| Vascular dementia（VD）is a heterogeneous group of brain disorders,in which cognitive impairment is attributable to cerebrovascular pathologies.VD is the second most common cause of dementia,causing around 15-20%of total dementia cases.However the treatment strategies for VD are more focus on controlling of underlying cardiovascular,cerebrovascular risk factors and treatment of associated symptoms.There didn’t have effective treatment for VD before.Therefore,finding a high potency drug has become a key of medical issue.Numerous studies have demonstrated that chronic cerebral hypoperfusion（CCH）is associated with the initiation and progression of VD.The deprivation of oxygen and glucose in brain tissue after CCH could result in a serious of homeostasis alterations,including oxidative stress,apoptosis and autophagy.A recent report showed that CCH resulted in rat hippocampal neuronal apoptosis,enhancement and redistribution of autophagy.Moreover,consistent with the previous report,our previous studies also had found that oxidative stress injury was induced in VD animal models,and apoptosis activated simultaneously with autophagy to accelerate neuronal death.Inhibition of activation of autophagy and apoptosis in the central nervous system of VD rats may play a neuroprotective role.Molecular hydrogen could rapidly diffuse across all the cell membranes.And it can easily penetrate the blood-brain barrier.In 2007,Ohsawa reported that gas hydrogen（H2）acts as a therapeutic and preventive antioxidant by selectively reducing highly strong oxidants such as hydroxyl radical（·OH）and peroxynitrite（ONOO-）in cells,and H2 exhibits cytoprotective effects against oxidative stress.Multiple lines of evidence have shown that lots of disease models and human diseases were benefit from hydrogen and an increasing number of studies revealed the protective effects of hydrogen in various models of brain injury.Molecular hydrogen has been intensively studied in the past ten years and it arouses wide concern due to its anti-oxidative,anti-apoptotic,and anti-inflammatory activities.Recently,there are more and more researches on the relationship of hydrogen and autophagy.However,the role of hydrogen in autophagy remains controversial.Some studies show that hydrogen can down-regulation of autophagy,and some studies demonstrated the opposite.Furthermore,no literature highlighted the protective effects of hydrogen on VD.Therefore,the role of hydrogen in CCH and the underlying mechanism still needs to be further investigated.Based on the above research background,in the current study,a widely used model of CCH induced by permanent bilateral common carotid arteries occlusion（2-vessel occlusion,2VO）in rats was used to explore:（1）the changes in cognitive function in rats with administration of hydrogen-rich water（HRW）,（2）the effects of HRW on oxidative stress in hippocampus,（3）the effect of HRW on the morphology of hippocampal neurons,（4）the effects of HRW on apoptosis and autophagy-related protein expression,（5）the relationship between the neuroprotective effect of HRW and related signaling pathway.Part oneEffects of Hydrogen-rich Water on Cognitive Function ofVascular Dementia Rats Induced by chronic cerebral hypoperfusionObjective:To establish a VD rat model induced by chronic cerebral hypoperfusion through 2VO and observe the effects of HRW on spatial learning and memory changes of VD rats.Methods:1.Forty-eight 3-month-old male Sprague–Dawley rats weighing200-250g were randomly divided into four groups:Sham group,model group,Hydrogen-rich water group（HRW）,donepezil group（Don）.n=12.2.Permanent bilateral common carotid artery occlusion（two-vessel occlusion,hereafter referred to as 2VO）was used to establish VD model of rats.From the next day of the operation,the rats in HRW group were gavaged with HRW 3ml a day,sham group and model group were provided with an equal volume of double steamed water,while Don group were treated with donepezil（1mg/kg 3ml/d）through oral treatment.Each group was investigated for 4 weeks postoperative.3.The next day after the final gavage administration,all rats were evaluated spatial learning and memory abilities by the Morris water maze test.The first 5 days the rats were tested for Place navigation test and on the sixth day,the rats were tested on a spatial probe trial.Results:1.Place navigation testThere was a significant overall group difference among four groups.With the increase of MWM training days,the escape latencies of four groups of rats gradually shortened.Compared with the sham group,rats in the model group showed significant longer mean escape