Alberto Javier Ramos

Intermittent hypoxia during sleep induces reactive gliosis and limited neuronal death in rats: implications for sleep apnea.

J. Neurochem. (2010) 112, 854–869.

The 5HT1A receptor agonist, 8-OH-DPAT, protects neurons and reduces astroglial reaction after ischemic damage caused by cortical devascularization.

Serotonin 1A (5HT1A) receptor agonists have shown neuroprotective properties in different models of central nervous system injury. Activation of neuronal 5HT1A receptors appears to be involved in the neuroprotective effects. It remains to be elucidated if astroglial cells are responsive to the 5HT1A neuroprotective effects. The participation of astroglial S100B trophic factor has been proposed since 5HT1A activation leads to S100B release and nanomolar concentration level of this molecule showed pro-survival activity in neuronal cultures. Using the cortical devascularization model (CD; unilateral pial disruption), a procedure that results in localized ischemia without producing direct physical damage to brain tissue, we tested the effects of a full 5HT1A agonist, 8-OH-DPAT, or the antagonist WAY-100635 on cortical neuronal survival, astroglial cell response and S100B expression. Wistar rats were subjected to CD lesion which consisted of a craniotomy followed by physical damage to the underlying pial blood vessels. Two and twenty-four hours after the CD lesion, animals received intraperitoneally 8-OH-DPAT (1 mg/kg), WAY-100635 (1 mg/kg) or vehicle (sterile saline). At 3, 7 or 14 days post-lesion, animals were sacrificed and their brains processed for immunohistochemistry to detect GFAP, vimentin, MAP-2, S100B and nuclear Hoechst staining. S100B level in the brain cortex and serum was quantified by an ELISA assay. Serum S100B was considered an index of S100B release. 8-OH-DPAT treatment reduced neuronal death, dendrite loss, astroglial hypertrophy and hyperplasia. In contrast, WAY-100635 treatment increased these parameters of damage. S100B intracellular immunoreactivity in astrocytes and total S100B level showed long-lasting changes after the CD lesion and subsequent treatments depending on the 5HT1A activity. The level of serum S100B was increased in 8-OH-DPAT-treated animals. Increased damage observed in WAY-100635-treated animals supports the hypothesis that the protective 8-OH-DPAT action may be mediated by specific 5HT1A receptors. The reduction in astroglial hypertrophy and hyperplasia as well as long-term changes in S100B immunoreactivity and increased S100B release that we observed allows us to hypothesize that astroglial cells may play an important role in 5HT1A-mediated neuroprotection.

Neuronal and Glial Expression of the Multidrug Resistance Gene Product in an Experimental Epilepsy Model

Abstract1. Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood. It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy.2. Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy. Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p. for either 4 days (MP-4) or 7 days (MP-7). Control rats received an equivalent volume of vehicle. One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170. As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group.3. Increased Pgp-170 immunoreactivity was detected in MP-treated animals. Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats. Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution. MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling.4. Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures.5. The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.

Neuronal cytoskeleton and synaptic densities are altered after a chronic treatment with the cannabinoid receptor agonist WIN 55,212-2.

Cannabinoid CB1 receptors are the most abundant G-protein-coupled receptors in the brain. Its presynaptic location suggests a role for cannabinoids in modulating the release of neurotransmitters from axon terminals by retrograde signaling. The neuroprotective effects of cannabinoid agonists in animal models of ischemia, seizures, hypoxia, Multiple Sclerosis, Huntington and Parkinson disease have been demonstrated in several reports. The proposed mechanism for the neuroprotection ranges from antioxidant effects, reduction of microglial activation and anti-inflammatory reaction to receptor-mediated reduction of glutamate release. In the present work, we analyzed the morphological changes induced by a chronic treatment with the synthetic cannabinoid receptor agonist, WIN 55,212-2, in four brain regions where the CB1 cannabinoid receptor is present in high density: the CA1 hippocampal area, corpus striatum, cerebellum and frontal cortex. After a twice-daily treatment for 14 days with the cannabinoid receptor agonist (3 mg/kg sc, each dose) to male Wistar rats (150-170 g), the expression of neurofilaments (Nf-160 and Nf-200), microtubule-associated protein-2 (MAP-2), synaptophysin (Syn) and glial fibrillary acidic protein (GFAP) was studied by immunohistochemistry and digital image analysis. Ultrastructural study of the synapses was done using electron microscopy. After the treatment, a significant increase in the expression of neuronal cytoskeletal proteins (Nf-160, Nf-200, MAP-2) was observed, but we did not find changes in the expression of GFAP, the main astroglial cytoskeletal protein. In cerebellum, there was an increase in Syn expression and in the number of synaptic vesicles, while, in the hippocampus, an increase in the Syn expression and in the thickness of the postsynaptic densities was observed. The results obtained from these studies provide evidences on the absence of astroglial reaction and a sprouting phenomena induced by the WIN treatment that might be a key contributor to the long-term neuroprotective effects observed after cannabinoid treatments in different models of central nervous system (CNS) injury reported in the literature.

