| ObjectiveNowadays neural plasticity and repair after cerebral ischemia is a hot issue in neuroscience. Nonetheless, what kind of animal model of stroke and neuroprotective agents are critical questions. This study would like to introduce a reproducible focal cerebral ischemia/ reperfusion animal model in mice. To study the protection and their mechanism of rosuvastatin, erythropoietin(EPO) ,carbamylated erythropoietin (CEPO) on ischemic brain injury. To observe the effect of multidrug resistance protein (P-glycoprotein,P-gp) on the concentration in brain and neuroprotection of FK506. To investigate the effects and health economic evaluation of rehabilitation procedure on patients with stroke.MethodsFocal cerebral ischemia/reperfusion was induced by occlusion of the middle cerebral artery(MCA) using the intraluminal filament technique. The whole mice brain were stained with TTC, and the slices of brain tissue stained with cresyl-violet. The cerebral blood flow were monitored with laser-Doppler flow metry(LDF). The cerebral volume of infarction and edema were quantified with ImageJ software. The expression of endothelial NO synthase(eNOS) and activated caspase-3 were detected with western blot. The inducible NO synthase(iNOS) positive cells were observed with immunohistochemistry. Theexpression of P-gp was detected with immunohistochemistry and western blot. The concentration of FK506 in blood and brain tissue were determinated with ELISA. The apoptotic cell was detected with TUNEL staining. 42 patients were randomly divided into two groups, rehabilitation group and control group. Both groups were given early rehabilitation during the first 21 days following the stroke. Then, the rehabilitation group went on the same rehabilitation in the rehabilitation center for 2 months ,and then go to community medical service or go home with rehabilitation for 3 months. Whereas the control group were given self-training at home. Outcome measures were National Institute of Health Stroke Scale ( NIHSS), Fugl-Mayer Assessment(FMA), Modified Barthel Index(MBI) and the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). The health economic evaluation was made by using cost-effectiveness analysis and increment analysis.Results1. A reproducible focal cerebral ischemia/ reperfusion animal model in mice: (1) The cerebral blood flow droped sharply after MCAO, and jumped rapidly after reperfusion. (2) After cerebral ischemia, the infarct area was very clear, and the volume of infarction and edema were stable. (3) In group ischemia 30 min / reperfusion 24h, the volume of infarction and edema were significantly smaller than those in group ischemia 90 min / reperfusion 24 h (P<0.001) . (4)After cerebral ischemia 30 min / reperfusion 24 h and reperfusion 72 h, remarkable edema appeared (61.6±3.5 mm~3,72.6±4.3 mm~3 respectively) (P<0.001) , whereas the volume of edema was only 50.9±4.1 mm~3 after reperfusion 7 d. However, after reperfusion 30 d, the volume of whole brain shrinked, and the volume of edema came to negative value (-20.1±1.8 mm~3) 2. The protection and its mechanism of rosuvastatin on ischemic brain injury :(1) rosuvastatin 20 mg/kg could remarkably decrease infarct volume and cerebral edemaafter MCAO 90 min/reperfusion 24 h. (2) Western blots showed the expression of eNOS incerebral cortex before and after ischemia were 100±43.3%, 1668.9±112.2% respectively( P<0.001 ) , rosuvastatin significantly up-regulated the expression of eNOS innon-ischemic cortex from 100±43.3% to 511.4±68.7% (P<0.001), whereas in ischemic cortex rosuvastatin did not up-regulated the expression of eNOS (rosuvastatin vs NS: 1678.8±121.3% vs 1668.9±112.2%) (P>0.05). (3) There was no expression of activated caspase-3 in non-ischemic cortex, nonetheless the expression of activated caspase-3 gained 88.3±15.6% after ischemia, rosuvastatin significantly diminished it to 42.1±11.2% (P<0.01) (4) Immunohistochemistry revealed no iNOS positive cells in non-ischemic brain area, while in ischemic brain area the number of iNOS positive cells go up 2.6±1.8 cells/sq., rosuvastatin 20mg/kg reduced them to 0.6±0.3 cells/sq (P<0.05).3. Potentiating the neuroprotection of FK506 on ischemic brain injury by inhibiting P-glycoprotein: (1) The expression of P-gp in cortex and striatum started to rise after MCAO 30 min reperfusion 3 h. Until reperfusion 24 h, the expression of P-gp in cortex and striatum increased 55.3% and 67.9% respectively (P<0.05); However, after reperfusion 