| Malignant cerebral ischemic stroke which are always caused by middle cerebral arteries and internal carotid occlusion is a kind of acute cerebrovascular disease. It could lead to brain tissues ischemia, edema and necrosis in the large areas. Middle cerebral artery infarctions are one of the most highoccurrence diseases, with a mortality of up to80%in untreated patients. Thus, how to treat the malignant cerebral ischemic stroke, and protect the brain cells to reduce the lethality and disability rate has been the focus all over the world. Thrombolytic therapy is the most effective method for the treatment of acute cerebral infarction, but the short Time Window limits (4.5h) the clinical use of it. Although the survival benefit from hemicraniectomy is undisputed, the functional outcome of surviving patients treated with this procedure is variable. There are hundreds of pharmacological approaches to prevention and management of the developing brain ischemia, and tens of drugs were used in randomized clinical trials, but their efficacy has not yet been proven. Thus, nonpharmacotherapies of neuroprotection are important for the treatment of this disease.The recent introduction of therapeutic hypothermia has improved acute stroke therapy considerably. The brain protection of hypothermia has been studied all over the world, and it has been shown that hypothermia has definite neuroprotection. At present, systemic hypothermia is the main cooling method, but poor efficiency and severe complications limit its use and weak its neuroprotection. Selective brain cooling (SBC) is different from systemic cooling because it just cooled the temperature of brain, even the lesion area in brain, which avoids kinds of side effects and improves the efficiency of cooling. Intracarotid infusion which reduces the encephalic blood temperature directly, may be the fastest method for inducing hypothermia. The previous studies have proved that intracarotid cold saline infusion (ICSI) is an effective treatment for ischemic stroke. Furthermore, a clinical pilot study has shown that the feasibility, safety and physiological responses of ICSI are satisfactory. However, what is the relationship between the neuroprotection and parameter setting of ICSI? where is the neuroprotection of ICSI from, and how much the proportion is shared by the different elements in ICSI for neuroprotection in different situations? How to overcome the drawbacks of ICSI which includes hematocrit (Hct) drop and short duration?In these studies, firstly, to choose an appropriate animal model of ICSI, we studied the variability of model of middle cerebral artery occlusion (MCAO) in New Zealand rabbits and Sprague-Dawley (SD) rats by comparing the coefficient of variation of them. Secondly, the security and feasibility of ICSI with different parameters were studied. The neuroprotective effects of ICSI, ice cap and systemic cooling in rats with early cerebral ischemia were compared. Additionally, we assessed the relative weight of hypothermia and cerebral artery flushing in neuroprotection in a rat ischemia model. Finally, we describe and investigate the effects of interrupted ICSI relative to the traditional ICSI. This novel method could overcome the negative effects of ICSI to a certain extent.Chapter1Study on the animal model of focal cerebral ischemiaAnimal model is important and basal for a fine experimental research. Rabbit is more appropriate for studying ICSI than rat because the proportion of rabbit brain in their weight is far smaller than rat and human. Due to transient focal ischemia model in rabbits is seldom seen, the variation of this model was observed, and compared to the same model in rats.Firstly, for selecting sensitive quantitative electroencephalograph (qEEG) indexes for brain injuries, the qEEG were used during the whole ischemia. There are the positive correlation between infarction and the decline of the band, the upper and lower bound of amplitude-integrated electroencephalography (aEEG), and the absolute band power P, and there are the positive correlation between edema and the decline of the band, the upper and lower bound. But there is no correlation between the neurological severity scores and all indexes of EEG. The regression equation between the infarction (y) and the decline of the lower bound of aEEG (x) is y=-10.514+43.345x (F=16.602; P=0.001; R2=0.510), and between the edema (y) and the decline of the band of aEEG (x) is y=-1.943+18.563x (F=9.191; P=0.010; R2=0.434)Then, the variability of cerebral infarct in rabbit temporary