Hypo-Osmolar Stress Induces p75NTR Expression by Activating Sp1-Dependent Transcription

Injury-induced expression of the p75 neurotrophin receptor (p75NTR) in the CNS facilitates neuronal apoptosis and prevents neuronal regrowth, but the mechanisms regulating p75NTR expression are poorly characterized. In this study, we showed that hypo-osmolarity induces p75NTR expression in primary neurons, and, using a comparative genomics approach, we identified conserved elements in the 25 kb upstream sequences of the rat, mouse, and human p75NTR genes. We found that only one of these, a proximal region rich in Sp1 sites, responds to changes in hypo-osmolarity. We then showed that Sp1 DNA binding activity is increased in cells exposed to hypo-osmolarity, established that hypo-osmolarity enhanced Sp1 binding to the endogenous p75NTR promoter, and showed that Sp1 is required for p75NTR expression induced by hypo-osmolarity. We examined how Sp1 is regulated to effect these changes and established that Sp1 turnover is strongly inhibited by hypo-osmolarity. We propose that stress-induced Sp1 accumulation that results from reductions in Sp1 turnover rate contributes to injury-induced gene expression.

S100B alters neuronal survival and dendrite extension via RAGE-mediated NF-κB signaling

J. Neurochem. (2011) 117, 321–332.

Neuroglial interactions in a model of para-chlorophenylalanine-induced serotonin depletion

Serotonin (5HT) is involved in the development and plasticity of the CNS through the release of S-100beta, a glial trophic factor which stabilizes synapses and neuronal cytoskeleton and promotes neuronal development. S-100beta is released from glial cells after activation of glial 5HT(1A) receptors. We present in this paper the effects upon neurons and glia of a 5HT depletion induced by 14 days of treatment with para-chlorophenylalanine (PCPA) in adult rats. S-100beta, 5HT, 5HT-transporter (5HT-T) and neurofilaments (Nf-200 and Nf-68) expressions were studied by immunohistochemistry and image analysis in striatum, hippocampus, parietal and frontal cortex. Immediately after ending PCPA treatment we found increased intracellular S-100beta immunoreactivity in glial cells, reduced 5HT immunolabelling, reduced density of 5HT-T, Nf-200 and Nf-68 fibers and morphological alterations in neuronal cytoskeleton. One week after PCPA treatment S-100beta immunoreactivity decreased towards control levels, 5HT was normalized in dorsal raphe nucleus, but not in innervation areas; 5HT-T, Nf-200 and Nf-68 fiber densities increased but some neuronal cytoskeletal alterations were still present in striatum. Two weeks after PCPA treatment S-100beta had returned to control levels in most studied regions; 5HT immunoreactivity was normalized, meanwhile 5HT-T, Nf-200 and Nf-68 fiber densities increased reaching values over the control level. We propose that S-100beta could be accumulated in glial cells during the 5HT depletion period, to be released once 5HT levels have recovered. Neuronal cytoskeletal alterations and reduced fiber density may be the expression of decreased extracellular availability of S-100beta. Conversely, increased 5HT-T, Nf-200 and Nf-68 expressions, once S-100beta is normalized, may be the biological response to the growth factor release.

