RESUMO
The purpose of the present study was to investigate balance alterations and the possible role of the cholinergic neurons in the pedunculopontine nucleus (PPN) in the early stages of a progressive animal model of Parkinson's disease (PD). Twenty-eight middle-aged (8-9 months) male Wistar rats received 4 or 10 subcutaneous vehicle (control, CTL) or reserpine (RES) injections (0.1 mg/kg). The animals were submitted to different behavioral tests. Forty-eight hours after the 4th injection, half of the animals of each group (n = 7) were perfused and submitted to immunohistochemical analysis for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT). The remaining animals (n = 7 per group) were killed 48 h after the 10th injection. RES group presented motor deficits in the catalepsy and open field tests starting at days 12 and 20 of treatment, respectively (only for the animals that received 10 injections). On the other hand, dynamic and static balance changes were observed at earlier stages of RES treatment, starting at days 6 and 4, respectively. At this point of the treatment, there was no decrease in the number of TH immunoreactivity neurons in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and dorsal striatum (DS). However, a decrease was observed in SNpc and dorsal striatum of animals that received 10 injections. In contrast, there was a decrease in the number of ChAT immunoreactive cells in PPN concomitantly to the balance alterations at the early stages of treatment (after 4 RES injections). Thus, by mimicking the progressiveness of PD, the reserpine model made it possible to identify static and dynamic balance impairments prior to the motor alterations in the catalepsy and open field tests. In addition, changes in balance were accompanied by a reduction in the number of ChAT immunoreactive cells in NPP in the early stages of treatment.
Assuntos
Transtornos Parkinsonianos , Animais , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/metabolismo , Masculino , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/tratamento farmacológico , Ratos , Ratos Wistar , Substância Negra/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The cholinergic system is present in both bacteria and mammals and regulates inflammation during bacterial respiratory infections through neuronal and non-neuronal production of acetylcholine (ACh) and its receptors. However, the presence of this system during the immunopathogenesis of pulmonary tuberculosis (TB) in vivo and in its causative agent Mycobacterium tuberculosis (Mtb) has not been studied. Therefore, we used an experimental model of progressive pulmonary TB in BALB/c mice to quantify pulmonary ACh using high-performance liquid chromatography during the course of the disease. In addition, we performed immunohistochemistry in lung tissue to determine the cellular expression of cholinergic system components, and then administered nicotinic receptor (nAChR) antagonists to validate their effect on lung bacterial burden, inflammation, and pro-inflammatory cytokines. Finally, we subjected Mtb cultures to colorimetric analysis to reveal the production of ACh and the effect of ACh and nAChR antagonists on Mtb growth. Our results show high concentrations of ACh and expression of its synthesizing enzyme choline acetyltransferase (ChAT) during early infection in lung epithelial cells and macrophages. During late progressive TB, lung ACh upregulation was even higher and coincided with ChAT and α7 nAChR subunit expression in immune cells. Moreover, the administration of nAChR antagonists increased pro-inflammatory cytokines, reduced bacillary loads and synergized with antibiotic therapy in multidrug resistant TB. Finally, in vitro studies revealed that the bacteria is capable of producing nanomolar concentrations of ACh in liquid culture. In addition, the administration of ACh and nicotinic antagonists to Mtb cultures induced or inhibited bacterial proliferation, respectively. These results suggest that Mtb possesses a cholinergic system and upregulates the lung non-neuronal cholinergic system, particularly during late progressive TB. The upregulation of the cholinergic system during infection could aid both bacterial growth and immunomodulation within the lung to favor disease progression. Furthermore, the therapeutic efficacy of modulating this system suggests that it could be a target for treating the disease.
