BACKGROUND： Nikethamide, a respiratory center stimulant, is widely used in China. However, its effects on the central nervous system and medullary respiratory center remain poorly understood. OBJECTIVE： To investiga...BACKGROUND： Nikethamide, a respiratory center stimulant, is widely used in China. However, its effects on the central nervous system and medullary respiratory center remain poorly understood. OBJECTIVE： To investigate the influence of nikethamide on inspiratory neuron discharge in the medial region of the nucleus retrofacialis in neonatal rats, based on the observations addressing rhythmic respiratory discharge generated by the basic medullary respiratory center and various respiration neuron discharges in brain slices. DESIGN, TIME AND SETTING： A controlled, observational study utilizing in vitro neuroelectrophysiology was performed at the Department of Physiology in Southern Medical University between September and December in 2007. MATERIALS： Nikethamide was purchased from Sigma, USA; BL-420E biological signal collection and manaclement system was provided by Chengdu TME Technology, China.METHODS： Isolated medulla-spinal cord preparations were collected from neonatal Sprague Dawley rats, aged 1-3 days. Tissues were divided to include the medial region of the nucleus retrofacialis, ventral respiratory, and dorsal respiratory groups. Subsequently, modified Kreb＇s solution and 5 μg/mL nikethamide-containing modified Kreb＇s solution were consecutively perfused into the medial region of the nucleus retrofacialis in neonatal rat brain slices. MAIN OUTCOME MEASURES： Hypoglossal nerve root respiratory-related rhythmic discharge activities and inspiratory neuron discharges were recorded with an adsorption electrode and microelectrode. RESULTS Nikethamide resulted in prolonged inspiratory neuron discharge time, shortened respiratory cycle and expiratory time. Nikethamide intervention resulted in enhanced integral amplitude of some inspiratory neurons with no changes in discharge frequency or increased discharge frequency in remaining inspiratory neurons with no changes in integral amplitude. CONCLUSION： Nikethamide excites inspiratory neurons in the basic rhythmic respiration and medullary respiratory center, in addition to increased inspiratory neuron and neural network excitability.展开更多
BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors...BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome. OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006. MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）. METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days. MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure. RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles. CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.展开更多
The electrical excitability of neural networks is influenced by different environmental factors. Effective and simple methods are required to objectively and quantitatively evaluate the influence of such factors, incl...The electrical excitability of neural networks is influenced by different environmental factors. Effective and simple methods are required to objectively and quantitatively evaluate the influence of such factors, including variations in temperature and pharmaceutical dosage. The aim of this paper was to introduce ‘the voltage threshold measurement method＇, which is a new method using microelectrode arrays that can quantitatively evaluate the influence of different factors on the electrical excitability of neural networks. We sought to verify the feasibility and efficacy of the method by studying the effects of acetylcholine, ethanol, and temperature on hippocampal neuronal networks and hippocampal brain slices. First, we determined the voltage of the stimulation pulse signal that elicited action potentials in the two types of neural networks under normal conditions. Second, we obtained the voltage thresholds for the two types of neural networks under different concentrations of acetylcholine, ethanol, and different temperatures. Finally, we obtained the relationship between voltage threshold and the three influential factors. Our results indicated that the normal voltage thresholds of the hippocampal neuronal network and hippocampal slice preparation were 56 and 31 m V, respectively. The voltage thresholds of the two types of neural networks were inversely proportional to acetylcholine concentration, and had an exponential dependency on ethanol concentration. The curves of the voltage threshold and the temperature of the medium for the two types of neural networks were U-shaped. The hippocampal neuronal network and hippocampal slice preparations lost their excitability when the temperature of the medium decreased below 34 and 33°C or increased above 42 and 43°C, respectively. These results demonstrate that the voltage threshold measurement method is effective and simple for examining the performance/excitability of neuronal networks.展开更多
基金the National Natural Science Foundation of China,No.30570670the Natural Science Foundation of Guangdong Province,No.5004714
文摘BACKGROUND： Nikethamide, a respiratory center stimulant, is widely used in China. However, its effects on the central nervous system and medullary respiratory center remain poorly understood. OBJECTIVE： To investigate the influence of nikethamide on inspiratory neuron discharge in the medial region of the nucleus retrofacialis in neonatal rats, based on the observations addressing rhythmic respiratory discharge generated by the basic medullary respiratory center and various respiration neuron discharges in brain slices. DESIGN, TIME AND SETTING： A controlled, observational study utilizing in vitro neuroelectrophysiology was performed at the Department of Physiology in Southern Medical University between September and December in 2007. MATERIALS： Nikethamide was purchased from Sigma, USA; BL-420E biological signal collection and manaclement system was provided by Chengdu TME Technology, China.METHODS： Isolated medulla-spinal cord preparations were collected from neonatal Sprague Dawley rats, aged 1-3 days. Tissues were divided to include the medial region of the nucleus retrofacialis, ventral respiratory, and dorsal respiratory groups. Subsequently, modified Kreb＇s solution and 5 μg/mL nikethamide-containing modified Kreb＇s solution were consecutively perfused into the medial region of the nucleus retrofacialis in neonatal rat brain slices. MAIN OUTCOME MEASURES： Hypoglossal nerve root respiratory-related rhythmic discharge activities and inspiratory neuron discharges were recorded with an adsorption electrode and microelectrode. RESULTS Nikethamide resulted in prolonged inspiratory neuron discharge time, shortened respiratory cycle and expiratory time. Nikethamide intervention resulted in enhanced integral amplitude of some inspiratory neurons with no changes in discharge frequency or increased discharge frequency in remaining inspiratory neurons with no changes in integral amplitude. CONCLUSION： Nikethamide excites inspiratory neurons in the basic rhythmic respiration and medullary respiratory center, in addition to increased inspiratory neuron and neural network excitability.
文摘BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome. OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006. MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）. METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days. MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure. RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles. CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.
基金supported by the National Natural Sciences Foundation of China,No.61534003,61076118the Innovation Foundation for State Key Laboratory of the Ministry of Science and Technology,China,No.2016-2018a grant from the Open Projects of Key Laboratory of Child Development and Learning of the Ministry of Education of China,No.CDLS201205
文摘The electrical excitability of neural networks is influenced by different environmental factors. Effective and simple methods are required to objectively and quantitatively evaluate the influence of such factors, including variations in temperature and pharmaceutical dosage. The aim of this paper was to introduce ‘the voltage threshold measurement method＇, which is a new method using microelectrode arrays that can quantitatively evaluate the influence of different factors on the electrical excitability of neural networks. We sought to verify the feasibility and efficacy of the method by studying the effects of acetylcholine, ethanol, and temperature on hippocampal neuronal networks and hippocampal brain slices. First, we determined the voltage of the stimulation pulse signal that elicited action potentials in the two types of neural networks under normal conditions. Second, we obtained the voltage thresholds for the two types of neural networks under different concentrations of acetylcholine, ethanol, and different temperatures. Finally, we obtained the relationship between voltage threshold and the three influential factors. Our results indicated that the normal voltage thresholds of the hippocampal neuronal network and hippocampal slice preparation were 56 and 31 m V, respectively. The voltage thresholds of the two types of neural networks were inversely proportional to acetylcholine concentration, and had an exponential dependency on ethanol concentration. The curves of the voltage threshold and the temperature of the medium for the two types of neural networks were U-shaped. The hippocampal neuronal network and hippocampal slice preparations lost their excitability when the temperature of the medium decreased below 34 and 33°C or increased above 42 and 43°C, respectively. These results demonstrate that the voltage threshold measurement method is effective and simple for examining the performance/excitability of neuronal networks.
