| ObjectiveTo establish a selective brain hypothermia model induced by intra-carotid infusion of cold saline in rats and evaluate its safety on local brain tissue and physiological variables. To explore the effect of selective brain hypothermia on brain infarct volume, neurological functional outcomes and brain water content after brain ischemia.This study was performed to determine the neuroprotective potentials of intra-carotid magnesium sulfate infusion to ischemic stroke and find the optimal protective dose of magnesium sulfate for local infusion using the filament model of transient MCAO in rats.The aim of the study was to evaluate the protective effect of combination therapy with selective brain hypothermia and intra-carotid magnesium sulfate and determine whether intra-carotid infusion of cold magnesium sulfate would provide better neuroprotection against cerebral ischemia injury than cold saline infusion alone. MethodsFifty-eight male rats were randomly assigned into following groups:sham operated group (n=5), normal infusion group (n=5), stroke group (n=12), local hypothermic group (n=12), local normothermic group (n=12), systemic infusion group (n=12). Rats in normal infusion group and local hypothermic group received intra-carotid infusion of cold saline at the rate of0.4ml/min for20min. Both of brain and rectal temperature were monitored before, during and after cold infusion until the brain temperature returned to normal. Physiological variables including heart rate, mean arterial pressure, heamatocrit and blood gases were measured too. Brain sections were stained with HE and TTC to observe the morphological changes of brain tissue. Brain water content and neurological deficits were also evaluated. Rats with focal cerebral ischemia were given various infusions treatments following3h ischemia according to different groups. Physiological variables at different time point were compared among experimental groups. Brain infarct volume and cerebral water content were analyzed48h after MCAO. Neurological deficits were assessed using the mNSS24and48h after MCAO.Seventy-eight rats were randomly divided into following groups:sham operated group (n=6), stroke group (n=12), saline group (n=12), magnesium sulfate group A (n=12), magnesium sulfate group B (n=12), magnesium sulfate group C (n=12), and magnesium sulfate group D (n=12). There were four dose of magnesium sulfate (60mg/kg,90mg/kg,120mg/kg,150mg/kg) were used in the study. Each animals in local infusion groups was given an intra-carotid magnesium sulfate or saline infusion overe a period of20min at the initiation of reperfusion (180-200min after onset of ischemia). Before reperfusion was established,8ml of magnesium sulfate or saline was injected slowly and continuously via a catheter, using an infusion pump to control the infusion rate at0.4ml/min. Physiological variables including heart rate, mean arterial pressure, blood gases, glucose and hemotocrit were measured before MCAO, before and after reperfusion. Rectal temperatures were continuously monitored until48h after MCAO. Infarct volume and brain water content were measured48h after MCAO. Neurological examinations were performed24h and48h after MCAO using the mNSS.One hundred and fifty-two male rats were randomly assigned into following groups:sham operated group (n=8), stroke group (n=16), normothermic saline group (n=16), normothermic magnesium group (n=16), hypothermic saline group (n=48) and hypothermic magnesium group (n=48). Animals in hypothermic saline group and hypothermic magnesium group divided into three subgroups according to different initiation time of cold infusion. Each animal in local infusion groups was given an intra-carotid magnesium sulfate (15℃or37℃) or saline (15℃or37℃) infusion over a period of20min starting at the initiation of reperfusion (180-200min after onset of ischemia). Before reperfusion was established,8ml of magnesium sulfate (15℃or37℃) or saline (15℃or37℃) was injected slowly and continuously via a catheter, using an infusion pump to control the infusion rate at0.4ml/min for20min. Infarct volume and brain water contents were measured48h after MCAO. In a blinded manner, animals were examined for neurological deficit48h after MCAO using the mNSS. Evans blue (EB) concentrations in brain tissue were measured48h after MCAO. Concentrations of serum MMP-9were also measured48h after MCAO using Elisa.ResultsSignificant differences were found in brain temperatures among different time points (F=2035.541, P<0.001). There was no significant difference in rectal temperatures among different time points (F=1.448, P>0.05). At each time point after cold infusion, brain temperature was significantly lower than