The Neuroprotective Effect And Manchanism Of Methylene Blue On Traumatic Brain Injury

Backgrounds:Traumatic brain injury (TBI) is a serious disease with high morbidity and causes high disability rate and mortality around the world. It is estimated that over10million people worldwide suffering from traumatic brain injury annually, leading to many deaths, many survivors are subject to catastrophic complications, including neurological deficit and motor dysfunction.Although the survival rate of TBI has significantly improved in the last few decades due to advances in emergency and intensive care medicine, there are no effective proactive treatment options for augmenting functional recovery or preventing long-term neuropsychiatric complications post-TBI.A series of studies have shown that the severity of brain injury damages not only related to the primary injury, but also related to the secondary injury, caused by neuroinflammation, cerebral edema and blood-brain barrier breakdown. Secondary brain injury lasts from a few hours to several months or even years, ultimately leading to neuronal death or neurodegeneration. As the primary injury was unable to intervene in clinic, research has focused on the secondary brain injury mechanisms or pathways, and attempts to discover new drugs to block or attenuate it.Methylene blue (MB) is a phenothiazine compounds, with redox function. It has been used in clinic for more than120years. In the past, MB is used for diagnostic procedures and for treating methemoglobinemia, malaria, cyanide, carbon monoxide poisoning, cardiopulmonary bypass, and septic shock. As it can be selectively absorbed in neural tissues, recent studies have shown that MB also has beneficial effects on the central nervous system. For example, it may have neuroprotective effect against ischemic stroke, amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, Leber optic neuropathy and other neurodegenerative diseases. As many pathophysiological mechanisms involved in traumatic brain injury are similar to other neural system diseases, such as ischemic stroke and neurodegenerative diseases. Thus, we assume that methylene blue may have the neuroprotective effect against traumatic brain injury.This study intends to test the neuroprotective effect and mechanism of methylene blue on traumatic brain injury using a controlled cortical impact injury model with ICR mice. Part1. Neuroprotective effects of Methylene blue for traumatic brain injuryAims:To investigate the neuroprotective effect of methylene blue against traumatic brain injury using a controlled cortical impact injury model with ICR mice.Methods:(1) Ascertain the optimal dose of methylene blue:Mice were divided into five groups:sham operation group (Sham), normal saline treated group (NS), low-dose MB treated group (i.p.0.1mg/kg), median-dose MB treated group (i.p.1mg/kg), and high-dose MB treated group (i.p.10mg/kg). The optimal dose of MB was determined according to the neurological function and brain water content24h post-TBI.(2) Mice were divided into three groups:Sham group, NS group and MB group (using the optimal dose). Base on the previous studies, we evaluated the brain water content, injury volume, neurological function and number of neurons24h and72h post-TBI. The injury volume and neurological function were also measured14days post-TBI.(3) The modified neurological severity score (mNSS) was used to examine the effects of MB on neurological deficits. The brain water content (BWC) was calculated as follows:(wet weight-dry weight)/wet weight×100%. Injury volume was evaluated using HE staining and then analyzed using ImageTool. The numbers of neurons in injury cortex were detected by immunohistochemical staining and data were analyzed using Image-Pro Plus.Results:(1) The brain water content and mNSS were significantly increased after TBI compared to Sham group (P<0.05).MB with median-dose (i.p. lmg/kg), significantly reduced neurological deficits and brain water content relative to NS group24h post-TBI(P<0.05). However, this effect was not found in the low-dose MB group (i.p. O.lmg/kg) and high-dose MB group (i.p. lOmg/kg). Therefore, the optimal dose used in the following studies was median-dose (i.p. lmg/kg).(2) The injured hemisphere BWC was significantly elevated24h post-TBI; MB attenuated it significantly (P<0.05). However, the BWC of contralateral hemisphere, cerebellum, and brain stem was not significantly different among three groups. At72h post-TBI, the BWC in the injury hemisphere decreased, and the BWC of the NS and MB groups were not statistically different (P>0.05).(3) The NeuN staining used to verify the effect of MB in preventing neuronal death revealed that there were significantly more neurons in the MB group than in the NS group both in24h and in72h post-TBI.(4) The TBI resulted in neurological functional deficits. Compared with the NS group, mNSS scores were significantly lower in the MB group24h and72h post-TBI (p<0.05).However, there was no significantly difference between NS group and MB group on mNSS scores14days post-TBI (p>0.05).(5) We evaluated the cerebral lesion volume to confirm the neuroprotective effect of MB in TBI. The lesion volume of MB group was significantly smaller than that of the NS group24h,72h and14days post-TBI (p<0.05)Conclusions:Methylene blue has neuroprotective effect against traumatic brain injury including alleviated cerebral edema, decreased neurological functional deficits, inhibited neuron cell death caused by TBI and reduced cerebral lesion volume. Part2. The possible manchanisms of methylene blue against traumatic brain injuryAims:To investigate the possible manchanisms of methylene blue against traumatic brain injury using a controlled cortical impact injury model with ICR mice.Methods:(1) Mice were divided into three groups:Sham group, NS group and MB group. Base on the previous studies, we evaluated the release of proinflammatory cytokines, the activation of autophagy and microglia24h and72h post-TBI. The activation of microglia was also detected14days post-TBI.(2) The activation of autophagy was detected by immunohistochemical staining of Beclin-1and western blot analysis of Beclin-1and LC3. The Iba-1staining was used to evaluate the activation of microglia. Moreover, the release of proinflammatory cytokines, such as IL-6, IL-iβ was detected by western blot.(3) The change of mTOR/p70S6K pathway was also detected by western blot.Results:(1)Based on the increased Beclin1and LC3-II/LC3-I ratio, we found that autophagy was significantly activated post-TBI. Compared to the NS group, autophagy in the MB group was more obvious at24h and72h post-TBI (p<0.05).(2)The release of IL-6andlL1β increased significantly after TBI (p<0.05)., and MB decrease the level of IL-6, ILlβ compared to the NS group at24h and72h post-TBI (p<0.05).(3) The number of microglia was low at24h post-TBI, but obvious activation was seen at72h, persisting until14days post-TBI (p<0.05). Although the Iba-1-positive cell numbers in the MB and NS groups at24h post-TBI were not significantly different, there was obvious inhibition of microglial activation at72h and14days post-TBI in MB-treated mice (p<0.05). Image-Pro Plus identification of the Iba-1optical density of the two groups yielded similar results.(4) The levels of mTOR and P70S6K were not significantly different among three groups (p<0.05). However, the ratio of p-mTOR/mTOR and p-P70S6K/P70S6K increased significant after TBI (p<0.05), and MB attenuated the ratio of p-mTOR/mTOR and p-P70S6K/P70S6K significantly compared to the NS group (p<0.05).Conclusions:(1) We found that MB exerts a neuroprotective effect by enhancing autophagy, reducing the reasese of proinflammatory cytokines and inhibiting microglial activation, which reduce cerebral lesion volume, neuronal death, brain edema and neurological deficit.(2) The present study demorstrated that the inactivation of mTOR/p70S6K pathway by MB may plays a neuroprotective role in traumatic brain injury.