Cognitive Dysfunction Associated With Synapse-Related Proteins Expression In Rats Following Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a type of acute cerebrovascular disease with incidence only lower than cerebral infarction and cerebral hemorrhage. Most SAH are caused by rupture of cerebral aneurysms and its mortality is decreased by 17% over the past 30 years, the survival rate reaching 65%, while 50-60% of patients still have different levels of cognitive dysfunction and behavioral changes mainly in memory, execution, and language skills, which affect their work and life and hence bring a heavy burden on society and family. Cognitive dysfunction is consider as the most devastating complication following SAH beside rehaemorrhagia and cerebral vasospasm, and is partly responsible for the low curative effect of the treatment of cerebral vasospasm with Clazosentan, an endothelin receptor antagonist. However, the mechanism by which SAH leads to cognitive dysfunction is still unclear.(?)rbo M. et al. noted that the amount of bleeding and the severity of SAH were not the determinant factors for cognitive dysfunction, while its complications such as delayed seizures and intracranial hypertension might exert greater effect. Haug T. et al. observed that cognitive dysfunction had no association with cerebral vasospasm after SAH, considering that cognitive dysfunction occurred in the patients who have recovered from cerebral vasospasm for one year. Other studies showed that comprehensive brain injury, including intracranial hypertension, blood-brain barrier damage, cerebral edema, decreased cerebral blood flow, oxygen free radicals, etc, and chronic inflammatory mechanism were involved into SAH. As such, the pathophysiological mechanism underlying SAH needs to be further clarified. Little animal study has been conducted until now. Jeon H et al. confirmed that some hippocampal neurons have been damaged after subarachnoid hemorrhage, primarily manifested as apoptosis, and most neurons maintained intact with light damage. It is insufficient to explain the cognitive damage caused by SAH. Therefore, we hypothesize that synapses that are closely associated with neuron functions may play a crucial role in cognitive damage after SAH.Hippocampal synaptic plasticity is the biological basis of learning and memory. Tariq A, et al. found that after SAH, hippocampal long-term potentiation (LTP) was lost in CVS, synaptic transmission in CA3-CA1 was damaged, and synaptic plasticity was reduced, suggesting that these alterations were linked to memory decline after SAH. Synapse-related proteins are executor of synaptic transmission, synaptic plasticity and other functions, and are an important molecule in learning and memory. These proteins and there phosphorylation levels are associated with many factors that affect learning and memory. Synaptic transmitter release from vesicles in nerve ending s is precisely regulated process, wherein a variety of proteins that interact in a network are involved. Every step in the process, including protein complex assembly and conformation regulation, the transportation and dorking of synaptic vesicles, membrane fusion, neurotransmitter release, reuptake of membrane protein etc., needs the participation of interaction among various proteins. Bolay H. et al. observed that synaptic transmission was blocked in ischemic injury, which was associated with the selective decrease of phosphorylized synapsin IIn recent years, more and more proteomic approaches were widely used in neuroscience research especially in neural degeneration. Researches on cerebral vascular disease have started. Honer WG et al. have found a variety of abnormally expressed synaptic proteins in Alzheimer’s disease using proteomic approaches, including synaptotagmin, synapsin, rab3a, AP2 and so on. However, there is litter report about different expression of synapse-related proteins after SAH.This study was to establish delayed cerebral ischemia models in rats after SAH, detect ultrastructural changes of synapse, and conduct Morris water maze as behavioral test. Immunohistochemistry and westernplot were used to detect dynamic changes of synapsin I and its phosphorylated form. Synaptosome in rats brains were extracted using sucrose gradient centrifugation and underwent proteomic approach to compare its expression of synapse-related proteins before and after SAH for exploring the molecular mechanisms in cognitive impairment after SAH and providing cue for new targets in drug therapy. This study consists