RESUMO
After different types of acute central nervous system insults, including stroke, subarachnoid haemorrhage and traumatic brain and spinal cord injuries, secondary damage plays a central role in the induction of cell death, neurodegeneration and functional deficits. Interestingly, secondary cell death presents an attractive target for clinical intervention because the temporal lag between injury and cell loss provides a potential window for effective treatment. While primary injuries are the direct result of the precipitating insult, secondary damage involves the activation of pathological cascades through which endogenous factors can exacerbate initial tissue damage. Secondary processes, usually interactive and overlapping, include oxidative stress, neuroinflammation and dysregulation of autophagy, ultimately leading to cell death. Resveratrol, a natural stilbene present at relatively high concentrations in grape skin and red wine, exerts a wide range of beneficial health effects. Within the central nervous system, in addition to its inherent free radical scavenging role, resveratrol increases endogenous cellular antioxidant defences thus modulating multiple synergistic pathways responsible for its antioxidant, anti-inflammatory and anti-apoptotic properties. During the last years, a growing body of in vitro and in vivo evidence has been built, indicating that resveratrol can induce a neuroprotective state and attenuate functional deficits when administered acutely after an experimental injury to the central nervous system. In this review, we summarize the most recent findings on the molecular pathways involved in the neuroprotective effects of this multi target polyphenol, and discuss its neuroprotective potential after brain or spinal cord injuries.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Lesões Encefálicas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Resveratrol/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Lesões Encefálicas/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Humanos , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Resveratrol/farmacologia , Traumatismos da Medula Espinal/metabolismoRESUMO
Inflammatory pain associates with spinal glial activation and central sensitization. Systemic administration of IMT504, a non-CpG oligodeoxynucleotide originally designed as an immunomodulator, exerts remarkable anti-allodynic effects in rats with complete Freund´s adjuvant (CFA)-induced hindpaw inflammation. However, the anti-nociceptive mechanisms of IMT504 remain unknown. Here we evaluated whether IMT504 blocks inflammatory pain-like behavior by modulation of spinal glia and central sensitization. The study was performed in Sprague Dawley rats with intraplantar CFA, and a single lumbosacral intrathecal (i.t.) administration of IMT504 or vehicle was chosen to address if changes in glial activation and spinal sensitization relate to the pain-like behavior reducing effects of the ODN. Naïve rats were also included. Von Frey and Randall-Selitto tests, respectively, exposed significant reductions in allodynia and mechanical hypersensitivity, lasting at least 24 h after i.t. IMT504. Analysis of electromyographic responses to electrical stimulation of C fibers showed progressive reductions in wind-up responses. Accordingly, IMT504 significantly downregulated spinal glial activation, as shown by reductions in the protein expression of glial fibrillary acidic protein, CD11b/c, Toll-like receptor 4 (TLR4) and the phosphorylated p65 subunit of NFκB, evaluated by immunohistochemistry and western blot. In vitro experiments using early post-natal cortical glial cultures provided further support to in vivo data and demonstrated IMT504 internalization into microglia and astrocytes. Altogether, our study provides new evidence on the central mechanisms of anti-nociception by IMT504 upon intrathecal application, and further supports its value as a novel anti-inflammatory ODN with actions upon glial cells and the TLR4/NFκB pathway. Intrathecal administration of the non-CpG ODN IMT504 fully blocks CFA-induced mechanical allodynia and hypersensitivity, in association with reduced spinal sensitization. Administration of the ODN also results in downregulated gliosis and reduced TLR4-NF-κB pathway activation. IMT504 uptake into astrocytes and microglia support the concept of direct modulation of CFA-induced glial activation.