latencies from the beginning day 1 to day 5（day 1-5:P<0.01）.Compared with model group,rats in the HRW group and Don group took shorter time to find the platform,especially from the 3rd day to the 5th day（day 1:P<0.05;P>0.05,respectively;day 2:P<0.05;P>0.05,respectively;day 3-5:P<0.01;P<0.01,respectively）.Meanwhile,during the five days,the mean escape latencies were not significant different between rats treated with HRW and Donepezil（day 1-5:P>0.05）.2.In the spatial probe trial,there were significant differences in the percentage of time that the rats spent in the target quadrant among all groups（P<0.01）.Rats in the 2VO group stayed in the target quadrant for significantly less time than in the sham group（P<0.01;P<0.01;P<0.01,respectively）.Compared with the model group,rats that treated with HRW increased the ratio of time spent in the target quadrant（P<0.01）.Rats in Don group had a similar trend（P<0.01）;however,no statistical difference was found between the HRW group and the Don group（P>0.05）.3.Changes in the swimming trajectory of the four groups of ratsThe initial method of seeking platform is random,and the swimming trajectory is mostly marginal.However,as the number of training days increased,the rats in the sham-operated group,HRW group,and donepezil group gradually changed the method,（a trend or even a straight line）,especially in the sham group.The rats in the model group were mostly swimming marginally or tendency to the platform.Summary:The rats induced by chronic cerebral hypoperfusion through2VO had obvious cognitive dysfunction.To some extent,2VO rat model,similar to the pathogenesis of clinical VD,is an ideal animal model of VD.HRW attenuates not only the spatial learning impairment but also the memory impairment that induced in 2VO rats,which plays a neuroprotective effect in the learning and memory abilities.Part two The Effect of Hydrogen-rich Water on Hippocampal Neuronal Cells in Rats with Vascular Dementia and Its Effect on Preventing Oxidative Stress InjuryObjective:To observe the patho-morphological changes of hippocampal CA1 region.To evaluate the malondialdehyde（MDA）content and Superoxide Dismutase（SOD）activity changes in hippocampus of rats 4 weeks after 2VO.And further to explore whether HRW has the effect of correcting the abnormal expression to reduce oxidative stress damage.Methods:1.Shortly after the behavioral tests,three rats chosen randomly from each group were anesthetized and perfused with 4%paraformaldehyde.Then the brains were embedded in paraffin.Coronal brain sections were cut and underwent Nissl staining.The histopathological changes of the hippocampal CA1 area were observed under the light microscope.Furthermore,three rats from each group were randomly chosen to be perfused with Electron microscopy perfusion fixative under anesthesia.The electron microscopy specimens of hippocampal CA1 area were observed through the electron microscopy.2.Furthermore,another six rats chosen randomly from each group were fully anesthetized.The hippocampi were quickly dissected and homogenated.MDA content and SOD activity were evaluated according to the manufacturer’s instructions in different groups.Results:1.Nissl staining results of neurons in hippocampal CA1 region of rats in four groups.In the sham group,there was no damage of the pyramidal neurons in the hippocampal CA1 region.However,obvious pathological changes were exhibited in hippocampal CA1 area,including hippocampal atrophy,significant neuronal shrinkage,neuronal loss,light color staining,loosely arranged neurons in the model group.Administration of HRW,like treatment with donepezil,markedly reversed the morphologic changes mentioned above.2.Results of electron microscopy on ultrastructure damage in hippocampal CA1 region in four groups in 2VO rats.The sham-operated rats had complete ultrastructure,and autophagosomes were founded in a little of neurons.There were more autophagosomes,swelling mitochondria,loose endoplasmic reticula and less nuclear border in the model group.In HRW group and Don group,the number of autophagosomes decreased in varying degree and the damage of nucleus and organelle microstructure was ameliorated when compared with the model group.However,unlike the Don group,mitochondrial swelling was not significantly improved in the HRW group.3.The content of MDA in the hippocampus of four groups.The content of MDA in the 2VO groups（model group,HRW group,donepezil group）was higher than that in the sham group.The increase in the model group was the most obvious（P<0.05）.The content of the MDA in the HRW group were lower than that in the model group,but it