Assuntos
Sistema Colinérgico não Neuronal/fisiologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/patologia , Acetilcolina/metabolismo , Animais , Colina O-Acetiltransferase/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Inflamação/metabolismo , Inflamação/patologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Antagonistas Nicotínicos/farmacologia , Sistema Colinérgico não Neuronal/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Regulação para Cima/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Barckground Alzheimer's disease (AD) is mainly caused by cellular loss and dysfunction of the basal forebrain cholinergic neurons and cholinergic axons in the cortex leading to slowly progressive decline in learning and memory performance. Unfortunately, no definitive treatment to halt neural cell loss exists to date. Therefore, it is necessary to obtain an unlimited source of cholinergic neurons for future pharmacological applications in AD. Human mesenchymal stromal cells (hMSCs) represent a unique source of cholinergic-like neurons (ChLNs). New method hWJ-MSCs were incubated with Cholinergic-N-Run medium for 4 and 7 days. Results hWJ-MSCs cultured with Cholinergic-N-Run medium differentiated into ChLNs in 4 days as evidenced by high levels of protein expression of the neuronal markers ChAT, VAChT, AChE, MAP2, ß-Tubulin III, NeuN, TUC-4, NF-L and no expression of the immature marker SOX2, the dopaminergic marker TH, GABAergic marker GAD67 and glial marker GFAP. Comparison with existing method(s) The hWJ-MSCs form ChLNs (e.g., â¼26% IF+) within 20 days by using complex conditioned mediums that are expensive and time-consuming. We report for the first time, to our best knowledge, a direct method of hWJ-MSCs transdifferentiation into ChLNs (â¼76% ChAT /VAChT assessed by immunofluorescence microscopy and flow cytometry) in an economic, efficient and timely fashion. Conclusions The fastest method to obtain ChLNs from hWJ-MSCs takes only four days using the one-step incubation medium Cholinergic-N-Run.
Assuntos
Técnicas de Cultura de Células/métodos , Transdiferenciação Celular , Neurônios Colinérgicos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/metabolismo , Meios de Cultura , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Regulação para CimaRESUMO
The aim of this study was to conduct cytoarchitectonic studies and choline acetyltransferase (ChAT) immunohistochemical analysis to delimit the cholinergic groups in the encephalon of the rock cavy (Kerodon rupestris), a crepuscular Caviidae rodent native to the Brazilian Northeast. Three young adult animals were anesthetized and transcardially perfused. The encephala were cut in the coronal plane using a cryostat. We obtained 6 series of 30-µm-thick sections. The sections from one series were subjected to Nissl staining. Those from another series were subjected to immunohistochemistry for the enzyme ChAT, which is used in acetylcholine synthesis, to visualize the different cholinergic neural centers of the rock cavy. The slides were analyzed using a light microscope and the results were documented by description and digital photomicrographs. ChAT-immunoreactive neurons were identified in the telencephalon (nucleus accumbens, caudate-putamen, globus pallidus, entopeduncular nucleus and ventral globus pallidus, olfactory tubercle and islands of Calleja, diagonal band of Broca nucleus, nucleus basalis, and medial septal nucleus), diencephalon (ventrolateral preoptic, hypothalamic ventrolateral, and medial habenular nuclei), and brainstem (parabigeminal, laterodorsal tegmental, and pedunculopontine tegmental nuclei). These findings are discussed through both a functional and phylogenetic perspective.
Assuntos
Encéfalo/citologia , Neurônios Colinérgicos/citologia , Animais , Encéfalo/metabolismo , Forma Celular/fisiologia , Colina O-Acetiltransferase/metabolismo , Neurônios Colinérgicos/metabolismo , Feminino , Imuno-Histoquímica , Masculino , RoedoresRESUMO
The aim of this study was to verify the effects of ovariectomy (OVX) and/or vitamin D supplementation (VIT D) on inflammatory and cholinergic parameters in hippocampus, as well as on serum estradiol and VIT D levels of rats. Ninety-day-old female Wistar rats were randomly divided into four groups: SHAM, OVX, VIT D or OVX + VIT D. Thirty days after OVX, VIT D (500 IU/kg/day) was supplemented by gavage, for 30 days. Approximately 12 h after the last VIT D administration, rats were euthanized and hippocampus and serum were obtained for further analyses. Results showed that OVX rats presented a decrease in estradiol levels when compared to control (SHAM). There was an increase in VIT D levels in the groups submitted to VIT D supplementation. OVX increased the immunocontent of nuclear p-NF-κB/p65, TNF-α and IL-6 levels. VIT D partially reversed the increase in p-NF-κB/p65 immunocontent and IL-6 levels. Regarding cholinergic system, OVX caused an increase in acetylcholinesterase activity without changing acetylcholinesterase and choline acetyltransferase immunocontents. VIT D did not reverse the increase in acetylcholinesterase activity caused by OVX. These results demonstrate that OVX alters inflammatory and cholinergic parameters and that VIT D supplementation, at the dose used, partially reversed the increase in immunocontent of p-NF-Kb/p65 and IL-6 levels, but it was not able to reverse other parameters studied. Our findings may help in the understanding of the brain changes that occurs in post menopause period and open perspectives for futures research involving VIT D therapies.
