| Traumatic brain injury(TBI)is the leading cause of death and disability in the world of modern life.It is also a major public health problem worldwide unresolved.Mechanical damage neurons in the body to trigger a series of reactions leading to nerve cell death,brain edema and neurological deficits.However,cell death can be divided into three types: necrosis,apoptosis and autophagy.Some studies suggest that autophagy is activated after TBI,the expression of LC-3 and Becline-1 is up-regulated in the hippocampus and cortex damage.Other studies have shown that inhibition of autophagy relieve TBI and improve neurological recovery.Right Dexmedetomidine is a highly selective a2 receptor agonists,hypnotic,sedative,analgesic,anxiolytic,sympatholytic effect without significant respiratory depression generation.Thus,the right dexmedetomidine widely used in operating rooms and intensive care anesthesia and sedation.Recently,many studies have shown that the right dexmedetomidine ischemic brain injury may have neuroprotective effects.Right dexmedetomidine has also been reported to alleviate cerebral ischemia / reperfusion injury in animal brain.According to reports,the right dexmedetomidine can improve the anti-apoptotic factor BCL-2 levels and ERK1 / 2 phosphorylation is involved in cell survival,thereby inhibiting apoptosis and improved neuronal survival.Currently,PI3K/ Akt / mTOR has been widely reported to be apoptosis and autophagy activation of signaling pathways classic.Based on these findings,we assume that a given application dexmedetomidine can activate PI3K/ Akt / mTOR pathway inhibition of hippocampal neurons autophagy of TBI rats play a strong neuroprotective effect.The first part Effect of dexmedetomidine on recovery of neurological function following traumatic brain injury in ratsObject:The experimental study on the male SD rats(270g-290g),non traumatic brain injury model of open rats,and to verify the recovery of dexmedetomidine on rat neural function in rats after traumatic brain injuryMethod: Rat model of brain injury was made by using the modified rat model of traumatic brain injury.The rats were randomly divided into three groups(n=48): control group(Group sham),traumatic brain injury group(group TBI)and dexmedetomidine group(group Dex).Methods: 1 Wet and dry specific gravity method was used to detect the water content of brain tissue;2 Morris water maze test to detect changes of the ability of learning and memory in space;3 Nerve motor function score.4 In open field test.Experimental results obtained in this experiment using Excel established,as mean ± standard deviation reflect,using SPSS 16.0 will be random data obtained ANOVA,Between the two groups were compared using qq test way,with P <0.05 indicates a statistically significant difference.Result:1.Light microscopy after traumatic brain injuryAfter traumatic brain injury,you can see both subarachnoid hemorrhage whole,but also a small part of intraventricular hemorrhage.In the brain parenchyma to spotting mainly,through observation,can be seen at the surface of the brain in rats have significant vascular congestion,and did not find the local brain trauma situation.2.Brain water content was measured by wet/dry method relustThere was no difference in brain tissue water content in Sham group at all time points;TBI group was significantly higher than that in Sham group(P < 0.05);the water content in Dex group was significantly lower than that in TBI group(P < 0.05).3.Morris water maze resultMorris water maze tests showed that obvious space leaming and memorizing obstruction existed in rats after injury.Delitescence prolonged obviously 8 to 9 days later.Search moving tracks showed that search strategy altered more ideally with time in the Dex group than in TBI group(P<0.05).4.Recommending score of neural functionSham group,TBI group and Dex group each had a score of 24,7-9,15-18 respectively.As a result,there was significant difference between TBI group,Dex group and sham group(P<0.05);there was also significant difference between TBI group and Dex group(P<0.05).5.In open field test resultThe rats in the sham group the total walking distance,the central lattice activity time,frequency and vertical behavior of the least number of rats.In the TBI group the total walking distance,the central lattice activity time,frequency and the number of upright behavior than the sham group(P<0.05);Dex group than TBI group in the total Walk,central cell activity time,frequency and the number of upright behavior decreased,but higher than that of sham group(P<0.05).Summary:The rat brain injury model meet the basic requirements of the experiments;through the detection of relevant experiments on the behavior of the data in rats after traumatic brain injury of dexmedetomidine to a certain extent to the recovery of neurological function.The second part The effect of dexmedetomidine