rectal temperature (P>0.05). There was no correlation between brain temperature and rectal temperature during the monitoring periods (r=0.220, P=0.152). Physiological variables including heart rate, mean arterial pressure, blood gases, glucose and hemocrit were not significantly different among each time point. Moreover, there were no correlation between brain temperature and all physiological variables. No significant morphological abnormal was found in brain sections stained with TTC and HE. Animals received local cold infusion did not show neurological deficits48h after infusion. All baseline data was balanced between different groups (all P>0.05). Brain temperatures were significantly different before and after local infusion among groups (F values were386.698and5480.737, both P=0.000). In the local hypothermic group, brain temperature was reduced to33-34℃within5-10min and significantly lower than that in other groups after5min of commencing infusion, and this significantly low temperature was maintained to nearly60min after infusion continued. There was no significant difference in rectal temperatures among groups. A significant difference was found in infarct volume among groups (F=124.402, P<0.001). Animals treated with local cold infusion had a significantly smaller infarct volume compared with other groups (P<0.05). Both of24h and48h mNSS were significantly different among all groups (F values were9.296and10.303, all P=0.000). Both24h and48h mNSS in local hypothermic group was significantly lower than that in other groups. There was a significant difference between24h and48h mNSS in local hypothermic group. Brain water contents were significantly different among all goups (F=24.087, P=0.000). Local hypothermic group siginificantly decreased brain water contents compared to stroke group (P<0.05).Heart rate of the animals differ significantly during the period of experiment among six study groups (F=6.185, P=0.000). After reperfusion, a significant difference was found in heart rate among study groups (F=9.588, P=0.000).Heart rate in magnesium sulfate group D decreased significantly as compared to other groups (all P<0.05). Mean arterial pressure did not significantly different during the monitored period among the study group (F value was1.555and1.613, P was0.220and0.187). After reperfusion, there was a significant difference in mean arterial pressure among groups (F=4.533, P=0.003). Mean arterial pressure in magnesium sulfate group D was significantly lower than that in stroke group, saline group, magnesium sulfate group A and B (all P<0.05). Although a significant difference in rectal temperatures was found during the monitoring period, there was no significant difference between the experimental group at each time point. The other physiological parameters of the animals were kept within normal physiological limits during the course of experiments and did not differ significantly among the study groups. There was a significant difference in infarct volume among all groups (F=53.723, P=0.000). Magnesium sulfate group C revealed the smallest infarct volume. Infarct volume in all treatment groups were significantly smaller than that in stroke group (all P<0.05). Animals treated with intra-carotid magnesium sulfate revealed decrease infarct volume compared with saline infusion (all P<0.05). Infarct volume in magnesium sulfate group C significantly smaller than that in stroke group, saline group, magnesium sulfate group A and B (all P<0.05). There was no significant difference between magnesium sulfate group C and D (P>0.05). There was a significant difference in24h and48h mNSS among all groups (F value was3.014and6.984, P was0.025and0.000). Magnesium sulfate group C revealed the least mNSS in24h and48h after MCAO. At24h after MCAO, mNSS in magnesium sulfate group C significantly less than that in stroke group (all P<0.05). At48h after MCAO, magnesium sulfate group C significantly less than stroke group, saline group and magnesium sulfate group A (all P<0.05). There was a significant difference in cerebral water contents among all groups (F=12.518, P=0.000). Brain water contents in stroke groups were significantly higher than that in sham-operated group (all P<0.05). Magnesium sulfate group C demonstrated significantly reduced brain edema compared to the stroke group (P<0.05).Baseline data among groups were balanced (all P>0.05). Brain temperatures were significantly different during the monitoring periods among all the groups (F=447.427, P=0.000). At five minutes after local infusion, brain temperatures in cold infusion groups significantly lower than that in stroke group, this significantly low temperature maintained until60min after