Assuntos
Sensibilização do Sistema Nervoso Central , Hiperalgesia , Animais , Hiperalgesia/tratamento farmacológico , Inflamação , Oligodesoxirribonucleotídeos , Dor , Ratos , Ratos Sprague-Dawley , Medula EspinalRESUMO
Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a frequent and devastating side effect of cancer therapy. No preventive strategies are currently available. We investigated the use of resveratrol (RESV) in the prevention of CIPNP and evaluated key components of the antioxidant defense system and neuroinflammatory factors as possible mediators contributing to RESV actions. Male rats were injected with oxaliplatin (OXA) and received daily oral RESV. Paw mechanical and thermal allodynia, oxidative stress, antioxidant, pro-inflammatory and neuronal injury/activation markers were evaluated in the sciatic nerve (SN), lumbar dorsal root ganglia (DRG) and spinal cord (SC). OXA-injected animals developed mechanical and thermal allodynia, while those receiving OXA + RESV showed patterns of response similar to control animals. Higher TBARS levels and lower GSH/GSSG ratios were observed in the SN of animals receiving OXA. The mRNA levels of the transcription factor NFκB and the pro-inflammatory cytokine TNFα were found to be upregulated both in lumbar DRG and SC. In addition, the antioxidant enzymes NQO-1 and HO-1 and the neuronal injury marker ATF3 showed increased levels of expression in lumbar DRG. In the dorsal SC the neuronal activation marker c-fos and the transcription factor Nrf2, main regulator of antioxidant defenses, were found to be upregulated. RESV early and sustained administration prevented NFκB, TNFα, ATF3 and c-fos upregulation, while increasing the expression of Nrf2, NQO-1, HO-1 and the redox-sensitive deacetylase SIRT1. RESV treatment was also able to restore TBARS levels and GSH/GSSG ratio. Thus, RESV administration resulted in the upregulation of antioxidant mediators, suppression of pro-inflammatory parameters and prevention of OXA-induced mechanical and thermal allodynia.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Hiperalgesia/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Resveratrol/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Citocinas/metabolismo , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Masculino , Oxaliplatina/efeitos adversos , Medição da Dor , Ratos , Resveratrol/farmacologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
Single ligature nerve constriction (SLNC) of the rat sciatic nerve triggers neuropathic pain-related behaviors and induces changes in neuropeptide expression in primary afferent neurons. Bone marrow stromal cells (MSCs) injected into the lumbar 4 (L4) dorsal root ganglia (DRGs) of animals subjected to a sciatic nerve SLNC selectively migrate to the other ipsilateral lumbar DRGs (L3, L5 and L6) and prevent mechanical and thermal allodynia. In this study, we have evaluated the effect of MSC administration on the expression of the neuropeptides galanin and NPY, as well as the NPY Y(1)-receptor (Y(1)R) in DRG neurons. Animals were subjected to a sciatic nerve SLNC either alone or followed by the administration of MSCs, phosphate-buffered saline (PBS) or bone marrow non-adherent mononuclear cells (BNMCs), directly into the ipsilateral L4 DRG. Seven days after injury, the ipsilateral and contralateral L4-5 DRGs were dissected out and processed for standard immunohistochemistry, using specific antibodies. As previously reported, SLNC induced an ipsilateral increase in the number of galanin and NPY immunoreactive neurons and a decrease in Y(1)R-positive DRG neurons. The intraganglionic injection of PBS or BNMCs did not modify this pattern of expression. In contrast, MSC administration partially prevented the injury-induced changes in galanin, NPY and Y(1)R expression. The large number of Y(1)R-immunoreactive neurons together with high levels of NPY expression in animals injected with MSCs could explain, at least in part, the analgesic effects exerted by these cells. Our results support MSC participation in the modulation of neuropathic pain and give insight into one of the possible mechanisms involved.
Assuntos
Galanina/biossíntese , Neuropeptídeo Y/biossíntese , Receptores de Neuropeptídeo Y/biossíntese , Nervo Isquiático/lesões , Células Estromais/fisiologia , Animais , Células da Medula Óssea , Constrição Patológica/metabolismo , Ratos , Neuropatia Ciática , Transplante de Células-Tronco , Resultado do Tratamento , Ferimentos e Lesões/metabolismoRESUMO
We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.