after the rats traumatic brain injury to neuronal autophagyObject: This part of the experiment in the studies :Dexmedetomidine can have an impact on traumatic brain injury in rats neurons autophagy situation.Method: The fabrication of rat brain impact injury model in rats.The rats were randomly divided into two groups(n=48):Normal control group;brain trauma group;3-MA autophagy inhibitor group;dexmedetomidine group.Respectively post-traumatic 6h,12 h,24h,48 h four time points tested.Detection method:1.HE staining;2.Immunochemical staining assay LC-3 and Beclin-1 of protein localization.3.Western-blot assay LC-3 and Beclin-1 protein quantitation situation.Immunochemical staining results using Image Proplus 6.0 analysis system determined by quantitative analysis.Western-blot using image analysis software ImageJ protein bands were the reactions semi-quantitative analysis,obtain optical density(OD),in OD value than the internal reference protein(?-actin)OD value ratio,as a basis for comparison for comparison.Experimental results obtained in this experiment using Excel established,as mean ± standard deviation(1)reflect,using SPSS 16.0 will be random data obtained ANOVA,Between the two groups were compared using qq test way,with P <0.05 indicates a statistically significant difference.Result:1.Light microscopy after traumatic brain injuryAfter traumatic brain injury,you could see both subarachnoid hemorrhage whole,but also a small part of intraventricular hemorrhage.In the brain parenchyma existing spots mainly,through observation,the surface of the brain in rats had significant vascular congestion,and the partial brain trauma cases could not be seen.2.HE test resultsHE staining showed that in the normal control group the organizational structure of the brain has not changed,the lumen of the blood vessel was not unusual,vascular wall formation,nerve cell morphological rules;TBI group neuron cell body had changed shape,cytoplasmic staining decreased,depth stained nuclei appeared.Between the cells there was a clear gap,and accompanied by necrotic neurons appear.Dex group compared to the TBI group structure and neuronal cells staining depth improved.3.Immunohistochemical detection result of Beclin-1Beclin-1 appeared brown granules positive expression.The expre-ssion amount of Beclin-1 was seen in the control group,and immune expression is weak.Compared with the control group,wounds Group Beclin-1 positive cells were significantly increased,enhanced immune response.And at 6h expression trend gradually increased after trauma,at 24 hours reached the maximum,and 48 hours fell.Compared with TBI group,expression trend at each time point of Dex group has not changed.Peak expression still in 24 h,but the overall expression was significantly decreased.4.Immunohistochemical detection result of LC-3LC-3 appeared brown granules were positive expression.The expression amount of LC-3 was seen in the control group,and immune expression is weak.Compared with the control group,wounds Group LC-3 positive cells were significantly increased,enhanced immune response.And at 6h expression trend gradually increased after trauma,at 24 hours reached the maximum,and 48 hours fell.Compared with TBI group,expression trend at each time point of Dex group has not changed.Peak expression still in 24 h,but the overall expression was significantly decreased.5.Western blot analysis of Beclin-1The molecular weight of Beclin-1 is 60 KD.Beclin-1 protein in the control group had basal expression trace and expression at each time point were no significant trends.Compared with the control group,the trauma group Beclin-1 protein at each time point showed a significant increase in the expression of elevated(P<0.05).Changing trends of expression presentation,at 6h expression trend gradually increased after trauma,at 24 hours reached the maximum,and 48 hours fell.But still higher than the control group at the same time of the basis expression(P<0.05).Compared with TBI group,expression trend at each time point of Dex group has not changed,peak expression still in 24 h,but the overall expression significantly decreased.6.Western blot analysis of LC-3The results showed the different shades of dual-band LC-3? and LC-3?.The molecular weight of LC-3? is 15KD;the molecular weight of LC-3? is 17 KD.LC-3 protein in the control group had basal expression trace,and expression at each time point showed no significant trends.Compared with the control group,the trauma group LC-3 protein at each time point showed a significant increase in the expression of elevated(P<0.05).Changing trends of expression presentation,at 6h expression trend gradually increased after trauma,at 24 hours reached the maximum,and 48 hours fell.But still higher than the control group at the same time of the basis expression(P<0.05).Compared with TBI group,expression trend at each time point of Dex