| BackgroundVertigo is a common clinical symptom and is a moveability or positional illusion which is caused by a balanced system dysfunction. Vertebrobasilar insufficient is one of the most common reason of senile vertigo. Vertebrobasilar insufficiency (VBI) refers to a temporary set of symptoms due to decreased blood flow in the posterior circulation of the brain. VBI is considered with varied clinical symptoms, including vertigo, vision disorders, hearing loss and balance disorders, etc. However, about how ion channel attributes to the medial vestibular nucleus (MVN) neuronal activity in VBI. Vertebrobasilar insufficient vertigo is the first and the most common clinical manifestations symptoms. It is of high incidence, onset rapid and complex causes and may seriously affect the work and the life quality of patients. The diagnosis and treatment of the disease has been confused with Clinical doctors. It has become an important topic in the field of clinical interventions and scientific research. At present,although there are many methods for dizziness, but poor curative effect. The reason may still a lack of a through study on the exact mechanism of vertigo. Little is known, however,about how ion channel attributes to the vestibular nucleus neuronal activity in VBI. Therefore, to further explore the neural mechanism of vertigo is the key to improve the effect of treatment.Vestibular system is one of the important part of the body’s balance system, and plays a key role in processing of neural signals related to gaze and postural stabilization. Vestibular Nuclear complex is the largest of the brain stem nuclei, and it is the relay station of vestibular system and the main station in vestibular signal. In the vestibular nuclear complex, medial vestibular nucleush as a unique anatomical features, and can play an important role in nerve electrophysiology. Thus it becomes the preferred nuclei with vertigo mechanism study. It is particularly sensitive to hypoxia or ischemia stimulation. The functions of the vestibular system are closely related to the vestibular nuclei neurons electrophysiological characteristics. However, the mechanisms underlying MVN neurons abnormal firing in hypoxia has yet to be determined. It has important practical significance to clarify vertigo disease mechanism by further study of its electrophysiological regulatory mechanism.Large conductance Ca2+activated K+channels (BKCa channels) are widely found in a variety of excitable cells and show a high density of its expression. It has high conductivity, strong voltage dependence, high sensitivity to Ca+and the unique character of pharmacological effects, thus plays an important role in biological systems. Dysfunction of BKCa channel can cause hearing loss, hypertension, asthma and brain ischemia, and it has potential clinical significance in some diseases of drug targets. BKCa channel is the key control components of the cell membrane potential. They are activated by both intracellular calcium increased and membrane potential depolarization. BKCa channels are a key determinant of neuronal excitability via their effects on action potential shape and firing rate. Reduction of BKCa channels can cause neuronal hyper excitability. MVN neurons fire both spontaneously and evoked action potentials. BKCa channels activation may serve as the "emergency brake" to protect neurons via inhibiting membrane depolarization and a large calcium influx. On the other hand, BKCa channels inhibitor may act as a neuro protective agent via decreasing excessive potassium efflux. BKCa channel a subunit has its uniqueness of the structure and function, the BKCa functionsare closely tied to the expression of a subunit. However, it remains unclear of the changes of BKCa channels and BKCa channel a subunit in MVN neurons following hypoxia.BKCa channel also had a key influence on the nerve cell electrophysiological activity followed by hypox and ischemic injury. Hypoxia and ischemic injury are both characterized by reduced oxygen availability in the tissue, which is detrimental for the brain because of its high metabolic rate. Sodium channels, Ca2+channels, small conductance Ca2+-activated K+channels (SK channels)and large conductance Ca2+-activated K+channels (BK channels) have been found in the MVN neurons. However, it is not clear whether BKCa channel regulation can be used as MVN nerve protective mechanism against hypoxic damage. Therefore, it is particularly urgent to discuss BKCa contribute to neuronal abnormal firing in the medial vestibular nucleus following hypoxia.To address this issue, we performed whole-cell patch clamp recording, quantitative polymerase chain reaction and immunohistochemistry to explore the changes of MVN neuronal activity by hypoxia, and the contribution of BKCa channels. The study will further reveals the treatment of vertebral-basilar artery insufficiency vertigo