The Dynamic Changes Of Voltage-gated Sodium Channel And The Role Of Neuroglia In Salicylate-induced Tinnitus

Nervous system was mainly comprised of neurons and glial cells.Neurons can form complex neural networks through synaptic connection.Glia regulate many aspects of synaptic function,nourish neurons and maintain homeostasis.Voltage-gated sodium channels are crucial for the generation and propagation of action potentials in neurons.In general,the Nav1.1,Nav1.2,Nav1.3 and Nav1.6 are more widely distributed in the central nervous system,but their electrophysiological characteristics,expression patterns and locations are distinct from each other.In this study,we established two tinnitus models(imitate acute and chronic tinnitus)via salicylate administration in rats.To explore the mechanism of acute and chronic tinnitus,we focus on the changes of neurons,glial cells and VGSCs in A1 and MGB.The results are shown as below:1.The results of GPIAS and PPI indicate that rats will generate tinnitus-like behavior via acute or long-term chronic treatment of salicylate.Compared with rats from control group,rats from three groups(2h,4h and 8h after injection)showed tinnitus-like behavior at 12 k Hz and 16 k Hz,but not at 6 k Hz background sounds following acute salicylate treatment.Chronic salicylate treatment can also arise tinnitus-like behavior in rats at 12 k Hz and 16 k Hz,but not at 6 k Hz,and rats can recover to normal level after 7-days recovery.2.Protein expression of Nav1.1 and Nav1.2 were significant increase in the MGB after acute salicylate treatment(8h after injection),and the expression was mainly upregulated in the m MGB and v MGB.No significant differences were found in expression of Nav1.3 and Nav1.6.Protein expression of Nav1.1 and Nav1.2 were also significant increase in the MGB after chronic salicylate treatment,both in m MGB,v MGB and d MGB.The expression of Nav1.3 and Nav1.6 were familiar with control group.3.The protein expression of Nav1.1,Nav1.2 and Nav1.6 were up-regulated in the A1 cortex after acute salicylate treatment.The increase was more significant in layer II/III and layer IV in expression of Nav1.1 and Nav1.2,and the expression of Nav1.6was mainly up-regulated in layer II/III than in other layers.No significant differences were detected in expression of Nav1.3.The expression of four VGSCs were significant increase in the A1 cortex after chronic salicylate treatment.The increased expression of Nav1.1 was more significant in layer II-layer V.For Nav1.2,the expression was more significant increase in layer IV-layer VI.All layers show up-regulated expression of Nav1.3,but the up-regulated expression of Nav1.6 was only detected in layer II/III and layer V.4.The expression of excitatory neuron in the A1 cortex was significantly increased at 2h after acute salicylate treatment,and still keep high expression until 8h.The expression of inhibitory neuron in the A1 cortex was significantly decreased at 8h after acute salicylate treatment.The expression of excitatory neuron was significantly increased and the expression of inhibitory neuron was significantly decreased in the A1 cortex after chronic salicylate treatment,and both of them can recover to normal level after 7-days recovery.5.Acute salicylate treatment can result in up-regulated expression of Nav1.3 and Nav1.6 on excitatory neurons,and down-regulated expression of Nav1.2 on inhibitory neurons in the A1 cortex.Chronic salicylate treatment can also result in up-regulated expression of Nav1.3 and Nav1.6 on excitatory neurons,but result in down-regulated expression of Nav1.1 and Nav1.2 on inhibitory neurons in the A1 cortex.6.The expression of GFAP in the A1 cortex significantly increased in 4h after acute salicylate treatment,and the up-regulated was more significant in layer V and layer VI than other layers,accompany with increased number and length of branches in astrocyte.The expression of Iba1 in the A1 cortex significantly increased in 4h after acute salicylate treatment,and still keep high expression after the recovery of tinnitus(24h after injection).The morphology change was not detected in microglial after acute salicylate treatment.After chronic salicylate treatment,the expression level of GFAP and Iba1 in the A1 cortex significantly increased,together with the rising number of branches in astrocyte but no significant change was found in microglial.The expression of GFAP and Iba1 were recover to control level after 7-days recovery.7.The expression of GFAP in the MGB significantly increased in 4h after acute salicylate treatment,and the up-regulated was only detected in m MGB.The morphology change was not detected in astrocyte after acute salicylate treatment.The expression of Iba1 in the MGB significantly increased after acute salicylate treatment(2h after injection),and still keep high expression after the recovery of tinnitus(24h after injection),accompany with increased number and length of branches in microglial.After chronic salicylate treatment,the expression level of Iba1 in the MGB significantly increased,together with the rising length of branches in microglial.No significant change of expression level or morphology was detected in astrocyte.8.The expression level of IL-1β in the A1 cortex significantly increased at 4h after acute treatment of salicylate,and keep high expression after the recovery of tinnitus.But after chronic treatment of salicylate,the elevated IL-1β expression in the A1 cortex was tend to normal level following 7-days recovery.In the MGB,the expression level of IL-1β increased at 4h,recovered to control level at 8h after acute treatment of salicylate,but that was almost constant after chronic treatment of salicylate.In conclusion,acute or chronic salicylate treatment may result in tinnitus with different mechanism.Changes in cortex and thalamus show different tendency of protein expression.Collaboration of four subtypes of VGSCs in A1 inhibit the activity of inhibitory neurons and activate the excitatory neuron,resulted in hyperexcitability of auditory central system.Meanwhile,the up-regulated expression of astrocyte may disturb the synthesis of GABA and affect the inhibitory synaptic transmission.The increased expression of microglia can promote the release of inflammatory factors,show the tinnitus may accompanied with neuroinflammation.