Assuntos
Células da Medula Óssea/fisiologia , Globinas/biossíntese , Degeneração Neural/patologia , Regeneração Nervosa/fisiologia , RNA Mensageiro/biossíntese , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Animais , Western Blotting , Movimento Celular/fisiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Imuno-Histoquímica , Masculino , Proteína Básica da Mielina/metabolismo , Ensaios de Proteção de Nucleases , Peptídeos/química , Nervos Periféricos/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células de Schwann/fisiologia , Nervo Isquiático/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TripsinaRESUMO
The characterization of the functional interactions between the metabotropic glutamate receptors (mGluR) and the dopaminergic (DR) receptors in the corticostriatal projections may provide a possible interpretation of synaptic events in the basal ganglia. It has been suggested that presynaptic D2-type receptor located on glutamatergic corticostriatal neurons regulates the release of glutamate. In a first approach we have studied the cellular distribution of the D4R and the mGluRs in cerebral cortex and striatum employing immunocytochemistry. D4R positive neurons were particularly numerous in medial prefrontal cortex mainly occupying layers II and III. An even distribution was found on small round-shaped neurons in the striatum. Group I mGluR1alpha-like immunoreactivity (mGluR1alpha-LI) was found in medial and deep layers of the cerebral cortex while group III mGluR4a labeled more superficial layers; group II mGluR2/3 signal was intense on fine fibers with a punctate appearance. In the striatum, mGluR1alpha and mGluR2/3 stained mainly fibers while mGluR4a labeled round shaped cell bodies. After lateral ventricular injection of colchicine, an axonal transport and firing activity blocker, D4R labeling significantly increased in cerebral cortex and decreased in the striatum. mGluR1alpha and mGluR4a signal decreased in cerebral cortex and only mGluR4a signal decreased in the striatum. These results support previous reports indicating a presynaptic localization of D4R in the striatum. In contrast, striatal mGluR1alpha appears to be a postsynaptic receptor probably synthesized in situ. Our results do not support the hypothesis of a colocalization of D4 receptor and one or more of the metabotropic glutamatergic receptors studied here.
Assuntos
Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Colchicina/administração & dosagem , Imuno-Histoquímica , Injeções Intraventriculares , Masculino , Ratos , Ratos Wistar , Receptores de Dopamina D4RESUMO
The coexistence of vasopressin (VP), oxytocin (OXY), galanin (GAL) and cholecystokinin (CCK) and the synthesis of GAL and CCK during neuritic regeneration was investigated in cultured magnocellular neurons, isolated from adult rat supraoptic nuclei. Double-labelling immunofluorescence was performed after 7 days of culture using primary antibodies for VP, OXY, GAL and CCK (paired in all possible combinations) and secondary antibodies labelled with either fluorescein or rhodamine. Confocal laser scanning microscopy revealed the coexistence of the mentioned peptides in all possible combinations, an unexpected result considering that the only combinations observed in tissue sections are VP-GAL and OXY-CCK. Freshly dispersed cells were devoid of any neuritic processes and showed a very poor immunocytochemical staining reaction for GAL and CCK. In contrast, neurons cultured for 7, 12 and 21 days showed many neurites and a strong immunoreactivity for GAL and CCK indicative of an increased synthesis of both peptides in the regenerating neurons. This increased synthetic activity is consistent with transient upregulation of these peptides observed in situ after hypophysectomy by other authors. The results suggest that the upregulation of GAL and CCK is functionally related to the neuronal regeneration processes observed during culture and that the 'uncommon' coexistences as well as the prolonged sythesis of GAL and CCK may be due to the lack of environmental inputs, which normally regulate the expression and up- and downregulation of these peptides in vivo.