did not reach statistical difference.4.The activity of SOD in the hippocampus of four groups.Compared with the sham group,the SOD activity in the model group was significantly decreased（P<0.05）,while the activity of SOD in the HRW group was significantly higher than that in the model group（P<0.05）.It was close to or even higher than that in the sham group（P>0.05）.Although the SOD activity in the donepezil group was slightly higher than that in the model group,there was no statistical difference between the two groups（P>0.05）.Summary:1.CCH injury can cause damage to neurons in the hippocampal CA1region and induce apoptosis in rats.2VO activates the free radical production system and gradually enhances the oxidative stress response.Oxidative stress may be one of the mechanisms by which CCH causes VD.2.HRW can improve the neuronal cell damage in CA1 area of hippocampus caused by CCH,reduce the number of cell death,reduce the number of autophagosome,maintain the integrity of cell morphology,and normal structure.The protective effect of HRW on mitochondria is not sufficient to improve mitochondrial swelling.3.HRW treatments can play the neuroprotective effect by improving the activities of SOD and reducing the content of MDA,which lead to inhibit the oxidative stress damage.Hydrogen has better effect of anti-oxidative stress than positive drug groups.Part threeThe influence of Hydrogen-rich Water on the expression of apoptosis related proteins in vascular dementia rats.Objective:To observe the effects of HRW on the expression of apoptosis-associated proteins:Cleaved cysteinyl aspartate specific proteinase-3（cleaved caspase-3）,B-cell lymphoma-2（Bcl-2）,Bcl-2 associated X protein（Bax）and explore the effect of regulating neuronal apoptosis and the neuroprotective mechanisms of HRW.Methods:1.Three rats chosen randomly from each group were anesthetized and perfused with 4%paraformaldehyde.Then the brains were embedded in paraffin.Coronal brain sections were cut and immunohistochemically stained for cleaved caspase-3（1:500）.The expression of cleaved caspase-3 positive cells in CA1 region of hippocampus in each group was observed under light microscope.2.Furthermore,another six rats chosen randomly from each group were fully anesthetized.The hippocampi were quickly dissected and homogenated to detect the protein expression of Bcl-2 and Bax by western blot.Results:1.Expression of cleaved caspase-3 immunoreactive cells in CA1 region of hippocampus in four groups of rats.Four weeks after 2VO,the neurons in the hippocampal CA1 region of rats showed positive expression of cleaved caspase-3.The positive products were brown particles and were mainly located in the nucleus of the neurons.2.The effect of HRW on expression of cleaved caspase-3immunoreactive cells in hippocampal CA1 region of VD rats.The IOD/area value of cleaved caspase-3 in CA1 region was significantly different among all groups（P<0.01）.And compared with the sham group,cleaved caspase-3 was dramatically increased in the model group（P<0.01）.Besides,compared with the model group,the IOD/area value of cleaved caspase-3 was significantly reduced in the rats treated with HRW（P<0.01）and donepezil（P<0.01）.However the reduction measured in the HRW group was more than that measured in the donepezil group（P<0.01）3.The effect of HRW on Bcl-2 and Bax protein expression in hippocampus of VD rats.There were significant differences in the expressions of Bcl-2 and Bax as well as Bcl-2/Bax ratio among four groups（P<0.01）.Compared with the sham group,the expression of Bcl-2 was significantly decreased in model group（P<0.05）while Bax was significantly increased（P<0.01）.Thus the ratio of Bcl-2/Bax was markedly reduced in the rats of model group（P<0.01）.However,rats drank HRW simultaneously enhanced the expression of Bcl-2（P<0.01）and decreased the expression of Bax（P<0.01）which led to a significant increase of Bcl-2/Bax ratio（P<0.01）.The Bcl-2 relative expression in Don group was as similar as in HRW group（P>0.05）,but the reduction of Bax was more than that in the HRW group（P<0.05）,so that,the ratio of Bcl-2/Bax was obviously less than that in the HRW group（P<0.05）.Summary:1.Apoptosis of rat hippocampal neurons was activated 4 weeks after2VO,which mediates programmed cell death.2.HRW attenuates neuronal apoptosis in hippocampal CA1 area in rats subjected to chronic cerebral hypoperfusion and then improves hippocampal tissue damage.3.The effect of HRW on oxidative stress plays an important role in the mechanism of anti-neuronal apoptosis.Part fourThe influence of Hydrogen-rich Water on Autophagy of hippocampal neurons in rats