of three parts.Part I Delayed cerebral ischemia models in rats following subarachnoid hemorrhage and cognitive function evaluationObjectiveTo study regional cerebral blood flow, the dynamic changes of behavior and synaptic test ultrastructural changes in double blood injection model of SAH to evaluate the degree of cognitive impairment and explore the feasibility of establishing cognitive impairment model.MethodsSAH model was built using double blood injection into cisterna magna with arterial blood. Healthy adult Wistar rats were randomly divided into control group, sham operation group and SAH model group, and each group was subdivided into five subgroups in time series (1h, 1d,3d,5d,14d). Basilar artery diameter was measured using stereomicroscope and dynamic loop detection. rCBF was observed using laser Doppler flowmetry under control of stereotactic instrument. Perfusion fixation through hearts was conducted with 2% paraformaldehyde and 1.25% glutaraldehyde. Brains were sampled and cut into slices. Using transmission electron microscope, interface structure of synapses (synaptic interface curvature, sudden contact gap width, and the thickness of postsynaptic density quality) was measured. Morris water maze was taken for behavioral test for five days, calculating the average escape latency and the percentage of swimming time in platform in total swimming time as a measure of memory to detect the different grades in water maze.Results 1. Basilar artery diameterBasilar artery diameters of SAH3d,5d rats were significantly narrowing than that of controls (P<0.01); SAH14d BA diameter contracture was not obvious.2. Dynamic changes of regional cerebral blood flow in parietal cortexAfter double injections, rCBF in SAH model group was rapidly decreased, reached the lowest value at 1h, about 51.8% -55.7% of preoperative value. Then, rCBF recovered at 1d, about 93.8% of preoperative value, yet declined at 3d and 5d. At 14d, it did not restore to controls. Compared with sham operation group, SAH group has a significantly different blood flow at 1h,3d and 5d (P<0.01).3. Transmission electron microscope (TEM) resultsCompared with controls, rats in SAH group had fewer vesicles in presynaptic membrane in hippocampal neurons. In SAH group rats, synaptic membrane was blurred, cleft was widened, postsynaptic dense material was thinning, some mitochondria disappeared, rough endoplasmic reticulum and myelin broke down. Synaptic active zone length, the width of synaptic cleft and PSD thickness were significantly less in SAH 14d group than in controls (p<0.05).4. Morris water maze testIn navigation test, SAH3d rats had a significant long average latency at day 4 compared with sham operated rats, as well as SAH5d and SAH14d rats at day 2 (P<0. 05). In space exploration experiments, SAH5d and SAH14d rats had a significant shorter platform swimming time than sham operated rats (p<0.01).ConclusionSAH models using double blood injection into cisterna magna into cistern present characteristics of delayed cerebral vasospasm and cognitive impairment and provide solid basis for further exploration of SAH pathophysiological mechanism and drug treatment.Part II The Dynamic Changes of Synapsin I And its Phosphorylated Form in Rat Brain Following Subarachnoid HemorrhageObjectiveTo study the dynamic changes of synapsin I and its phosphorylated form and to explore its role in delayed cerebral ischemia after SAH and the development of cognitive impairment.Methods1. SAH model was built using double blood injection model. Healthy adult Wistar rats were randomly divided into sham operation group and SAH model group, and each group was subdivided into five subgroups in time series (1h, 1d,3d,5d,14d). After perfusion fixation, brains were separated and cut into slices. Using immunohistochemistry and fluorescent chemical staining, the location of synapsin I and its phosphorylated form in brain tissues was observed in optical microscopy and laser confocal microscope.2. After double injections of fresh autologous arterial blood into cistern, healthy adult Wistar rats were randomly divided into control group, sham operation group and five SAH model group (1h, 1d,3d,5d,14d). After rats were sacrificed, synapses in hippocampus and cortex were extracted using sucrose gradient centrifugation and were treated with lysis for western blot to analyze the difference of synapsin I and its phosphorylated form between these groups.ResultsThe microscopic observation showed that synapsin I and its phosphorylated form were mainly located in