Methylene Blue Treat Hypoxia-ischemic Brain Injury In A Neonatal Rat Model

Objective:Methylene Blue(MB)is a widely used and long-standing ancient drug,especially for neuropathic diseases,with active neuroprotective effects.Hypoxic-ischemic(HI)brain injury is an important reason for neonatal death and is also one of the main causes of disability in newborns.The survivors with HI-induced neonatal brain damage often have permanent neurological disorders.Whether MB can effectively treat neonatal HI brain damage,is still unclear.Therefore,this study is intended to clarify whether MB treatment has a neuroprotective effect on HI-induced neonatal brain damage,and also to investigate the underlying mechanisms of MB treatment on neonatal HI brain damage,providing more new treatment methods for HI brain injury and providing theoretical foundation and basis for clinical use of MB to treat neonatal HI brain injury.Materials and Methods:Firstly,based on the successful establishment of animal models in our previous studies,we established neonatal HI brain damage to the new 10-day Sprague-Dawley(SD)rats.Experimental rats were randomly divided into three groups,namely a sham-surgical control group(Sham),HI group(HI),HI plus with MB treatment group(HI + MB).In HI + MB group,we treated rats with MB through Alzet capsules permeable pressure at a dose of 0.5 mg/kg/day for 7 days.In the first part of the study,we observed the effect of MB on HI-induced brain tissue loss by Cresyl violet(CV)staining.Double staining of NeuN(a neuron-specific marker)and F-Jade C(a marker of neuronal degeneration)was conducted to determine the efficacy of MB treatment on neuronal damage.Then,the effects of MB on the functional outcome of HI rats were assessed by behavioral tests.Hippocampal-dependent spatial reference learning and memory were evaluated by the Barnes Maze test,and the motor coordination was evaluated by the balance beam test.In the second part,we investigated the underlying mechanism of MB therapy in neonatal HI brain injury from the following four aspects,such as mitochondrial function,oxidative stress,inflammatory response,and blood-brain barrier(BBB)integrity.1.Immunofluorescence with the mitochondrial membrane antigen TOM20 staining under a common focus microscope was used for the observation of mitochondrial defect.TUNEL staining was next utilized to evaluate neuronal death.Therefore,we evaluated if MB treatment could attenuate mitochondrial dysfunction in neonatal HI brain injury.2.DHE staining was performed for the evaluation of the total level of ROS production in the brain,and protein carbonyls levels in sample homogenate were measured to determine the subsequent oxidative damage to the brain.So,it is evaluated whether MB protected neonatal HI brain injury by regulating oxidative stress.3.GFAP and IBA1 staining were performed to examine astrocyte and microglia activation respectively.And Typical pro-inflammatory cytokines,IL-1β and TNF-α were measured afterward with high-sensitivity ELISA technique.Thus,we evaluated whether MB protects neonatal HI brain injury by suppressing HI-induced neuroinflammation.4.To evaluate whether MB can fight against neonatal HI brain injury by preserving BBB integrity,we intravenously injected Na F dye and Evans Blue(EB)dye,and separately examined their extravascular infiltration in the cortex and hippocampus by IHC staining.To,infiltration through BBB was next assessed through intravenous injection.Finally,all the data were analyzed with Graph Pad Prism 7 software,and pictures were generated using Graph Pad Prism 7.0 software.Results:In the functional research part,HI caused robustly increased brain loss in neonatal rats as compared to the sham group,the brain loss ratio is 19.20 ± 4.82%,while the MB treatment can effectively reduce the brain loss,and the proportion of substantial loss of MB treated rats has dropped to 11.33 ± 2.50%.We found a robust elevation of F-Jade C intensity in both cerebral cortex and hippocampal CA1 region in HI rats,compared with Sham group rats,which was strongly decreased by MB treatment.These results demonstrated MB treatment can strongly attenuate HI-induced brain loss and neuronal damage in the cortex and hippocampus of neonatal rats.On behavioral tests,the Barnes Maze test showed HI rats exhibited significantly increased escape latency to find the hidden chamber in the second day and third day of training trial,decreased quadrant occupancy,and increased searching error in probe trial as compared to Sham group.Whereas,HI rats with MB treatment displayed improved cognitive function,as evidenced by decreased escape latency and searching error,as well as increased quadrant occupancy compared with HI rats.Similar escape velocities among the groups suggested differences in escape latency were not due to speed variations.Balance beam test showed HI rats spent more time to cross the balance beam and had more slips times as compared to Sham rats.Interestingly,MB treatment can reverse the motor deficit by significantly decreasing the cross time and feet slip times.This revealed that MB significantly improved the spatial reference memory and motor coordination of neonatal HI rats.In the mechanism research part,our study investigated four different mechanisms behind neuroprotective actions of MB for HI brain injury,including mitochondrial function,oxidative stress,neuroinflammation response,and BBB integrity.First,TOM20 staining results showed that a dramatic increase of mitochondrial fragmentation value in the cortex and hippocampal CA1 after HI,as compared to Sham control.In contrast,MB treatment significantly decreased the mitochondrial fragmentation for both cortex and hippocampal CA1.TUNEL staining showed MB treatment strongly reduced numbers of TUNEL-positive neurons in both cortex and hippocampal CA1 region of HI rats.This suggested that MB strongly preserved mitochondrial function by repressing HI-induced mitochondrial fragmentation,and the following neuronal death in the cortex and hippocampus.Second,DHE staining and protein carbonyls levels showed that DHE intensities and protein carbonyls levels were significantly increased in both cerebral cortex and hippocampal CA1 region of HI rats as compared to the Sham group,while DHE levels and protein carbonyls levels in these brain regions were significantly downregulated by MB treatment,suggesting a remarkable inhibition on ROS generation is another mechanism of MB treatment on neonatal HI brain injury.Third,GFAP and IBA1 staining revealed the robustly elevated GFAP and IBA1 levels in the HI cortex and hippocampal CA1 region were significantly decreased by MB treatment in both cortex and hippocampal CA1.As expected,HI caused significantly increased levels of IL-1β and TNF-α productions in both cortex and hippocampus of HI rats as compared to sham.In contrast,MB administration can significantly suppress these cytokines generation.This suggested that MB profoundly inhibited microglia and astrocyte activation and the pro-inflammatory cytokines production in neonatal cortex and hippocampus after HI,which is another mechanism of MB treatment on neonatal HI brain injury.Fourth,after intravenous injection of Na F dye and EB dye,the results showed Na F contents were strongly increased in both cortex and hippocampus CA1 and CA3 region of HI rats,indicating disrupted BBB integrity following HI.In contrast,MB treatment robustly decreased Na F levels in all of these brain regions.EB levels were highly increased in the ipsilateral hemisphere of HI rats compared with the contralateral hemisphere.Whereas MB treatment efficiently reduced EB contents in the ipsilateral hemispheres compared with their contralateral levels and their relative changes versus the Sham group in ipsilateral hemispheres.No differences were found between the two hemispheres of rats from both sham and MB treatment groups.This result provides evidence that MB treatment was able to significantly reduce BBB disruption against neonatal HI brain injury.Conclusion:These findings demonstrate that the mechanisms behind neuroprotective actions of methylene blue are multifactorial,including restoration of mitochondrial function,suppression of oxidative stress and neuroinflammation,as well as attenuation of blood-brain barrier disruption.Our study might provide further directions for MB as a promising option in neonatal HI encephalopathy therapy.