with vascular dementia and its Effect on Fox01-mediated autophagy pathway.Objective:To observe the effects of HRW on the expression of autophagy-associated proteins 4 weeks after 2VO,and explore the effect of regulating neuronal autophagy and the influence of FoxO1-mediated autophagy pathway.Furthermore,we explore the neuroprotective mechanism of HRW.Methods:1.Three rats chosen randomly from each group were anesthetized and perfused with 4%paraformaldehyde.Then the brains were embedded in paraffin.Coronal brain sections were cut and immunohistochemically stained for Microtubule-associated protein 1 light chain 3（LC3）.Expressions of LC-3positive cells in CA1 region of hippocampus in each group were observed under light microscope.2.Furthermore,another six rats chosen randomly from each group were fully anesthetized.The hippocampi were quickly dissected and homogenated to detect the protein expression of LC3B,B-cell lymphoma-2 interacting protein 1（Beclin 1）,p62,Forkhead box proteins 1（FoxO1）and autophagy associated gene 7（Atg7）by western blot.Results:1.Expression of LC3-II immunopositive cells in hippocampal CA1region in four groups of rats.The expression of LC3 immunopositive cells in the CA1 region of the hippocampus in the model group rats was positive.The positive products were brown granules and were mainly located in the cytoplasm of neurons.The IOD/area value of LC3-II among all groups was significantly different（P<0.01）.CCH increased the IOD/area value of LC3 in CA1 region relative to the sham group（P<0.01）.After four week administration of HRW,the IOD/area value of LC3 was significantly decreased compared to that in the model group,as the same as administration of donepezil（P<0.01）.2.Effect of HRW on the expression of Beclin1,LC3 and p62 in hippocampi of VD rats.Obvious increase of the relative expression of Beclin 1 was found in the model group when compared with the sham group（P<0.01）,while they were significantly ameliorated in the HRW and Don group（P<0.01）.In addition,an obviously decline of LC3-I after four weeks suffered 2VO surgery when compared with the sham group（P<0.01）,whereas the expression of LC3-II slightly increased（P>0.05）and the LC3-II/I ratio increased significantly（P<0.01）.HRW and donepezil significantly reduced the elevated expression of the ratio of LC3-II/I,（P<0.01;P<0.01,respectively）,whereas there was not a significant difference between the two groups（P>0.05）.Additionally,the relative expression of p62 was obviously decreased in the model group as compared with the sham group（P<0.01）,while that in the HRW group showed a salient enhancement when compared with the model group（P<0.01）.Furthermore,the p62 level in the Don group were significantly higher than that in model group（P<0.01）,however,there did not show a significant difference between the HRW group and the Don group（P>0.05）.3.HRW decrease the expressions of FoxO1 and Atg7 proteins in the hippocampus.When compared with the sham group,the expressions of FoxO1 and Atg7 in hippocampus were significantly increased in model group（P<0.01;P<0.01,respectively）.However the elevations in the 2VO groups were significantly decreased in the HRW group（P<0.01;P<0.01,respectively）.The levels of FoxO1 and Atg7 were sharply decreased in the HRW group compared with that in the Don group.Meanwhile,there were not differences between the Don group and the model group（P>0.05;P>0.05,respectively）.Summary:1.Autophagy in neurons of hippocampi is activated.2.HRW suppresses the activation of autophagy in hippocampus 4 weeks after the animals suffered 2VO surgery.3.FoxO1 may be a key target for regulating autophagy.HRW treatment could increase FoxO1-mediated autophagy induced by CCH in rats,at least in part.Conclusions:1.The rats induced by CCH through 2VO had obvious cognitive dysfunction.CCH injury can cause oxidative stress reaction to neurons in the hippocampal CA1 region and induce apoptosis and autophagy which mediates programmed cell death.To some extent,2VO rat model,similar to the pathogenesis of clinical VD,is an ideal animal model of VD.2.HRW has effects of anti-oxidative stress,anti-apoptosis and anti-autophagy.It reduces programmed cell death,reduce pathological changes in hippocampus of VD rats.HRW attenuates not only the spatial learning impairment but also the memory impairment that induced in 2VO rats,which plays a neuroprotective effect in the learning and memory abilities.3.FoxO1 may be a key target for regulating autophagy.HRW treatment could increase FoxO1-mediated autophagy induced by CCH in rats,at least in part. |
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