| Background:It has been estimated that1–6%of the world population may harbor anintracranial aneurysm and that each year～10/100,000people suffer from ananeurysmal subarachnoid hemorrhage (SAH). Despite major advances in surgicaltechniques, radiology, and anesthesiology, the mortality and morbidity rates afterspontaneous SAH have not changed in recent years. Early brain injury includingelectrophysiological disorders, rather than cerebral vasospasm, may contribute tothe high mortality and morbidity rate of SAH. Electrophysiological disorders suchas "cortical spreading depolarization," first appeared at the early stage after SAH.This kind of neuronal hyperexcitability originates from a temporary disruption oflocal ionic homeostasis and ultimately contributes to delayed ischemic neurologicaldeficit in patients with SAH.The hyperpolarization-activated/cyclic nucleotide (HCN)-gated channels are amixed-cation conductance, encoded by4genes (HCN1–4), and widely distributedin peripheral and central neurons.An abundance of evidence has demonstrated thatHCN1channels are critical for the regulation of neuronal excitability inhippocampus CA1region and neocortex. In these brain areas, HCN1subunits aredensely distributed and expressed on the dendritic spines of pyramidal neurons,and are involved in the integration of excitatory synaptic input and therebyinfluences the excitability of neural network. Interestingly, cortical spreadingdepolarization is more readily provoked in hippocampus CA1sector and neocortex,and its generation as well as propagation is also greatly dependent on the apicaldendrites of pyramidal neurons. Therefore, we speculate that HCN1channels arepotential regulative targets which contribute to the formation of neuronalhyperexcitability after SAH. Methods:1. The endovascular perforation model of SAH was established in vivo. Firstly,the mortality rat and extent of SAH were observed. Then, HE staining was appliedto observe the blood clots distributed within the cerebral ventricles nearhippocampus tissue at24and72h after SAH. Furthermore, the extent of Hbpenetrated into hippocampus tissue around cerebral ventricles was evaluated bythe method of immuno-histochemistry.2. Western Blot and RT-PCR were applied to detect the changes of expression ofHCN1in hippocampus CA1region at24and72h after SAH.3. For patch-clamp studies, Sprague–Dawley rat pups (p21-24) were used forslice preparation. Whole-cell recordings of HCN channels and neural activity wereconducted in hippocampus CA1pyramidal neurons in the presence and absence ofhemoglobin (Hb)-containing artificial cerebrospinal fluid (CSF). Additionally, somecells were intracellularly labeled with biocytin (0.5%) to confirm morphologicalidentification.4. At1,2,3and4h after perfusion of hippocampus slices with Hb, the NOlevels of hippocampus CA1tissue were assayed according to the instructions in theNO detection kit. For patch-clamp studies, NO/Sp or L-NNA was applied duringthe perfusion of Hb on CA1pyramidal neurons before the excitatory effect of Hbevaluated. Simultaneouly, the influence of Hb on HCN channel was observedagain in the presence of NO/Sp or L-NNA.Results:1. The mortality rate in the SAH group was38%. None of the sham operatedcontrol animals died during experiment. At24h post hemorrhage, the SAH scorewas13±2out of a possible18in the SAH groups. HE staining showed dense redblood clots distributed within the cerebral ventricles near hippocampus tissue at24and72h after SAH. Furthermore, the immune-positive results of Hb demonstratedHb penetrated into hippocampus tissue around cerebral ventricles after SAH.Especially, the distribution of Hb in hippocampus tissue at72h is more extensivethan that at24h after SAH. 2. HCN1protein expression in SAH group was significantly reducedcompared with control group, and the decrease at the72h post-SAH point wasmore pronounced. Similar to the results of Western blot, HCN1mRNA expressionreduced obviously in SAH group. Concretely, HCN1mRNA expression reduced by60.7%±5.4%at24h post-SAH point and81.2%±4.1%at72h post-SAH point,respectively.3. Hippocampus CA1pyramidal neurons display electrophysiological featureof HCN currents. Hb induced a moderate fluctuation of membrane potential,accompanied with a rapid firing of action potentials (APs). Interestingly, Hbsynchronously produced a significant decrease in the amplitude of HCN currents.CsCl, a HCN blocker, was pretreated before Hb administration. The resultsindicated that there was no significant increase of spike firing between applicationof CsCl alone and CsCl/Hb combined. Additionally, all the biocytin-labeled cellshad morphological features of CA1pyramidal neurons as described previously.4. The level of NO released from hippocampus slices had a dramatic drop at1h after Hb perfusion, and then slowly decreased at2,3, and4h after Hb perfusion.Simultaneously, the protein expression of HCN1in CA1region decreasedmoderately at1and2h after Hb perfusion, and then had a significant decrease at3and4h after Hb perfusion. NO/Spermine, a controlled releaser of nitric oxide,attenuated neuronal excitability and enhanced HCN currents in CA1pyramidalneurons. L-NNA, an inhibitor of nitric oxide synthase (NOS) reduced the HCNcurrents. The inhibitory action of Hb on HCN currents was reversed by applicationof NO/Sp, which also reduced neuronal hyper-excitability.Conclusion:These observations demonstrated a reduction of HCN channels expressionafter SAH and Hb reduced HCN currents in hippocampus CA1pyramidal neurons.These results revealed a functional interaction between Hb and HCN channels afterSAH. Hb, released from blood clot after SAH, exhausted the NO signaling, thusinhibited HCN channels, and consequently induced or facilitated the formation ofneuronal hyperexcitability in hippocampus CA1region after SAH. In a word, thepresent results implied that the change of HCN channels may be a novel process involved in the formation of neuronal hyperexcitability after SAH, and mayprovide new therapeutic clues in patients with SAH.
|
PDF Full Text Request