| Although tremendous achievements have been made in the clinical and technological diagnosis of stroke in the past decade, the therapeutic outcome remains unsatisfactory. Stroke remains to be the second commonest cause of death and a leading cause of adult disability worldwide. According to recent estimates published by the World Health Organization, about 15 million people per year fall victim to stroke worldwide, of whom 5 million die and another 5 million are left permanently disabled. Stroke emerges as a condition associated with a combination of multiple risk factors including hypertension, hyperlipidemia and diabetes, of which hypertension is the most prevalent and powerful, across age, sex, and geographic regions.Individual susceptibility to stroke varies greatly among hypertensive patients. Interestingly, a similar variation in susceptibility to stroke was found in spontaneously hypertensive rats (SHR), and thus a sub-strain of rats was developed based on their susceptibility to stroke from SHR and named stroke-prone spontaneously hypertensive rats (SHR-SP). SHR-SP is a useful and unique animal model for studying the pathogenesis of stroke. These rats were subjected to a stroke at a mean age of 10 months for males and 14 months for females in our laboratory. We speculated that the difference between SHR-SP and SHR might imply some mechanisms underlying the pathogenesis and development of stroke, and that as SHR and SHR-SP are both hypertensive, a comparison of SHR-SP with SHR would provide new insights into the pathogenesis and development of stroke beyond hypertension.Traditionally, studies on stroke have been performed by one or a few factors in blood plasma/serum or tissues. These factors are usually preselected based on some expected hypothesis, and the aim is to either verify or disprove this hypothesis. However, as a means of unbiased global screening of physiological perturbations, these approaches are limited. This means that detection of unexpected or novel mechanistic phenomena or markers is almost impossible to obtain. For this purpose, a robust method that can simultaneously quantify and identify a large number (hundreds to thousands) of molecules is needed. The integrated multiple systems biology approach has been employed in recent years and has turned out to be an efficient approach for improving our understanding of systems as a whole.The main aspects of this study are as followings:1. To establish a hypothesis-free high-throughput strategy capable of resolving several thousands of individual protein spots on a single gel -- two dimensional fluorescent difference gel electrophoresis (2D-DIGE). Each soluble protein sample from SHR-SP and SHR was minimally labeled with 400pmol (1ul) CyDye DIGE fluors dissolved in 99.8% DMF per 50ug of protein (8 nM dye/mg protein), one SHR, SHR-SP and internal standard sample forming a set of Cy2, Cy3, and Cy5 labeled samples was combined and mixed to perform the first dimensional isoelectric focusing (IEF) and second dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We successfully acquired the image of differentially expressed protein profile of the brain of SHR-SP and SHR.2. To study the differentially expressed protein profile in the brain of SHR-SP and SHR by using 2D-DIGE and matrix assisted laser desorption /ionization- time of flight mass spectrometry (MALDI-TOF MS). Using 2D-DIGE approach, we comparatively analyzed the proteome of SHR-SP and SHR. We reproducibly separated over 2,000 polypeptides using two-dimensional electrophoresis (2DE) at PH range of 3-10. Using DeCyder software to process the 2D gel images, forty-five protein spots were differentially expressed in the brains of the two kinds of animals. Of these, nineteen highly expressed in SHR-SP, and twenty-five highly expressed in SHR. Twenty proteins were identified with high confidence by MALDI-TOF/TOF MS. Our data suggested that some anti-oxidant proteins including glutathione S-transferase (GST) pi2 and GST A5 were down-regulated in the brain of SHR-SP.3. Since the anti-oxidant proteins were down-regulated in SHR-SP when compared with SHR, oxidative stress indicators including total anti-oxidant capacity (TAC), glutathione peroxidase (GPx) activity and maleic dialdehyde (MDA) were measured (n=10 in each group). The results showed that TAC level and GPx activity in brain of SHR-SP were significantly lower than those in SHR (12.47±3.02 vs 22.73±3.00, P<0.01; 891.81±108.93 vs 1124.4±160.06, P<0.05), while MDA level in SHR-SP was significantly higher than that in SHR (1.11±0.11 vs 0.82±0.11, P<0.05). The results were the same in the serum. The TAC level and GPx activity in SHR-SP were significantly lower than those in SHR (61.28±15.16 vs 123.6±32.39, P<0.05; 12.14±1.77 vs 18.59±2.42, P<0.05), while MDA level was significantly higher than that in SHR (13.33±2.90 vs 5.89 ±1.21, P<0.05).4. The above results revealed that oxidative damage was more severe in SHR-SP than that in SHR. Furthermore, we investigated the pathological importance of oxidative stress in the pathogenesis and development of stroke in SHR-SP. Twenty male SHR-SP and SHR aged 5 months were subjected to middle cerebral artery occlusion (MCAO) and the infarct area was measured (n=10 in each group). TTC staining showed that the infarct area in SHR-SP was lager than that in SHR. Using the computerized image analysis system, the mean infarct area of SHR-SP and SHR was 31.6 %±5.4 % and 23.0 %±3.3 %, respectively. Unpaired t-test showed that the mean infarct area of SHR-SP was significantly larger than that of SHR (P=0.004).5. The infarct area of SHR-SP in our work was significantly larger than that of SHR, suggesting that the increase in the cerebral ischemia infarct area may be associated with a higher level of oxidative stress in SHR-SP. In order to confirm the association between high oxidative stress and increased infarct area of SHR-SP, the animals were treated with vitamins C and E to improve the antioxidant defense system. Fifty male SHR-SP were randomly divided into control group (n=25) and vitamins treatment group (vitamin E and C (100 + 200 mg / kg. d) by gavage for 4 weeks, n=25). After 4-week treatment, twenty SHR-SP were killed for detection of TAC, GPx and MDA (n=10 in each group). The results showed that TAC level and GPx activity in brain of vitamin treated group were significantly higher than those in control (25.57±6.68 vs 12.47±3.02, P<0.05; 1453.11±126.98 vs 891.81±108.93, P<0.01), while MDA level in vitamin treated group was significantly lower than that in control (0.80±0.05 vs 1.11±0.11, P<0.05). The results were the same in the serum. The TAC level and GPx activity in vitamin treated group were significantly higher than those in control (124.75±28.43 vs 61.28±15.16, P<0.05; 19.63±4.84 vs 12.14±1.77, P<0.05), while MDA level was significantly lower than that in control (7.31±0.86 vs 13.33±2.90, P<0.05). The remaining thirty SHR-SP underwent MCAO and the infarct area was measured. TTC staining showed that the infarct area of SHR-SP control and vitamin treated group was 32.0 %±4.9 % and 20.8 %±3.8 %, respectively. Unpaired t-test showed that the infarct area of VCE group was significantly smaller than that of the control group (P=0.003). In conclusion:1. We successfully established the technology of 2D-DIGE and with 2D-DIGE we can detect the differentially expressed protein successfully.2. With 2D-DIGE, we acquired the differentially expressed protein profiles of SHR-SP and SHR. Of the multitudinous proteins, we detected a significant decrease in anti-oxidative proteins (GST Pi 2, and GST A5) of SHR-SP when compared with SHR.3. Oxidative stress plays an important role in the pathogenesis and development of cerebral ischemia: On the one hand, oxidative damage was more severe in SHR-SP than that in SHR, and the infarct area after MCAO was significantly larger in SHR-SP than that in SHR. On the other hand, vitamins significantly decreased the oxidative stress level of SHR-SP, and the decreased oxidative stress resulted in a decrease in the infarct area in SHR-SP.
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