providing theoretical basis, and it will provide new ideas for effective prevention and control and new drug targets.ObjectiveThis study analyzes the BK channel current changes in the medial vestibular nucleus following hypoxia. It is aimed at exploring BKCa channel on electrophysiology excitatory regulation in MVN neuron following hypoxia. It was to clarify whether BKCa participate in nerve protective effect of MVN neuron followed hypoxia and to reveal its molecular mechanism.MethodsExperiments were performed on6to9-week-old male C57BL/6mice. Brief hypoxic stimuli (upon to10min) of the brain slices containing MVN were administrated by switching the normoxic artificial cerebrospinal fluid (ACSF) equilibrated with21%O2/5%CO2to hypoxic ACSF equilibrated with5%O2/5%CO2(balance N2). First of all to build MVNs hypoxia mice model. 1. Using immunohistochemical technique, we detect the changes of c-Fos in the hypoxic MVNs and the normoxia MVNs.2. Using immunohistochemical technique, we detect the changes of BKCa channel a subunit in the hypoxic MVNs and the normoxia MVNs.3. Using whole cell patch clamp experiment technology, we record the effect of hypoxia to the MVN abnormal neuronal firing.4. To determine whether BKCa channels attribute to hypoxia-induced MVN abnormal neuronal firing, NS1619, a selective opener of BKCa channel was used.5. To determine whether hypoxia affects BKCa channels in MVN neurons, IBTX (iberiotoxin), a selective BKCa blocker was used to test the change of the BKCa currents in MVN neurons.6. Using semi-quantitative PCR technique to detect BKCa mRNA level in MVN neurons.All data used SPSS17.0statistical software. The significant level of α=0.05, with P<0.05as the difference has statistical significance.Results1Changes in c-Fos immunoreactivity in the hypoxic MVNsC-Fos expression in the MVNs was observed in the normoxic and hypoxic group. C-Fos-positive nuclei were rarely observed in the MVNs from normoxic mice. By contrast, abundant c-Fos-positive nuclei were found in the MVNs from hypoxic mice. C-Fos expression was significantly increased in the MVNs after1hr hypoxia exposure (P<0.05)2Changes in BKCa channel a subunit immunoreactivity in the hypoxic MVNsThe expression of the BKCa channel a subunit were observed in the normoxic and hypoxic group in the MVNs.Under control conditions (21%O2), the greatest number of BKCa channel a subunit-positive cells was found in MVNs. Treated by hypoxic (5%O2) inhalation for1h caused a significance decrease in the number of positive cells.3Effects of hypoxia on MVN neuronal firingHypoxia (5%O2) can effect the abnormal neuronal firing in MVN neurons. The firing rate spontaneous firing MVN neurons showed an initial increase by brief hypoxia, followed by a decrease in all tested neurons and even disappearance in MVN neurons after10min exposure to hypoxia. And the resting membrane potential of this MVN neuron depolarized were observed after10min exposure to hypoxia.4Effects of BK channel activation on hypoxia-induced MVN abnormal neuronal firingNS1619pre-incubation postponed the hypoxia-induced MVN abnormal neuronal firing. After pre-incubation with10μM NS1619for2-4h,10-min-exposure to hypoxia (5%O2) depolarized the typical MVN neuron. NS1619pre-incubation postponed the maximum increase by hypoxia (P<0.05). NS1619pre-incubation decreased3-min-hypoxia-induced firing rate increase (P<0.05), while not10-min-hypoxia-induced changes of firing rate (P>0.05). NS1619pre-incubation alleviated10-min-hypoxia-induced depolarization (P<0.05), while not3-min-hypoxia-induced depolarization (P>0.05).5Changes in BKCa currents in the hypoxic MVN neurons10-min-perfusion with hypoxic ACSF significantly decreased (P<0.05) the total K+outward current in MVN neurons, compared with that in normoxic MVN neurons. And BKCa current was significantly reducedin MVN neurons by hypoxia, compared with that in normoxic MVN neurons (P<0.05).6qPCR analysis of BKCa mRNA level in the hypoxic MVN neuronsRelative BKCa mRNA levels showed a significant decrease in MVNs incubated with normoxic ACSF compared with those incubated with10-min hypoxic ACSF (P<0.05). ConclusionsThese findings suggest that acute hypoxia increases neuronal activity. Our data on MVN neuronal activity demonstrated that BKCa channel expression and activity is decreased in MVNs following hypoxia, and decreased MVN BKCa channels contribute to hypoxia-induced abnormal neuronal activity.The present study is one of the first to indicate that BKCa channels are likely responsible for the brief increase in MVN neuronal activity by hypoxia and to to indicate the neuroprotection role of BKCa activation during hypoxia, suggesting the potential target for treatment hearing loss and vertigo in VBI. The study will further provide new ideas and reveal the effective prevention and control of new drug targets. |
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