stopping of infusion. Brain temperatures did not differ significantly between two cold infusion groups at any time point. There was a significantly difference in infarct volume among all groups (F=48.007, P=0.000). Animals in hypothermic magnesium sulfate group revealed the least infarct volume. Infarct volume in hypothermic magnesium sulfate group was significantly smaller than that in stroke group and hypothermic saline group (P<0.05). There was a significant difference in neurological function among groups at24h and48h after MCAO (F value was11.827and17.500, all P=0.000). Rats in hypothermic magnesium group had a significantly less mNSS than other groups at48h after MCAO (P<0.05). Brain water contents were significantly different among all groups (F=9.438, P=0.000). Hypothermic magnesium group significantly decreased brain water contents compared to stroke group. Concentrations of EB was significantly different among groups (F=19.263, P=0.000). Rats in hypothermic magnesium group demonstrated less EB concentrations than stroke group (P<0.05). Serum MMP-9concentration were significantly different among groups (F=4.740, P=0.004). Serum MMP-9in hypothermic magnesium group was less than that in stroke group (P<0.05). At each treatment time point, there was a significant difference in infarct volume among all group (F value was69.682,51.564,24.107, all P=0.000). Both hypothermic saline and hypothermic magnesium significantly decreased infarct volume compared to stroke group when local infusion begined with reperfusion or1h after reperfusion (all P<0.05). At2h after reperfusion, only hypothermic magnesium group significantly reduced infarct volume compared to stroke group (P<0.05). At each treatment time point, neurological functions were significantly different among all groups at48h after MCAO (F value was25.451,11.172,5.572, P value was0.000,0.000,0.011). Both hypothermic saline group and hypothermic magnesium group significantly improved neurological outcome compared to stroke group when local infusion commenced at the beginning of reperfusion (P<0.05). Only animals treated with cold magnesium can significantly reduced mNSS compared with stroke group when treatments begin with1h and2h after reperfusion (P<0.05). A significant difference was found in brain water contents among groups at each treatment time point (F value was17.063,26.766,28.507, P=0.000). Brain water content in hypothermic magnesium group was significantly less than that in stroke group when local infusions given at reperfusion and1h after reperfusion (P<0.05). There was no significant difference in brain water content between stroke group and hypothermic groups when treatments delayed to2h after MCAO.Conclusion1. Selective brain hypothermia induced by intra-carotid cold saline infusion can quickly and effectively reduce the brain temperature, while maintain the rectal temperature within normal range.2. Intra-carotid cold saline infusion did not have a harm effect on brain tissue and did not affect physiological variables. Intra-carotid infusion of cold saline is a relatively safe cooling method.3. Intra-carotid cold saline infusion significantly reduced brain infarct volume and improved neurological functional outcomes after brain ischemia.4. Brain water content in all stroke groups was significantly higher than that in sham-operated group48h after MCAO. Intra-carotid cold saline infusion can effectively reduce the cerebral edema after brain ischemia.5. Intra-carotid magnesium sulfate infusion can effectively reduce infarct volume following acute focal cerebral ischemia.6. Intra-carotid infusion of magnesium sulfate improved short-term functional outcomes after middle cerebral artery occlusion.7. Intra-carotid magnesium sulfate infusion reduced cerebral edema48h after ischemic stroke.8. Neuroprotective effect of magnesium sulfate in the dose of60mg/kg-120mg/kg was dose-dependent in cerebral ischemia. The optimal protective dose was120mg/kg.9. Selective endovascular hypothermia combined with intra-carotid administration of magnesium sulfate can provide better protection against ischemic damage than either treatment alone.10. Combination therapy improved short-term neurological outcomes after focal cerebral ischemia.11. Selective brain hypothermia combined with intra-carotid infusion of magnesium sulfate can significantly reduce concentrations of MMP-9and Evans blue, decrease brain water content, protect the blood-brain barrier, and inhibit vasogenic edema48h after ischemia.12. Combined with intra-carotid magnesium sulfate could prolong the therapeutic time window of selective hypothermia for acute focal cerebral ischemia. |
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