72 h, the expression of P-gp went down to control level. It revealed the expression of P-gp transiently grew at the early stage of cerebral ischemia. (2) The inhibitor of P-gp(tariquidar,TQD) had no significant effect on the concentration of FK506 in blood and brain tissue before or after MCAO (P>0.05); After application of TQD, the level of FK506 in non-ischemic hemispheres had no markedly change following MCAO. While the level of FK506 in ischemic hemispheres rose significantly, the average of brain-to-blood-concentration ratio jumped from 6.3 to 42.2 (P<0.001) . It indicated that the expression of P-gp upregulated following cerebral ischemia, which prevented FK506 from entering brain tissue. TQD, which suppressed P-gp, increased the level of FK506 in brain tissue following cerebral ischemia. (3) The lower doses of FK506 (1 mg/kg 和 3 mg/kg) had no anti-apoptosis after MCAO, whereas a higher dose of FK506 (5 mg/kg) had anti-apoptosis, from 81.2+12.3 TUNEL(+)cells /square to 46.5±9.2 TUNEL(+)cells /square (P<0.001) TQD had no anti-apoptosis after MCAO. Fortunately, FK506 1 mg/kg had anti-apoptosis when TQD and FK506 were applied together following MCAO. (4) TQD or FK506 3mg/kg could not reduce the infarct volume following MCAO 90 min reperfusion 24 h, while infarct volume significantly fell from 113.5±11.1 mm~3 to 70.6±10.2mm3 when TQD and FK506 were applied together (P<0.01). It noted that TQD (the inhibitor of P-gp) could lower the neuroprotective threshold of FK506 from 5 mg/kg to 1 mg/kg.4. The protection and its mechanism of carbamylated erythropoietin on ischemic brain injury : (1) In terms of decreasing of neurological deficit scores , CEPO 50μg/kg and EPO(50μg/kg)had equal function except for EPO(5μg/kg); (2) In group NS EPO 5μg/kg EPO 50μg/kg CEPO 50μg/kg, the infarct volume was 103.2±10.1 98.7±11.2, 66.9±8.3 67.1±8.5 mm~3, respectively. The edema volume was 78.9±6.8 76.8±7.3 52.2±5.4 53.1±5.6 mm~3 , respectively ; (3) The apoptotic cells in ischemic cortex fell from 94.2±15.2/sq. in group NS to 40.5±9.8/sq. in group CEPO 50μg/kg (P<0.001), the anti-apoptosis of EPO 50μg/kg was similar to CEPO 50μg/kg; (4) The influence of CEPO and EPO on eNOS in ischemic cortex were not significantly different. However, the expression of activated caspase-3 markedly dropped from 95.4±16.7% in group NS to 43.5±13.1% in group CEPO 50μg/kg (P<0.001) , and 45.1±11.2% in group EPO 50μg/kg, instead of group EPO 5μg/kg; (5) Immunohistochemistry revealed iNOS positive cells in ischemic cortex was 3.1±1.9 cells/sq., CEPO and EPO remarkably reduced them to 0.7±0.2/sq. and 0.8±0.2/sq.,respectively. (P<0.05).5. Effects and health economic evaluation of three-stage rehabilitation procedure on patients with acute stroke: (1) At acute stage of stroke (within 21 d), the scores of NIHSS, FMA and MBI in two groups were not significantly different (P>0.05). However, the patients in the rehabilitation group significantly got much better scores of NIHSS, FMA and MBI than the control group in recovery stage (21 d later -6 months) after the onset of stroke (P<0.01). (2) The patients in the rehabilitation group got much better scores in all 8 dimensions of SF-36 than the control group after 6 months and 2 years following the onset of stroke, the difference was statistically significant (P<0.05). (3) When the NIHSS scores were reduced one point, FMA and MBI scores were improved one point, the cost in the rehabilitation group were 2412.5, 442.0, 332.1 yuan RMB, respectively; Whereas in the control group those were 3285.4, 637.8, 447.5 yuan RMB, respectively.Conclusion(1) This kind of cerebral ischemia/ reperfusion animal model in mice is reproducible and easy to operate. (2) The mechanisms of rosuvastatin's neural protection on ischemic brain injury are to enhance expression of eNOS, to inhibit expression of iNOS and activated caspase-3. (3) TQD, which is the inhibitor of P-gp, potentiates the neuroprotection of FK506 by means of increasing the level of FK506 and lowering the neuroprotective threshold of FK506. (4) Lower dose EPO (5μg/kg) has no brain protection. CEPO 50μg/kg and EPO 50μg/kg have equal role of decreasing infarct volume, diminishing brain edema, anti-apoptosis by means of inhibiting expression of activated caspase-3 and iNOS. (5) Three-stage rehabilitation scheme can obviously improve patients' general functional abilities, decrease the disability and increase patients' quality of life, and is more economical.
|
PDF Full Text Request