Neuronal mdr-1 gene expression after experimental focal hypoxia: A new obstacle for neuroprotection?

Neuronal damage after stroke-associated brain hypoxia is a leading cause of long-term disability and death. The refractoriness to therapeutic strategies for neuroprotection after 3 h post brain ischemia is poorly understood. P-glycoprotein (P-gp), the multidrug resistance gene (MDR-1) product is normally expressed at blood-brain-barrier. P-gp neuronal expression has been demonstrated in refractory epilepsy and after brain ischemia. In this report we investigated the hypoxia-induced neuronal P-gp expression after local injection of CoCl(2) (1-200 mM) in the fronto-parietal cortex of male adult rats (Bregma -1.30 mm) by stereotaxic surgery. P-gp immunostaining of brain slides was analyzed using specific monoclonal antibodies and double immunolabeling was done with specific astrocytic and neuronal markers. Five days after injection of 1 mM CoCl(2), P-gp expression surrounding the lesion site was observed in neurons, astrocytic end-foot on capillary blood vessels and endothelial cells on blood vessels. Higher CoCl(2) doses (200 mM) resulted in additional P-gp immunostaining of the entire astrocytic and neuronal cytoplasm. Electron microscopy (EM) studies showed alterations in neurons as early as 6 h after the CoCl(2) injection. P-gp expression in hypoxic neurons and astrocytic end-foot could potentially impair of drugs access to the brain parenchyma thus suggesting the presence of two P-gp-based pumping systems (one in astrocytes and other in the hypoxic neurons) that are able to behave as a previously unnoticed obstacle for pharmacological strategies of neuroprotection.

Immunofluorescence and glutaraldehyde fixation. A new procedure based on the Schiff-quenching method.

The immunofluorescence technique is one of the most useful methods for localizing antigens in several tissues, including the central nervous system. For immunohistochemical procedures, especially immunofluorescence methods, formaldehyde is commonly used as a fixative agent. But for some protocols, mainly in neurobiology, glutaraldehyde is necessary to recognize a number of small molecules (haptens) whose antisera have been raised using glutaraldehyde as the cross-linking agent. This is a severe limitation because glutaraldehyde gives rise to a strong autofluorescence on tissue that precludes the observation of specific immunofluorescence staining. In this paper we present a new method that allows the use of immunofluorescence techniques on glutaraldehyde-fixed tissues. The new method consists of a treatment of tissue sections with the Schiffs reagent (leucobasic fuchsin) followed by a reduction of the Schiff-dye with sodium borohydride. This reduced dye produces a quenching of glutaraldehyde-induced fluorescence on the tissue. The goal of the new method is to make possible the use of a great number of available glutaraldehyde-raised antisera for immunofluorescence techniques, a useful tool in both basic and clinical research.

Increased nitric oxide synthase activity in a model of serotonin depletion

Serotonin (5HT) containing cell bodies are localized in mesencephalic and rhombencephalic raphe nuclei. It has been proposed that 5HT could be involved in neuronal development and plasticity. In the central nervous system, nitric oxide (NO) has been postulated as a neurotransmitter and neuromodulator, and has been implicated in neurotoxicity as well as in neuroprotection. Using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) technique, NO synthesizing neurons were described in raphe nuclei. By immunohistochemistry, nitric oxide synthase (NOS) was found colocalized with 5HT in some dorsal raphe nucleus (DRN) neurons. In a model of inhibition of 5HT synthesis produced by daily administration of parachlorophenilalanine during 14 days, we have studied the relationship between 5HT and NO systems after 5HT depletion by histochemical and immunocytochemical methods. After the treatment, we observed an important reduction of 5HT immunostaining in the DRN and enhanced NOS activity demonstrated by NADPH-d technique, especially in the dorsomedial and ventromedial subgroups. In spite of the increased NOS activity, we could not observe significant changes in the NOS-immunoreactivity in the DRN after 5HT depletion. These results could indicate that 5HT depletion is concomitant with changes in NOS activity without affecting NOS expression in the DRN.