group has not changed,peak expression still in 24 h,but the overall expression significantly decreased.Summary: It has been demonstrated in rats after brain injury,the existence of neuronal autophagy,this part of the experiment further confirmed,Dexmedetomidine given that could reduce the expression of autophagy-related proteins,thus achieving neuroprotection,and to improve learning and memory in rats after traumatic brain injury in remission.The third part Dexmedetomidine activated PI3K/Akt/mTOR pathway on TBI rats neurons autophagy in the affectObject: This part of the experiment in the studies : dexmedetomidine whether through activation PI3K/Akt/ mTOR pathway in rats after traumatic brain neurons autophagy of affect.Method: The fabrication of rat brain impact injury model in rats.The rats were randomly divided into two groups(n=48): Normal control group;brain trauma group;dexmedetomidine group.Respectively post-traumatic 6h,12 h,24h,48 h four time points were tested.Detection method:1.HE staining;2.Immunochemical staining assay LC-3 and Beclin-1 of protein localization.3.Western-blot assay p-AKt and p-mTOR protein quantitation situation.Analysis and statistical the same with the second part.Result:1.Light microscopy after traumatic brain injuryAfter traumatic brain injury,you could see both subarachnoid hemorrhage whole,but also a small part of intraventricular hemorrhage.In the brain parenchyma existed spots mainly.Through observation,it could be seen that surface of the brain in rats had significant vascular congestion,and the partial brain trauma cases could not be seen.2.HE test resultsHE staining showed that in the normal control group the organizational structure of the brain has not changed;the lumen of the blood vessel is not unusual,vascular wall formation;nerve cell morphological rules;TBI group neuron cell body has changed shape,cytoplasmic staining decreased,depth stained nuclei appeared.Between the cells there was a clear gap,and accompanied by necrotic neurons appearing.Dex group compared to the TBI group structure and neuronal cells staining depth improved.3.Western blot analysis of p-AKtP-AKt protein in the control group had basal expression trace,and expression at each time point showed no significant trends.Compared with the control group,the trauma group p-AKt protein at each time point showed a significant increase in the expression of elevated(P<0.05),changing trends of expression presentation.At 6h expression trend gradually increased after trauma,at 24 hours reached the maximum,and 48 hours fell.But still higher than the control group at the same time of the basis expression(P<0.05).Compared with TBI group,expression trend at each time point of Dex group has not changed,peak expression still in 24 h,but the overall expression significantly decreased.4.Western blot analysis of p-mTORP-mTOR protein in the control group had basal expression trace,and expression at each time point showed no significant trends.Compared with the control group,the trauma group p-mTOR protein at each time point showed a significant increase in the expression of elevated(P<0.05),changing trends of expression presentation.At 6h expression trend gradually increased after trauma,at 24 hours reached the maximum,and 48 hours fell.But still higher than the control group at the same time of the basis expression(P<0.05).Compared with TBI group,expression trend at each time point of Dex group did not change,peak expression still in 24 h,but the overall expression significantly decreased.5.LC3 and NeuN(Neuronal marker)fluorescent double labeling results in hippocampal area: LC-3 for DyLight 594 labeled cell cytoplasm red fluorescence,neuron specific marker NeuN for DyLight 488 labeled cell cytoplasm green fluorescence.In hippocampal area of TBI group,we can see there were large number of overlapping of red and green fluorescent,presented as orange color at 24 h,suggesting that autophagy is located in neurons.While when treated with Dex group,red fluorescent was significantly weaker and the overlapping of red and green fluorescent decreased signifcantly,illustrating that the level of neuronal autophagy in hippocampal area decreased.Summary: It has been demonstrated in rats after brain injury,the existence of neuronal autophagy,this part of the experiment further confirmed,Dexmedetomidine given that could reduce the expression of autophagy-related proteins,thus achieving neuroprotection,and to improve learning and memory in rats after traumatic brain injury in remission.Conclusion: It is existence of neuronal autophagy in rats after brain injury,Dexmedetomidine given can activate PI3K/Akt/ mTOR pathway and reduce the expression of autophagy-related proteins,thus achieving neuroprotection and improve learning and memory in rats after traumatic brain injury in remission. |
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