Assuntos
Regeneração Nervosa/fisiologia , Neuropeptídeos/metabolismo , Núcleo Supraóptico/metabolismo , Animais , Células Cultivadas , Colecistocinina/metabolismo , Regulação para Baixo , Galanina/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Neurônios/metabolismo , Ocitocina/metabolismo , Ratos , Núcleo Supraóptico/citologia , Regulação para Cima , Vasopressinas/metabolismoRESUMO
In the present paper we first studied the brain distribution and the time and dose dependent effects of apotransferrin, after its intracranial injection into young rats and at different post-natal ages. Its action upon the transferrin receptor (TfR) and upon the expression of brain transferrin, as well as its effect on the proliferation and differentiation of oligodendroglial cells (OLGc) was one of the main objectives of our investigation. Total DNA and BrdU labeling, as an index of cellularity and proliferation, respectively, were the same in the control and experimental groups of rats. A significant increase in the MBP+ and CA II+ OLGc, and a decrease in the more immature (A2B5+) OLGc were found in the aTf injected rats. At 10 and 17 days of age, Tf-mRNA decreased to around 20% of the amount present in control animals. The TfR-mRNA in the animals receiving a single dose of aTf at 3 days of age showed an increase in its expression at 10 and 17 days of age, coincident with a higher immunoreactivity of the TfR itself of neurons, choroid plexus and brain capillaries in different brain areas. Although TfR+ OLGc were present up to 7 days of age in controls and in the Tf injected rats, no positive cells were observed at 17 days of age, even in the aTf injected rats. Our results give support to the hypothesis that aTf is an important factor necessary for the maturation of the OLGc, and that the effects that it produces in the OLGc-myelin unit after its intracranial injection in young rats are not due to an increase in proliferation, but to an accelerated differentiation of Tf-sensitive OLGc.
Assuntos
Apoproteínas/farmacologia , Encéfalo/metabolismo , Oligodendroglia/citologia , Transferrina/farmacologia , Animais , Apoproteínas/administração & dosagem , Apoproteínas/genética , Encéfalo/citologia , Diferenciação Celular , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Humanos , Injeções , Radioisótopos do Iodo/metabolismo , Masculino , Oligodendroglia/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Receptores da Transferrina/genética , Crânio , Fatores de Tempo , Transferrina/administração & dosagem , Transferrina/genéticaRESUMO
We have previously shown that the expression of perlecan, a heparan sulfate proteoglycan localized on the myoblast surface, is down-regulated during terminal differentiation of skeletal muscle myoblasts (Larraín et al. [1997] Exp. Cell Res. 234:405-412). In this study, we have evaluated the biochemical characteristics of perlecan, its association with the myoblast surface, and its involvement in C(2)C(12) myoblast adhesion to different substrates. Perlecan associated with myoblasts was solubilized by Triton X-100, whereas heparin, high salt, and RGD peptides were unable to solubilize perlecan. Pre-incubation of myoblasts with [(35)S]-Na(2)SO(4), followed by solubilization with Triton X-100 and immunoprecipitation with antibodies against murine perlecan, demonstrated that this proteoglycan present on the cell surface has a heterogeneous size profile with a K(av) value of 0.45, determined by Sepharose CL-4B chromatography. Myoblasts were found to adhere with decreasing affinities to collagen type IV, type I, laminin, fibronectin, perlecan, and matrigel. We found that cell adhesion to collagen type IV was inhibited by blocking this substrate with exogenous perlecan prior to cell plating, whereas no effect was observed for laminin. Furthermore, adhesion of myoblasts to collagen type IV was inhibited by the perlecan core protein obtained by treatment of perlecan with heparitinase, as well as by pre-incubation of the cells with antibodies against murine perlecan. These data support the idea that skeletal muscle cells interact with collagen type IV through the perlecan core protein present on the surface of undifferentiated myoblasts.