brain ischemia induced by transorbital approach was assessed by observing the relation between aEEG lower bound and infarct size. Results showed that the coefficient of variation (CV) of relative and absolute infarct volume were75.0%and68.6%, respectively. Three types of aEEG change were found during surgery, which were drop completely, drop incompletely and recovery following complete drop. The2,3,5-triphenyltetrazolium chloride (TTC) staining showed the complete infarct in MCA territory in rabbits with drop completely of aEEG lower bound, incomplete infarct in animals with drop incompletely of aEEG lower bound, and either normal or infarct were founded in the rabbits with recovery following complete drop of aEEG lower bound. The results implied there were different extents of cerebral collateral flow, which might be the material cause for significant variability of infarct size in rabbits temporary MCAO. Following, we observed the variability of SD rats with similar model, the changes of EEG which were similar with rabbits was not seen, and the CV of relative and absolute infarct volume were9.5%and10.7%, respectively. Thus, we chose rats in following studies.Chapter2Study on the parameters of intracarotid cold saline infusionThe deepness and duration of hypothermia are important for the neuroprotection of ICSI. First of all, we observe the feasibility and security of the SBC inducing by ICSI, which would be the early and basic research for the ICSI.10-13℃saline solution was injected in the certain velocity by a perfusion pump, and the brain temperature was dropped to deep (24-26℃), moderate (28-30℃), and mild (32-34℃) hypothermia for1h, as well as mild hypothermia for2h. After that, the physiological indexes, ischemia, morphology of brains and internal carotids, and mortality would be observed. The injecting velocity arriving at deep, moderate and mild hypothermia were15.4,15and10ml/100g/h in35.0±5.2,21.7±3.5and5.3±1.5min, respectively. Deep and moderate hypothermia for1h and mild hypothermia for2h dropped the Hct significantly, but it was not observed in mild hypothermia for1h. The glucose after cooling in hypothermia groups was lower than control group significantly. TTC and hematoxylin and eosin (H-E) straining showed no ischemia and unusual changes of microstructure of brain occurred after cooling. Mortality of deep and moderate hypothermia groups were63%and38%, but no animal died in mild group. Thus, the insecurity exists in deep and moderate SBC inducing by ICSI due to physiological indexes being influenced apparently. The mild SBC inducing by ICSI with10ml/100g/h in1h is feasible and safe.Then, we compared the neuroprotective effects of ICSI, ice cap and systemic cooling in rats with early cerebral ischemia. SD rats were randomly divided into the model group, ice cap group, systemic cooling group and ICSI group (n=13). Cerebral ischemia was induced by MCAO for2h. In the3hypothermic groups, the target temperature of33-34℃was achieved and maintained for20min. In each group, the neurological severity scores were recorded at48h after the reperfusion, and the brains were removed for measuring the infarct size using TTC. The brain water contents and serum levels of neuron-specific enolase (NSE), S100βand matrix metalloproteinase9(MMP9) were also measured. Compared with the model group, the ice cap, systemic cooling and ICSI groups all showed significantly reduced infarct size by27.4%(P<0.05),47.6%(P<0.01) and61.6%(P<0.01), respectively. The systemic cooling and ICSI groups, but not the ice cap group, had significantly lower brain water contents than the model group (P<0.05). Among the3hypothermic methods, only ICSI significantly improved the neurological severity scores (P<0.05) and reduced serum NSE and S100β levels (P<0.05). Of the3hypothermic groups, ICSI has the best neuroprotective effects, and systemic cooling produces better effect than ice cap in rats with ischemic stroke.Through the studies above, we obtained the safety use scope of ICSI. Meanwhile, we found, in these use scope, ICSI has better neuroprotective effects than systemic cooling and ice cap. However, the best duration of ICSI for treating the ischemia stroke is yet unknown, so we observed the neuroprotection of ICSI with different durations for the infarct size in2-h MCAO rats, we found ICSI with different durations could reduce the infarct size, but there are no significant differences among groups, which suggests except hypothermia, flushing in ICSI should not be neglected.ICSI protects against ischemic stroke not