neuropil, but not in nucleus of neurons, glial cells and blood vessels. The proteins were principally expressed in cerebral cortex and hippocampus, especially in hilus fascia dentate region. Immunohistochemistry and western blot showed that the expression synapsin I in cortex and hippocampus was lower in SAH rats than in sham operated rats at each time points, deceasing at day 1, reaching the lowest at day 3 (P<0.01) and recovering to the level of sham operation group approximately at day 14. Phosphorylated synapsin I in SAH rats also declined, but still had a significant difference from sham operated rats (P<0.05).Conclusion1. Synapsin I and phosphorylation levels of cerebral cortex and hippocampus in SAH rat were significantly reduced after SAH 3d to reduce the most significant, with SAH secondary to cerebral vasospasm of the same extent that there is a certain correlation between synapsin I and its phosphorylation and SAH delayed cerebral ischemia.2. There is a certain degree of phosphorylation dysfunction of synapsin I in rats’ cortex and hippocampus. The inhibition of phosphorylation of synapsin I may be one of pathophysiological mechanisms underlying cognitive impairment following SAH. The related phosphorylation sites in synapsin I may be a potential target for intervention treatment of cognitive impairment following SAH. Part III A Proteomic Study of Synaptosome Proteins in Rats with Cognitive Impairment Following Subarachnoid Hemorrhage ObjectiveTo compare synaptosome protein spectrum in rats with delayed cerebral ischemia with that in sham operation group to screen and identify proteins closely related to cognitive impairment following SAH, and to search new mechanisms underlying cognitive impairment.MethodsWistar rats that have undergone water maze behavioral test were selected and randomized into sham operation group and SAH5d group, with eight rats in each group. Hippocampal synaptosome proteins were separated in the first dimension with immobilized pH gradient isoelectric focusing (IPG-IEF), and then sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was chosen for the second dimension. Electrophoretograms were analyzed using image analysis software PDQUEST7.0 to find protein spots with meaningful difference. Mass spectrometry analysis was performed using ultraflex TOF/TOF MS. Protein information was obtained from NCBInr database.Results1.2-DE map of synaptosome-related proteins in SAH rats cerebral cortex and hippocampus was generated, and synaptosome-associated protein isoelectric points (PI) were between 4-7, suggesting that proteins were well separated. Eight differentially expressed protein spots were found in rats cortex and six protein spots in hippocampus.2. Compared with sham operation group, SAH5d group had an increased expression of centrosome protein 63 KD in rat hippocampus and a decreased expression of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), nebulin-related-anchoring protein isoform C (N-RAP), general control of amino-acid synthesis 1-like 1 (GCN1L1), aldehyde dehydrogenase 1 andβ-actin.3. Compared with sham operation group, SAH5d group had a upregulated expression of SH3-domain GRB2-like endophilin B2(SH3GLB2) in cerebral cortex and a downregulated expression of phosphatidylethanolamine-binding protein (PEEP1), UCH-L1, heat shock cognate 71 kDa protein (HSP71), Glyceraldehyde-3-phosphate dehydrogenase(GAPDH3), ATP synthase and its subtypes, and M-phase phosphoprotein 1.Conclusion1. There is different synaptosome-associated protein expression in rat hippocampus and cortex between SAH rats and sham operated rats.2. According to their biological function, these differentially expressed proteins can be categorized into four types:cytoskeletal proteins, energy metabolic enzymes, signal transduction proteins and chaperones. These proteins may play paramount roles in synaptic plasticity, microtubule transport, neurotransmission etc. It is helpful to further exploration in understanding of mechanism underlying impairment following SAH.RESEARCH SIGNIFICANCEWe established delayed cerebral ischemia rat models following SAH and performed Morris water maze test to evaluate cognitive ability. It is the first study which has explored the role of synapsinⅠphosphorylation in delayed cerebral ischemia following SAH. Furthermore, we also obtained 14 differentially expressed synaptosome-associated proteins in cognitive impairment following SAH. Our findings will be helpful to a further understanding of the underlying mechanisms and new target for clinical treatment.