Assuntos
Colágeno/metabolismo , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/metabolismo , Músculo Esquelético/metabolismo , Proteoglicanas/metabolismo , Animais , Anticorpos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia por Troca Iônica , Combinação de Medicamentos , Fibronectinas/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/farmacologia , Laminina/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Camundongos , Músculo Esquelético/citologia , Proteoglicanas/química , Proteoglicanas/farmacologiaRESUMO
In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI).In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed
Assuntos
Animais , Masculino , Feminino , Medula Suprarrenal , Anticorpos , Células Cromafins/química , Gânglios Simpáticos/citologia , Gânglios Simpáticos/química , Neuropeptídeo Y , Serpentes , TirosinaRESUMO
In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI).In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed
Assuntos
Animais , Masculino , Feminino , Medula Suprarrenal/química , Medula Suprarrenal/citologia , Anticorpos , Células Cromafins/química , Gânglios Simpáticos/química , Gânglios Simpáticos/citologia , Neuropeptídeo Y , Serpentes/anatomia & histologia , TirosinaRESUMO
Galanin (GAL) and nitric oxide synthase (NOS) have been implicated in the control of thermogenesis in mammals. An experimental protocol was designed to determine whether or not the expression of these molecules in the hypothalamus of the toad could be related to environmental temperature changes. Exposure of the animals to low temperature increased the number and intensity of NOS-positive neurons in the magnocellular hypothalamic region, in contrast to a weak immunoreactivity observed in control animals kept in a natural environment at a spring-summer temperature (23-27 degrees C). Also a significantly higher number of GAL-immunoreactive (-IR) cells was observed in the preoptic area as compared to that observed in controls, while no difference in the intensity of GAL immunostaining intensity was detected. These results show a temperature-related expression of GAL and NOS in the hypothalamus and preoptic area of the toad. The results suggest a possible role of GAL and NOS in the regulation of hibernation in these animals.
Assuntos
Aclimatação/fisiologia , Bufo arenarum/metabolismo , Galanina/metabolismo , Hipotálamo/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Contagem de Células , Hipotálamo/citologia , Hipotálamo/enzimologia , Imuno-Histoquímica , Masculino , TemperaturaRESUMO
In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI). In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed.
Assuntos
Medula Suprarrenal/citologia , Células Cromafins/química , Gânglios Simpáticos/citologia , Neuropeptídeo Y/análise , Serpentes/anatomia & histologia , Tirosina/análise , Medula Suprarrenal/química , Medula Suprarrenal/inervação , Animais , Anticorpos , Feminino , Gânglios Simpáticos/química , Masculino , Neuropeptídeo Y/imunologia , Tirosina/imunologiaRESUMO
Cultured magnocellular neurons, isolated from adult rat supraoptic nuclei, were characterized by immunocytochemistry, using the avidin-biotin-peroxidase complex and antisera to vasopressin, oxytocin, galanin and cholecystokinin. Light microscope examination of the immunostained cultures revealed the presence of vasopressin- and oxytocin-like immunoreactivity, as well as neurons containing either galanin- or cholecystokinin-like immunoreactivity. In contrast, no significant galanin- or cholecystokinin-like immunoreactivity could be observed in freshly dispersed cells. Correlative scanning electron microscopical observations in the secondary electron imaging mode revealed that the stained neurons appeared significantly brighter than the unstained structures. Complementary observations with toad brain sections (preoptic area), immunostained for galanin, led to the same result. Considering previous results, it is suggested that the presence of galanin- and cholecystokinin-like immunoreactivity in the cultured neurons and its virtual absence in freshly dispersed cells is indicating a participation of these peptides in the regenerative processes taking place during culture. It is further concluded that the avidin-biotin-peroxidase method is suitable for correlative light and scanning electron microscopical studies of smooth surfaces and cultured cells.