Assuntos
Acetilcolinesterase/metabolismo , Hipocampo/efeitos dos fármacos , Interleucina-6/metabolismo , Fator de Transcrição RelA/metabolismo , Vitamina D/farmacologia , Análise de Variância , Animais , Peso Corporal/efeitos dos fármacos , Calcifediol/sangue , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citocinas/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Suplementos Nutricionais , Ingestão de Alimentos/efeitos dos fármacos , Estradiol/sangue , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Ovariectomia , Ratos , Ratos WistarRESUMO
Tyrosinemia type II is an inborn error of metabolism caused by a deficiency in the activity of the enzyme tyrosine aminotransferase, leading to tyrosine accumulation in the body. Although the mechanisms involved are still poorly understood, several studies have showed that higher levels of tyrosine are related to oxidative stress and therefore may affect the cholinergic system. Thus, the aim of this study was to investigate the effects of chronic administration of L-tyrosine on choline acetyltransferase activity (ChAT) and acetylcholinesterase (AChE) in the brain of rats. Moreover, we also examined the effects of one antioxidant treatment (N-acetylcysteine (NAC) + deferoxamine (DFX)) on cholinergic system. Our results showed that the chronic administration of L-tyrosine decreases the ChAT activity in the cerebral cortex, while the AChE activity was increased in the hippocampus, striatum, and cerebral cortex. Moreover, we found that the antioxidant treatment was able to prevent the decrease in the ChAT activity in the cerebral cortex. However, the increase in AChE activity induced by L-tyrosine was partially prevented the in the hippocampus and striatum, but not in the cerebral cortex. Our results also showed no differences in the aversive and spatial memory after chronic administration of L-tyrosine. In conclusion, the results of this study demonstrated an increase in AChE activity in the hippocampus, striatum, and cerebral cortex and an increase of ChAT in the cerebral cortex, without cognitive impairment. Furthermore, the alterations in the cholinergic system were partially prevented by the co-administration of NAC and DFX. Thus, the restored central cholinergic system by antioxidant treatment further supports the view that oxidative stress may be involved in the pathophysiology of tyrosinemia type II.
Assuntos
Acetilcolinesterase/metabolismo , Antioxidantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Colina O-Acetiltransferase/metabolismo , Tirosina/toxicidade , Acetilcisteína/farmacologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Desferroxamina/farmacologia , Masculino , Memória/efeitos dos fármacos , Memória/fisiologia , Fármacos Neuroprotetores/farmacologia , Ratos WistarRESUMO
The recombinant C-terminal domain of tetanus toxin (Hc-TeTx) is a new non-toxic peptide of the tetanus toxin that exerts a protective action against glutamate excitotoxicity in motoneurons. Moreover, its efficacy as a neuroprotective agent has been demonstrated in several animal models of neurodegeneration. The eleven amino acids in the ß amyloid peptide (Aß25-35) mimic the toxic effects of the full ß amyloid peptide (Aß1-42), causing the impairment of the cholinergic system in the medial septum (MS) which, in turn, alters the septo-hippocampal pathway and leads to learning and memory impairments. The aim of this study was to examine the neuroprotective effects of the Hc-TeTx fragment against cholinotoxicity. The Hc-TeTx fragment (100 ng) was injected into the rats intercranially, with the Aß(25-35) (2 µg) then injected into their MS. The animals were tested for spatial learning and memory in the eight-arm radial maze. The brains were removed to assess cholinergic markers, such as choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and to explore neurodegeneration in the MS and hippocampus, using amino-cupric silver and H&E staining. Finally, capase-3, a marker of apoptosis, was examined in the MS. Our results clearly demonstrate that the application of Hc-TeTx prevents the loss of cholinergic markers (ChAT and AChE), the activation of capase-3, and neurodegeneration in the MS and the CA1 and CA3 subfields of the hippocampus. All these improvements were reflected in spatial learning and memory performance, and were significantly higher compared with animals treated with Aß(25-35). Interestingly, the single administration of Hc-TeTx into the MS modified the ChAT and AChE expression that affect cognitive processes, without inducing neurodegeneration or an increase in capase-3 expression in the MS and hippocampus. In summary, our findings suggest that the recombinant Hc-TeTx fragment offers effective protection for the septo-hippocampal pathway, given that it reduces the neurodegeneration caused by Aß(25-35) and improves learning and memory processes.