The Research Of The Expressions Of HIF-1α, SOCS-3and Caspase-3in Rats After Intracerebral Hemorrhage By Mild Hypothermia Therapy

Objective:By establishing the model of intracerebral hemorrhagein rats to observe the changes of neurological function, moisture of brain tissue,inflammatory reaction, and the expression of hypoxia inducible factor-1alpha(HIF-1α), suppressor of cytokine signaling-3(SOCS-3) and cysteine proteinase-3(Caspase-3) after systemic mild hypothermia therapy, to provide experimentalbasis for better protections on intracerebral hemorrhage. Methods:1.Toestablish the ICH model in the caudate nucleus of the rat brain by autologousblood injection methods.2. The90SD rats were randomly divided into the threegroups: sham-operation group, ICH group and mild hypothermia treatment group.The sham-operation group distributed6rats and injected without blood was onlyone time point with6hours after operation. And the other two group weredistributed42rats each, observed at six time points, which were6h,12h,1d,2d,3d,7d and14d after operation. And all the rats were killed at the correspondingtimes.3. The mild hypothermia intervention therapy: the rat of treatment groupwas treated by systemic mild hypothermia (32-33℃) with6hours immediatelyafter operation. The temperature was lowered to32-33℃in15minutes andmaintained by the ice, alcohol, and so on. And in the recovery time (generally3hours after operation) or if the rats shivered, the1%pentobarbital (20mg/kg) wasused again by intraperitoneal anesthesia. The temperature of ICH and sham-operated rats was maintained at36.5-37.5℃after operation. The rats oftreatment group were automatic rewarmed in20-25℃room temperature aftermild hypothermia treatment. The rectal temperature measured by medicalelectronic thermometer every15minutes until the hypothermia treatment overed.4. The succession of ICH model was estimated at6h after operation, and theneurologic deficiency scores assessed at the corresponding times.5. Themoisture of brain tissue was measured with wet-dry weight method.6. Using thehematoxylin-eosin staining to observed the morphological changes andinflammatory responses in brain tissue around the hematoma.7. Usingimmunohistochemical staining and the image analysis method to observed theexpression of Caspase-3protein in brain tissue around the hematoma.8. UsingSemi-quantitative RT-PCR to detect the expression of HIF-1α mRNA andSOCS-3mRNA in brain tissue around the hematoma. Results:1. Neurologicaldysfunction score: the sham-operated group rats had no neurological dysfunctionafter operation. The ICH group and treatment group showed varied degrees onneurological dysfunction after operation, compared with sham-operated group,which had obviously statistic significance (P<0.01); In the ICH and treatmentgroup, the score at3d was the lowest which was remarkably different from the6h,12h,1d,7d and14d (the ICH group, P<0.05；the treatment group, P<0.01),but without significant difference between3d and2d (P>0.05); The score oftreatment group was higher than the ICH group at each time point withsignificant difference after operation (6h,12h,1d,7d,14d, P<0.01;2d,3d, P<0.05).2. The moisture of brain tissue: The moisture of brain tissue of ICHgroup and treatment group was more than the sham-operated group at each timepoint, with significant difference after operation (P<0.01). In the ICH andtreatment group, the moisture at3d was the highest which was obviouslydifferent from the6h,12h,1d,7d and14d (P<0.01), but without significantdifference between3d and2d (P>0.05). The moisture of treatment group waslower than the ICH group at each time point with significant difference afteroperation(12h,1d,2d,3d, P<0.01;6h,7d,14d, P<0.05).3. Pathological changes:The pathological changes and inflammatory reaction of the brain tissue insham-operation group was not obvious. The nervous cells and structure wasclearly and arranged regularly integrity, without obvious swelling, karyopyknosisand karyolysis, and cell infiltration; In the ICH group and treatment group, thenervous cell degenerated and necrosis, and the inflammatory reaction wereclearly accompanied a large number of infiltration cells(acute period mainlyneutrophil, chronic period mainly lymphocytes). The most pathological changesoccurred in3d, and lightened after three days; The treatment group at eachtime point the pathological changes was better than ICH group.4.The expressionof Caspase-3protein: The expression of Caspase-3protein of sham-operated wasvery little while a large amount of Caspase-3protein expressed in ICH group andtreatment group at each time point after operation with remarkably difference(P<0.01); In the ICH and treatment group, the expression of Caspase-3protein at3d was the highest which was obviously different from the6h,12h,1d,7d and 14d (the ICH group, P<0.05; treatment group, P<0.01), but without significantdifference between3d and2d (P>0.05). The expression of Caspase-3protein intreatment group was less than the ICH group at each time point with significantdifference after operation (P<0.01).5. The expression of HIF-1α mRNA: TheHIF-1α mRNA expressed scarcely in the sham-operation group while a largequantity of HIF-1α mRNA expressed in ICH group and treatment group at eachtime point after operation with remarkably difference (P<0.01); In the ICH andtreatment group, the expression of HIF-1α mRNA at3d was the highest whichwas obviously different from the6h,12h,1d,7d and14d(P<0.01), but withoutsignificant difference between3d and2d (P>0.05). The expression of HIF-1αmRNA in treatment group was less than the ICH group at each time point withsignificant difference after operation (P<0.01).6. The expression of SOCS-3mRNA: The HIF-1α mRNA expressed scarcely in the sham-operation groupwhile a large quantity of SOCS-3mRNA expressed in ICH group and treatmentgroup at each time point after operation with remarkably difference (P<0.01); Inthe ICH and treatment group, the expression of SOCS-3mRNA at3d was thehighest which was obviously different from the6h,12h,1d,7d and14d(the ICHgroup, P<0.01; the treatment group, P<0.05), but without significant differencebetween3d and2d (P>0.05). The expression of SOCS-3mRNA in treatmentgroup was less than the ICH group at each time point with significant differenceafter operation (2d, P<0.05; other time point, P<0.01). Conclusion:1. The ICHmodel of rat established by autologous blood is very simple, and the pathophysiology in this model is nearly as same as human, thus it can be usedgenerally.2. After ICH, the increasing of HIF-1α can aggravate the expression ofCaspase-3, brain edema and inflammatory responses, promote apoptosis andneurological dysfunction, increased secondary brain injury.3. The expression ofSOCS-3increased after ICH, which can lighten the expression of Caspase-3andbrain edema and inflammation, inhibit apoptosis to reduce the neurologicaldysfunction and protect the secondary brain injury.4. After ICH, the mildhypothermia therapy can inhibit the expression of HIF-1α, increase theexpression of SOCS-3, and lighten the expression of Caspase-3, brain edema andinflammation, inhibit apoptosis to reduce the neurological dysfunction andprotect the secondary brain injury.