Assuntos
Colecistocinina/análise , Galanina/análise , Neurônios/química , Núcleo Supraóptico/química , Animais , Células Cultivadas , Técnicas Imunoenzimáticas , Masculino , Microscopia , Microscopia Eletrônica de Varredura , Neurônios/citologia , Neurônios/ultraestrutura , Ocitocina/análise , Ratos , Núcleo Supraóptico/citologia , Núcleo Supraóptico/ultraestrutura , Vasopressinas/análiseRESUMO
The distribution of the dopaminergic D4 receptor in rat brain was studied employing site directed polyclonal antibodies. Antisera were raised in rabbits to two oligopeptides corresponding to amino acids 160-172 of the second extracellular loop (P1) and amino acids 260-273 of the third intracellular loop (P2) of the D4 receptor sequence. Affinity-purified antibodies (anti-P1 and anti-P2) specifically recognized two major bands of 42-45 and 95 kDa in Western blots of denatured preparations of various rat brain areas. Immunocyto-chemistry studies showed that D4 receptor is widely distributed in rat central nervous system (CNS) showing higher labelling in the hippocampus (CA1, CA2, CA3 and dentate gyrus) frontal cortex, entorhinal cortex, caudate putamen, nucleus accumbens, olfactory tubercle, cerebellum, supraoptic nucleus and sustancia nigra pars compacta. In addition, anti-P1 decreased the binding of the antagonist [3H]YM-09151-2 selective for D2, D3 and D4 receptors but did not modify the binding of [3H]raclopride an antagonist selective for D2 and D3, in striatal synaptosomes. Anti-P2 did not modify the binding of these ligands. These results confirm the selectivity of the antibodies towards the D4 receptor and suggest that the binding site for the antagonists might be located at or close to the second extracellular loop of the protein sequence. D4 receptor protein is mainly expressed in plasma membranes and in the peripheral cytoplasm of neurons and is more widely distributed than was originally proposed based on mRNA localization, since it is present both in limbic, diencephalic and motor areas of rat brain.
Assuntos
Encéfalo/citologia , Neurônios/citologia , Receptores de Dopamina D2/análise , Sequência de Aminoácidos , Animais , Anticorpos , Especificidade de Anticorpos , Encéfalo/metabolismo , Química Encefálica , Membrana Celular/metabolismo , Antagonistas de Dopamina/metabolismo , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Masculino , Neurônios/metabolismo , Especificidade de Órgãos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Coelhos , Racloprida , Ratos , Ratos Wistar , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D4 , Salicilamidas/metabolismo , Sinaptossomos/metabolismoRESUMO
The aim of this work was to investigate the possible presence of DOPA decarboxylase (DDC) in endocrine cells of adult rat pancreas. Islet peptide hormones (insulin, glucagon, and somatostatin), as well as DDC, were detected immunohistochemically using the double-immunofluorescence technique and specific antibodies. DDC-like immunoreactivity was present in cytoplasmic granules within endocrine cells located at islet peripheries in a distribution consistent with islet localisation of A cells. Moreover, these same cells stained positively with glucagon antibody. As DDC is an enzyme specifically involved in catecholamine synthesis, insular cells must possess the capacity to elaborate this class of hormone at least up to the dopamine-decarboxylation step. Thus, after further metabolic processing either in A cells or elsewhere, endogenously-synthesised islet catecholamines may be released and participate in paracrine regulation of insulin secretion.
Assuntos
Dopa Descarboxilase/análise , Ilhotas Pancreáticas/enzimologia , Animais , Imunofluorescência , Glucagon/análise , Imuno-Histoquímica , Ilhotas Pancreáticas/citologia , Ratos , Ratos Sprague-Dawley , Somatostatina/análiseRESUMO
The expression of the neuropeptide galanin was analyzed by immunohistochemistry in magnocellular and preoptic hypothalamic neurons of toads following hypophysectomy (HPX) and pars distalectomy (PDX). There was a marked increase in the galanin-like immunoreactive expression in magnocellular hypothalamic cells 3 days after HPX, followed by a decrease to normal levels after 7 days. No changes in the expression of galanin were detected after PDX in these neurons when compared to controls. Moreover, 7 days after HPX or PDX the number of cells expressing galanin was significantly increased in the preoptic area, where numerous intraependymal cells were intensely immunoreactive. The hypophysis grafts into the hind limb in HPX or PDX animals prevented increased galanin-like immunoreactivity in preoptic cells but not in magnocellular neurons. Similarly, PDX toads given growth hormone showed no GAL-LI in the intraependymal preoptic cells. These results suggest the presence of a region regulation of galanin expression in the preoptic area by hypophyseal hormones, in particular growth hormone.
Assuntos
Bufo arenarum , Galanina/metabolismo , Hormônio do Crescimento/farmacologia , Hipofisectomia , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Animais , Masculino , Neurônios/metabolismo , Área Pré-Óptica/metabolismoRESUMO
A simple and efficient procedure for the dispersion and culture of magnocellular neurons of the adult rat hypothalamus was developed. The enzymatically and mechanically dispersed cells were highly viable and showed neurite outgrowth after 3 days of culture. The neurons could be maintained for more than 4 weeks without any sign of deterioration. Light-optic immunocytochemistry of the cultured cells revealed the presence of many oxytocin- and vasopressin-immunoreactive neurons. The results indicate that the method may be suitable for many experimental purposes.
Assuntos
Técnicas Citológicas , Neurônios/citologia , Núcleo Supraóptico/citologia , Animais , Comunicação Celular , Sobrevivência Celular , Células Cultivadas , Imuno-Histoquímica , Masculino , Neurônios/fisiologia , Ocitocina/metabolismo , Ratos , Ratos Endogâmicos , Vasopressinas/metabolismoRESUMO
The distribution of galanin (GAL)-like immunoreactivity (-LI) was studied in the CNS of the toad (Bufo arenarum Hensel). Tissue sections were incubated with antibodies directed toward rat or porcine GAL and processed either for the avidin-biotin complex, or for the indirect immunofluorescence techniques. In the telencephalon GAL-immunoreactive (-IR) perikarya were observed in the ventral part of the striatum and in the septal accumbens nuclei. Immunopositive neurons were also observed in the medial amigdala with some intermingled cells between the fibers of the anterior commissure. Numerous GAL-IR perikarya were present along the rostrocaudal medial preoptic nucleus. Occasionally lightly immunoreactive cells were detected in the magnocellular region. The most numerous accumulation of GAL-IR cells was present in the ventral hypothalamus around the infundibular region, in the posterior tubercle and in the nucleus of the paraventricular organ. Immunostained cells were also present in the pretectal gray, solitary nucleus, gracil nucleus and in the spinal cord in the intermediate gray and in large motoneurons of the ventral horn. The widespread distribution found of GAL-LI suggests that GAL in the toad, as well as in mammalian species, may serve a variety of functions with a preponderant role in neuroendocrine processes. A role for GAL as a trophic factor in the brain of the toad is also suggested.
Assuntos
Encéfalo/metabolismo , Bufonidae/metabolismo , Galanina/metabolismo , Animais , Mapeamento Encefálico , Galanina/imunologia , Técnicas Imunológicas , Masculino , Neurônios/metabolismoRESUMO
The presence and distribution of peptidergic nerve fibers were studied in the testis and mesorchium of the toad by means of immunohistochemistry. Cryostat sections of the testis and whole-mount preparations of mesorchia were immunostained with antisera to calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY). After leaving the mesorchium CGRP-immunoreactive (IR) fibers were seen predominantly running in between the seminiferous tubules. In addition, a small population of CGRP-IR nerve fibers formed thin plexuses around blood vessels. Conversely, NPY-like immunoreactivity predominated in nerve fibers that formed dense plexuses around vessels both in the mesorchium and testis. Additionally, some single NPY-IR nerve fibers could be seen in both structures studied. The functional significance of these peptidergic systems in the testis of the toad remains to be analyzed.