Caffeine Protects Against Hypoxia-ischemia Induced White Matter Injury By Promoting Histone Acetylation Of Oligodendrocytes In Neonatal Rats

Objectives:Cerebral white matter injury（WMI）is the most common form of premature brain injury.It is considered to be the leading contributor to the neurodevelopmental disabilities in premature infants.Periventricular leukomalacia（PVL）is a severe form of premature WMI.The neurological sequelae induced by WMI are diverse,including motor,cognitive,behavioral,intellectual,visual and auditory disorders,etc.The pathological changes of WMI are commonly believed to relate to oligodendrocytes injury and the subsequent myelination failure.However,the broad spectrum of the disabilities caused by premature WMI cannot be simply explained by hypomyelination.Synaptic injury may be related to the diversity of neurological sequelae in WMI survivors.Neuronal damage was found in brain tissue of neonates with WMI at autopsy,suggesting synaptic injury.Neurons in the thalamus are most commonly affected.However,studies on synaptic injury and the exploration of WMI treatment based on this pathological change are rare.The clinical manifestations of premature WMI are not typical and the diagnosis of the disease is often delayed.Moreover,there is no safe and effective treatment until now.It was found in a multicenter randomized controlled clinical study that early caffeine use to prevent and treat apnea in preterm infants reduced the incidence of bronchopulmonary dysplasia（BPD）and improved the long-term neurological outcome.However,it is not clear whether the improved neurological outcome related to caffeine is associated with premature WMI.Caffeine is a non-specific antagonism of adenosine receptor,particularly the A₁ and A_2AA receptors,which can affect the function of the respiratory center,stimulate the respiratory rhythm and eventually treat apnea of prematurity.However,the mechanism by which caffeine improves premature neurological outcomes is still unclear.In this study,we will investigate whether hypoxia/ischemia（HI）in neonatal rats results in synaptic abnormalities accompanied with myelination failure.We will also explore whether caffeine has neuroprotective effect in a neonatal rat model of HI-induced WMI and observe the specific effect of caffeine on both synaptic abnormalities and myelination failure.The optimal dosage of caffeine will be investigated.Finally,the mechanism of the neuroprotective effect of caffeine via improving the level of histone acetylation in oligodendrocytes and activating BDNF/TrkB pathway will be investigated.Methods:1.The 3-day-old（P3）Sprague Dawley（SD）rats were randomly divided into the control group and the hypoxia-ischemia（HI）group（n=82 each group）.The right common carotid artery ligation and subsequent hypoxia（8%oxygen for 2.5 hours）were performed after inhalation anesthesia in P3 SD rats of HI group.HE staining was performed to observe the pathological changes of brain tissue at 3,7,14 and 21 days after HI injury.Western blotting was performed to detect the expression changes of synaptophysin,PSD-95 and myelin basic protein（MBP）.Immunofluorescence was performed to detect expression of synaptophysin and MBP.Immunohistochemical examination was conducted to detect expression of psd-95.Immunofluorescent examination of Olig2,a pan-marker of oligodendrocytes,was performed to detect changes in the number of oligodendrocytes.The number and ultrastructure of synapses were observed by transmission electron microscopy.The learning and cognitive behavior in two groups was examined in the Morris water maze test.2.The P2 rats were randomly divided into 5 groups（n=16 each group）:control group,HI group,HI+caffeine（10mg/kg）group,HI+caffeine（20mg/kg）group and HI+caffeine（50mg/kg）group.Caffeine administration（intraperitoneal）was conducted daily from P2-P6.Hypoxia and ischemia were performed after inhalation anesthesia in P3 SD rats.Body weight and brain weight in each group were recorded.Western blotting was performed to detect the expression of MBP in each group at 14days after HI.The optimal caffeine dose was investigated and was selected for subsequent experiments.3.The P2 rats were randomly divided into 3 groups（n=18 each group）:control group,HI group and HI+caffeine（optimal dose）group.The time and method of caffeine administration were the same as the above,and hypoxia and ischemia were performed after inhalation anesthesia in P3 SD rats.The expression of synaptophysin and PSD-95 were detected by Western blotting at 14days after HI.Immunofluorescence was conducted to detect synaptophysin expression.Synaptic number and ultrastructural changes were detected by transmission electron microscopy.The learning and cognitive behavior of rats in each group was detected in the Morris water maze test.Immunohistochemical examination of MBP and transmission electron microscope of myelin sheath were conducted after the Morris water maze test.4.The P2 rats were randomly divided into 4 groups（n=16 each group）:control group,HI group,HI+caffeine group and HI+caffeine+ANA-12 group.The time and method of caffeine administration were the same as the above,and hypoxia and ischemia were performed after inhalation anesthesia in P3 SD rats.The administration time of ANA-12（TrkB antagonist）was consistent with that of caffeine.Double immunofluorescence staining of the acetylated histones 3（AH3）with Olig2 was conducted on the 7th day after HI injury.Immunofluorescence was performed to detect expression of BDNF,p-TrkB,PDGFR.On the 14th day after HI injury,expression was detected by immunofluorescence staining with MBP and PLP,markers of mature oligodendrocytes.Results:1.