only due to the resulting hypothermia, but also as a result of flushing of the cerebral arteries. In this study, we assessed the relative weight of hypothermia and cerebral artery flushing in neuroprotection in a rat ischemia model using hypothermic and normothermic saline infusions initiated over a range of times after the initiation of reperfusion. Ischemic strokes were induced in SD rats (n=115) by occluding the middle cerebral artery for2h using an intraluminal filament. In the hypothermic groups, the brain temperature was lowered to33-34℃for20min by ICSI at three time points (0,1and2h) after reperfusion. Correspondingly, in the normothermic groups, the brain temperature was maintained at normal levels during intracarotid normothermic saline infusion (INSI) for20min at the same time points.48h after reperfusion, infarct sizes and brain water contents were determined using TTC staining and the dry-wet weight method, respectively. Levels of NSE, S100β and MMP9in the serum were determined by enzyme-linked immunoassay (ELISA). Neurological deficits were also evaluated. Immediate infusion after the onset of reperfusion (0h) did not result in significant difference for reductions of infarct sizes, neurological deficits or S100β serum levels between ICSI and INSI groups, compared with the non-infusion group. However, brain water content and NSE serum level were significantly lower in the ICSI group than the non-infusion group. When the infusions were started1h after reperfusion, both ICSI and INSI infusions still reduced the infarct sizes, but only ICSI significantly decreased the brain water content, neurological deficits and S100β serum level. All therapeutic effects of INSI disappeared when infusions were started2h after reperfusion, whereas infarct size, neurological deficits and S100β serum level were still reduced significantly in ICSI group, compared with the non-infusion group. All in all, the neuroprotection of hypothermia and cerebral artery flushing in ICSI after ischemia weakens as the length of time between the reperfusion and infusion increases. The effect of cerebral artery flushing weakens faster than the effect of hypothermia.Chapter3Tentative exploration on interrupted pattern of intracarotid cold saline infusion ICSI is an effective method for protecting brain tissue, but its use is limited due to undesirable secondary effects, such as severe drops in Hct levels, as well as its relatively brief duration. In this study, we describe and investigate the effects of a novel ICSI infusion pattern (interrupted ICSI) relative to the traditional method (uninterrupted ICSI). Histology showed that interrupted ICSI did not affect neurons or fibers in rat brains, which suggests that this method is safe for brain tissues with ischemia. The duration of hypothermia induced by interrupted ICSI was longer than that induced by the traditional method, and the decrease in hematocrit levels was less pronounced. There were no differences in infarct size or brain water content between uninterrupted and interrupted ICSI, but NSE and MMP9serum levels were more reduced after interrupted ICSI than after the traditional method. In conclusion, interrupted ICSI has the potentional-improved neuroprotection for treating acute ischemic stroke, and has less detrimental side effects than the traditional ICSI method, thereby making it more attractive as an infusion technique in the clinic.Base on previous experimental data, we made a mathematical model for analyzing interrupted ICSI. We found if initial temperature is unchanged, the duration of intervals depend on the treatment section, and the temperature of the treatment section lower, the intervals shorter. Under the same initial temperature and treatment section, the fluid volume of cooling from initial temperature to target temperature, or from the upper bound of the treatment section to the lower bound is constant, and irrelevant with infusion speed. Under the same initial temperature, the duration of cooling has a negative correlation with infusion speed. While with the same infusion speed, the initial temperature has a positive correlation with the whole duration of cooling. When initial temperature is37℃, the treatment section is [34.5,35.5], and the target temperature is34.5℃,the volume is7.5ml, the longest duration of hypothermia (74min) can be obtained by the infusion speed of12ml/h, while when the duration of hypothermia is70min, the least volume (7.5ml) can be obtained by the infusion speed of9ml/h. |
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