5 prevented neurofilament (NF160/200) decrease in hippocampus cultures, whereas A and compound 5 partially prevented NF200 and NF160 decreases respectively in cerebral cortex cultures. A prevented microtubule associated protein (MAP 2b) decrease in cerebral cortex cultures, while in hippocampus cultures only compounds 3 and 5 had effect. All steroids prevented MAP 2c decrease in both brain regions.

Protective effects of the neurosteroid allopregnanolone in a mouse model of spontaneous motoneuron degeneration

Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by progressive death of motoneurons. The Wobbler (WR) mouse is a preclinical model sharing neuropathological similarities with human ALS. We have shown that progesterone (PROG) prevents the progression of motoneuron degeneration. We now studied if allopregnanolone (ALLO), a reduced metabolite of PROG endowed with gabaergic activity, also prevents WR neuropathology. Sixty-day old WRs remained untreated or received two steroid treatment regimens in order to evaluate the response of several parameters during early or prolonged steroid administration. ALLO was administered s.c. daily for 5days (4mg/kg) or every other day for 32days (3, 3mg/kg), while another group of WRs received a 20mg PROG pellet s.c. for 18 or 60days. ALLO administration to WRs increased ALLO serum levels without changing PROG and 5 alpha dihydroprogesterone (5α-DHP), whereas PROG treatment increased PROG, 5α-DHP and ALLO. Untreated WRs showed higher basal levels of serum 5α-DHP than controls. In the cervical spinal cord we studied markers of oxidative stress or associated to trophic responses. These included nitric oxide synthase (NOS) activity, motoneuron vacuolation, MnSOD immunoreactivity (IR), brain derived neurotrophic factor (BDNF) and TrkB mRNAs, p75 neurotrophin receptor (p75NTR) and, cell survival or death signals such as pAKT and the stress activated kinase JNK. Untreated WRs showed a reduction of MnSOD-IR and BDNF/TrkB mRNAs, associated to high p75NTR in motoneurons, neuronal and glial NOS hyperactivity and neuronal vacuolation. Also, low pAKT, mainly in young WRs, and a high pJNK in the old stage characterized WŔs spinal cord. Except for MnSOD and BDNF, these alterations were prevented by an acute ALLO treatment, while short-term PROG elevated MnSOD. Moreover, after chronic administration both steroids enhanced MnSOD-IR and BDNF mRNA, while attenuated pJNK and NOS in glial cells. Long-term PROG also increased pAKT and reduced neuronal NOS, parameters not modulated by chronic ALLO. Clinically, both steroids improved muscle performance. Thus, ALLO was able to reduce neuropathology in this model. Since high oxidative stress activates p75NTR and pJNK in neurodegeneration, steroid reduction of these molecules may provide adequate neuroprotection. These data yield the first evidence that ALLO, a gabaergic neuroactive steroid, brings neuroprotection in a model of motoneuron degeneration.

Reduction of the spinal nucleus of the bulbocavernosous volume by experimental diabetes.

The sexually dimorphic nuclei, spinal nucleus of the bulbocavernosus (SNB) and dorsolateral nucleus, are located at the lumbar segment of the rat spinal cord. These nuclei innervate perineal muscles involved in penile erection and ejaculation. Testosterone levels modulate their size in adult male rats. Because diabetes is associated with low levels of testosterone, we have evaluated morphological changes on spinal cord of diabetic animals. Significant reduction in the SNB volume was observed 4 weeks after diabetes induction accomplished by a reduction on the motoneuronal size. Insulin prevents the morphological alterations. No significant changes were observed on other dimorphic nucleus. The altered sexual behavior of diabetic rats could be consequence of the detected reduction in the SNB volume.

LXR ACTIVATION INCREASES THE EXPRESSION OF GNRH AND αMSH IN THE RAT HYPOTHALAMUS IN VIVO

Liver X receptors (LXR) are important transcription factors involved in the regulation of carbohydrate and lipid metabolism. Recently, we described LXR receptors expression in the hypothalamus but their function in this brain area remains unknown. Here, we evaluated the function of LXR on the expression of factors produced in the hypothalamus in vitro and in vivo by Western blotting and immunocytochemistry. More precisely we studied the expression of GnRH and GHRH, αMSH and NPY in male Sprague-Dawley rats. The effects of two synthetic LXR agonists, T0901317 and GW3965, were first tested in vitro. Hypothalamic explants were treated with either T0901317 or GW3965 (10μM) for 2, 4, 6 and 8h. As a positive control the cholesterol ABCA1 and glucose GLUT2 transporters were used. No changes were observed in the expression of the factors evaluated in vitro. The effects of the LXR agonists were then tested in vivo. Rats were injected ICV into the third ventricle with either T0901317 or GW3965 (2.5μg/5μL ICV) and after 3.5h or 24h the hypothalami were dissected out and rapidly frozen for analysis. αMSH and GnRH expression was significantly increased after 3.5h of T0901317 treatment. Anterior/posterior hypothalamic ratio increases for αMSH expression and decreases for GnRH expression after 24h of LXR activation. Altogether these results show that LXR activation affects the expression of GnRH and αMSH, suggesting that LXR in the hypothalamus is capable of modulating hypothalamic responses related to appetite, sexual behavior and reproductive functions.