（1）Oligodendrocytes reduction and MBP expression decrease induced by HI injury.Less oligodendrocytes in ipsilateral periventricular CC region were observed in HI group than control group at 7 d after HI exposure（P<0.001）.The length and density of myelinated fibers and the expression of MBP（P<0.01）in ipsilateral periventricular CC region all decreased in HI group at 14 d post HI injury;（2）Synapses reduction,synaptic ultrastructural abnormality and synaptic protein expression decrease induced by HI injury.At 14 d post HI injury,the number.of synapses decreased in ipsilateral thalamus in HI group（P<0.01）,accompanied with decreased length of active zone（AZ）and thickness of postsynaptic density（PSD）at ultrastructural level（P<0.05）.Furthermore,as detected in western blotting analysis,PSD-95 expression was significantly lower in HI rats than in control group from 14 d（P<0.01）to 21 d（P<0.05）post HI injury,while synaptophysin expression was significantly lower in HI group from7 d（P<0.05）to 14 d（P<0.01）post HI injury;（3）Impaired cognition after exposure to HI.As detected in the Morris water maze,significantly longer escape latency was observed in rats of HI group in the navigation test than the control group（P<0.05）.In addition,rats in the HI group passed less frequently through the platform location（P<0.01）and spent less time in the target quadrant（P<0.05）in the probe trial compared to the control group.2.（1）Changes of body weight and brain weight in rats after administration of different doses of caffeine.Compared with control group,decreased body weight and brain weigh was observed in rats of 50 mg/kg caffeine group at 14 d after HI injury（P<0.05）.At the same time,for rats of 20 mg/kg caffeine group,there were no brain weight and body weight loss;（2）Changes of MBP expression in rats after administration of different doses of caffeine.As detected in western blotting analysis,expression of MBP in ipsilateral periventricular CC region in rats of 20 mg/kg caffeine group at 14 d post HI injury was higher than HI group（P<0.01）.While the 10 mg/kg and 50 mg/kg caffeine group had no significant difference with HI group in MBP expression.The most significant improvement in the expression of MBP was detected in rats of 20mg/kg caffeine group.Therefore,we chose 20mg/kg as the optimal dose for this study and conducted following experiments.3.（1）Caffeine improved the cognitive and learning function impairment induced by HI injury.As detected in the Morris water maze,significantly shorter escape latency was observed in rats of HI+caffeine group in the navigation test than the HI group（P<0.05）.In addition,rats in the HI+caffeine group passed more frequently through the platform location（P<0.05）and spent more time in the target quadrant（P<0.05）in the probe trial compared to the HI group;（2）Effect of caffeine on MBP expression and myelin ultrastructural abnormality induced by HI injury.After caffeine administration,the improvement of length and density of myelinated neurofibers projected to the cortex was detected by immunohistochemical examination of MBP at 30 d after HI injury.At the ultrastructural level,more myelinated axons and thicker and regular myelin were detected in CC region in the control and HI+caffeine groups than in the HI group;（3）Effect of caffeine on synaptic protein expression and synaptic ultrastructural abnormality induced by HI injury.Higher number of synapses in thalamus was detected in HI+caffeine group than HI group（P<0.05）.However,there were no difference in AZ length,PSD thickness,synaptophysin and PSD-95 expression between HI group and HI+group.4.（1）Caffeine improved the expression of AH3,BDNF and p-TrkB in HI rats.Compared with the control group,the number of oligodendrocytes expressing AH3 in ipsilateral CC region declined at 7 d after HI injury（P<0.01）,accompanied by decreased expression of BDNF and p-TrkB（P<0.01）,which were considered as downstream protein of AH3.After caffeine intervention,compared with the HI group,AH3 expression in oligodendrocytes increased（P<0.05）,accompanied by improved expression of BDNF（P<0.01）and p-TrkB（P<0.001）;（2）Caffeine promote the differentiation and maturation of oligodendrocyte in HI rats.The expression of PLP（P<0.01）and MBP（P<0.001）decreased in HI group compared to the control group.While there was no significant change in the expression of PDGFR-αafter HI injury.After caffeine administration,the expression of PDGFR-α（P<0.01）,PLP（P<0.05）and MBP（P<0.01）increased in the HI+caffeine group compared to the HI group;（3）TrkB inhibitor ANA-12 weakened the effect of caffeine on promoting the differentiation and maturation of oligodendrocytes.Compared with the HI+caffeine group,the expression of p-TrkB decreased in HI+caffeine+ANA-12 group（P<0.01）,accompanied by the declined expression of PDGFR-α,PLP and MBP（P<0.05）.Conclusions:1.Hypoxia-ischemia induced WMI in newborn rat is not only limited to periventricular myelination disorder,synaptic ultrastructural abnormality in the thalamus is also involved,providing a more comprehensive pathological interpretation for premature WMI.2.Caffeine has a neuroprotective effect on hypoxia-ischemia induced WMI in newborn rat and improves the cognition and learning function by promotion of myelinogenesis.While caffeine has no effect on improvement of the impaired synaptic ultrastructure.Caffeine provides a novel direction for WMI treatment.3.The increased level of acetylated histone AH3 in oligodendrocytes of HI rats and subsequently improved expression of BDNF and p-Trkb,which will promote the differentiation of oligodendrocytes and myelinogenesis were thought to be a potential mechanism of the neuroprotective effect of caffeine.