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/farmacologia , Toxina Tetânica/farmacologia , Acetilcolinesterase/metabolismo , Animais , Caspase 3/metabolismo , Colina O-Acetiltransferase/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Nootrópicos/farmacologia , Fragmentos de Peptídeos/toxicidade , Distribuição Aleatória , Ratos Wistar , Septo do Cérebro/efeitos dos fármacos , Septo do Cérebro/metabolismo , Septo do Cérebro/patologia , Memória Espacial/efeitos dos fármacos , Memória Espacial/fisiologiaRESUMO
OBJECTIVES: (1) To describe the morphology of paravaginal ganglia and the neurochemical pattern of their neurons in virgin rabbits. (2) To analyze the effects of multiparity, primiparity and late pregnancy on morphometry of ganglia and their neurons. STUDY DESIGN: The morphology and neurochemical pattern of paravaginal ganglia were described in virgin nulliparous Chinchilla breed rabbits. Acetylcholinesterase histochemistry, Masson's trichrome, and immunohistochemistry for cholinergic and adrenergic neurons, and estrogen receptors (ERs) were undertaken. The area covered by ganglia (ganglionic area), the number of neurons, and the neuron soma area were measured in multiparas (4 consecutive and successive deliveries) and age-matched nulliparas. The same variables were measured in primiparous, late-pregnant, and age-matched nulliparous rabbits. RESULTS: Paravaginal ganglia were adjoined to the dorsolateral walls of the pelvic vagina. Their neurons were mostly cholinergic and expressed ERα and ERß. Multiparity increased the ganglionic area and reduced the number of ganglionic neurons. Late pregnancy transiently reduced the neuron soma area, which was coincident with a low expression of ERs. CONCLUSION: Multiparity but not primiparity affected the morphometry of ganglia. The hormonal state present in late pregnancy alters the neuron soma area and ER expression. Our findings support the notion that reproduction influences the morphometry of the pelvic plexus.
Assuntos
Gânglios/anatomia & histologia , Paridade , Parto , Vagina/inervação , Animais , Feminino , Gânglios/fisiologia , Gravidez , CoelhosRESUMO
Numerous functions have been attributed to the Edinger-Westphal nucleus (EW), including those related to feeding behavior, pain control, alcohol consumption and the stress response. The EW is thought to consist of two parts: one controls accommodation, choroidal blood flow and pupillary constriction, primarily comprising cholinergic cells and projecting to the ciliary ganglion; and the other would be involved in the non-ocular functions mentioned above, comprising peptide-producing neurons and projecting to the brainstem, spinal cord and prosencephalic regions. Despite the fact that the EW is well known, its connections have yet to be described in detail. The aim of this work was to produce a map of the hypothalamic sources of afferents to the EW in the rat. We injected the retrograde tracer Fluoro-Gold into the EW, and using biotinylated dextran amine, injected into afferent sources as the anterograde control. We found retrogradely labeled cells in the following regions: subfornical organ, paraventricular hypothalamic nucleus, arcuate nucleus, lateral hypothalamic area, zona incerta, posterior hypothalamic nucleus, medial vestibular nucleus and cerebellar interpositus nucleus. After injecting BDA into the paraventricular hypothalamic nucleus, lateral hypothalamic area and posterior hypothalamic nucleus, we found anterogradely labeled fibers in close apposition to and potential synaptic contact with urocortin 1-immunoreactive cells in the EW. On the basis of our findings, we can suggest that the connections between the EW and the hypothalamic nuclei are involved in controlling stress responses and feeding behavior.
Assuntos
Vias Aferentes/anatomia & histologia , Tronco Encefálico/anatomia & histologia , Hipotálamo/anatomia & histologia , Animais , Imuno-Histoquímica , Masculino , Ratos , Ratos Long-EvansRESUMO
Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer's Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels.