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Ischemic postconditioning decreases cerebral edema and brain blood barrier disruption caused by relief of carotid stenosis in a rat model of cerebral hypoperfusion.

Yang F, Zhang X, Sun Y, Wang B, Zhou C, Luo Y, Ge P - PLoS ONE (2013)

Bottom Line: ELISA was used to analyze the expression of MMP-9, claudin-5 and occludin.The activity and location of MMP-9 was analyzed by gelatin zymography and in situ zymography, respectively.The distribution of tight junction proteins claudin-5 and occludin was observed by immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, First Bethune Hospital of Jilin University, Changchun, China.

ABSTRACT

Background and purpose: Complications due to brain edema and breakdown of blood brain barrier are an important factor affecting the treatment effects of patients with severe carotid stenosis. In this study, we investigated the protective effects of ischemic postconditioning on brain edema and disruption of blood brain barrier via establishing rat model of hypoperfusion due to severe carotid stenosis.

Methods: Wistar rat model of hypoperfusion due to severe carotid stenosis was established by binding a stainless microtube to both carotid arteries. Ischemic postconditioning procedure consisted of three cycles of 30 seconds ischemia and 30 seconds reperfusion. Brain edema was evaluated by measuring cerebral water content, and blood brain barrier permeability was assayed by examining cerebral concentration of Evans' Blue (EB) and fluorescein sodium (NaF). ELISA was used to analyze the expression of MMP-9, claudin-5 and occludin. The activity and location of MMP-9 was analyzed by gelatin zymography and in situ zymography, respectively. The distribution of tight junction proteins claudin-5 and occludin was observed by immunohistochemistry.

Results: The increased brain water content and cerebral concentration of EB and NaF were suppressed by administration of ischemic postconditioning prior to relief of carotid stenosis. Zymographic studies showed that MMP-9 was mainly located in the cortex and its activity was significantly improved by relief of carotid stenosis and, but the elevated MMP-9 activity was inhibited markedly by ischemic postconditioning. Immunohistochemistry revealed that ischemic postconditioning improved the discontinuous distribution of claudin-5 and occludin. ELISA detected that the expression of up-regulated MMP-9 and down-regulated claudin-5 and occludin caused by carotid relief were all attenuated by ischemic postconditioning.

Conclusions: Ischemic postconditioning is an effective method to prevent brain edema and improve BBB permeability and could be used during relief of severe carotid stenosis.

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Related in: MedlinePlus

Measurment of gelatinase activity and expression of MMP-9.A, gel gelatinase activity of MMP-9 and statistical analysis. The gelatinase activity of MMP-9 in the stenosis relief group was 3.52±0.43 times higher than that in the carotid stenosis group at day 2 following relief of carotid stenosis, but it was alleviated significantly by ischemic postconditioning. B, in situ gelatinage activity of MMP-9. Relief of carotid stenosis made the FITC signal representing gelatinase activity become stronger when compared with that in the sham and carotid group. However, the increased signal intensity was inhibited markedly by ischemic postconditioning. C, ECL assay of MMP-9. The expressional levels of MMP-9 in the stenosis relief group were significantly higher than those in the sham and the carotid stenosis group. By contrast, this elevation was mitigated from day 1 to day 3 after application of ischemic postconditioning. *: p<0.01, versus carotid stenosis group; #: p<0.01, versus stenosis relief group.
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pone-0057869-g003: Measurment of gelatinase activity and expression of MMP-9.A, gel gelatinase activity of MMP-9 and statistical analysis. The gelatinase activity of MMP-9 in the stenosis relief group was 3.52±0.43 times higher than that in the carotid stenosis group at day 2 following relief of carotid stenosis, but it was alleviated significantly by ischemic postconditioning. B, in situ gelatinage activity of MMP-9. Relief of carotid stenosis made the FITC signal representing gelatinase activity become stronger when compared with that in the sham and carotid group. However, the increased signal intensity was inhibited markedly by ischemic postconditioning. C, ECL assay of MMP-9. The expressional levels of MMP-9 in the stenosis relief group were significantly higher than those in the sham and the carotid stenosis group. By contrast, this elevation was mitigated from day 1 to day 3 after application of ischemic postconditioning. *: p<0.01, versus carotid stenosis group; #: p<0.01, versus stenosis relief group.

Mentions: Previous studies have shown that MMP-9 played an active role in modulating the permeability of BBB [17]–[19], we thus speculated that stenosis relief might cause aberrant activity and expression in MMP-9. Because brain edema and the permeability of BBB reached peak value at day 2 when carotid stenosis was relieved (Figure 2), we chose this time point to assay MMP-9 activity in the rats treated with ischemic postconditioning or stenosis relief alone. As shown in figure 3 A, gelatin zymography revealed that the gelatinase activity of MMP-9 in the stenosis relief group was 3.52±0.43 times higher than those in the carotid stenosis group (P<0.01), but it was alleviated to 1.48±0.37 times by ischemic postconditioning. Similarly, in situ zymography showed that the FITC signal which represents gelatinase activity was weak in the sham and carotid stenosis group (Figure 3 B), but increased markedly in the stenosis relief group and was suppressed by ischemic postconditioning. To elucidate the factors affecting the increased activity of MMP-9, we analyzed its expressional level by ELISA. As figure 3 C showed, the expressional levels of MMP-9 in the stenosis relief group increased to 46.33±5.31, 61.2±8.65, and 62.1±8.11 at day 1, day 2 and day 3, respectively (P<0.01 versus carotid stenosis group at each scheduled time point). However, the elevation was mitigated from day 1 to day 3 after application of ischemic postconditioning was inhibited to 35.6±2.21, 46.2±3.18, 47.5±2.87 (P<0.01 versus stenosis relief group at each time point). These data indicated that expressional up-regulation underlies the increased activity of MMP-9 due to reestablishment of cerebral blood supply.


Ischemic postconditioning decreases cerebral edema and brain blood barrier disruption caused by relief of carotid stenosis in a rat model of cerebral hypoperfusion.

Yang F, Zhang X, Sun Y, Wang B, Zhou C, Luo Y, Ge P - PLoS ONE (2013)

Measurment of gelatinase activity and expression of MMP-9.A, gel gelatinase activity of MMP-9 and statistical analysis. The gelatinase activity of MMP-9 in the stenosis relief group was 3.52±0.43 times higher than that in the carotid stenosis group at day 2 following relief of carotid stenosis, but it was alleviated significantly by ischemic postconditioning. B, in situ gelatinage activity of MMP-9. Relief of carotid stenosis made the FITC signal representing gelatinase activity become stronger when compared with that in the sham and carotid group. However, the increased signal intensity was inhibited markedly by ischemic postconditioning. C, ECL assay of MMP-9. The expressional levels of MMP-9 in the stenosis relief group were significantly higher than those in the sham and the carotid stenosis group. By contrast, this elevation was mitigated from day 1 to day 3 after application of ischemic postconditioning. *: p<0.01, versus carotid stenosis group; #: p<0.01, versus stenosis relief group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585273&req=5

pone-0057869-g003: Measurment of gelatinase activity and expression of MMP-9.A, gel gelatinase activity of MMP-9 and statistical analysis. The gelatinase activity of MMP-9 in the stenosis relief group was 3.52±0.43 times higher than that in the carotid stenosis group at day 2 following relief of carotid stenosis, but it was alleviated significantly by ischemic postconditioning. B, in situ gelatinage activity of MMP-9. Relief of carotid stenosis made the FITC signal representing gelatinase activity become stronger when compared with that in the sham and carotid group. However, the increased signal intensity was inhibited markedly by ischemic postconditioning. C, ECL assay of MMP-9. The expressional levels of MMP-9 in the stenosis relief group were significantly higher than those in the sham and the carotid stenosis group. By contrast, this elevation was mitigated from day 1 to day 3 after application of ischemic postconditioning. *: p<0.01, versus carotid stenosis group; #: p<0.01, versus stenosis relief group.
Mentions: Previous studies have shown that MMP-9 played an active role in modulating the permeability of BBB [17]–[19], we thus speculated that stenosis relief might cause aberrant activity and expression in MMP-9. Because brain edema and the permeability of BBB reached peak value at day 2 when carotid stenosis was relieved (Figure 2), we chose this time point to assay MMP-9 activity in the rats treated with ischemic postconditioning or stenosis relief alone. As shown in figure 3 A, gelatin zymography revealed that the gelatinase activity of MMP-9 in the stenosis relief group was 3.52±0.43 times higher than those in the carotid stenosis group (P<0.01), but it was alleviated to 1.48±0.37 times by ischemic postconditioning. Similarly, in situ zymography showed that the FITC signal which represents gelatinase activity was weak in the sham and carotid stenosis group (Figure 3 B), but increased markedly in the stenosis relief group and was suppressed by ischemic postconditioning. To elucidate the factors affecting the increased activity of MMP-9, we analyzed its expressional level by ELISA. As figure 3 C showed, the expressional levels of MMP-9 in the stenosis relief group increased to 46.33±5.31, 61.2±8.65, and 62.1±8.11 at day 1, day 2 and day 3, respectively (P<0.01 versus carotid stenosis group at each scheduled time point). However, the elevation was mitigated from day 1 to day 3 after application of ischemic postconditioning was inhibited to 35.6±2.21, 46.2±3.18, 47.5±2.87 (P<0.01 versus stenosis relief group at each time point). These data indicated that expressional up-regulation underlies the increased activity of MMP-9 due to reestablishment of cerebral blood supply.

Bottom Line: ELISA was used to analyze the expression of MMP-9, claudin-5 and occludin.The activity and location of MMP-9 was analyzed by gelatin zymography and in situ zymography, respectively.The distribution of tight junction proteins claudin-5 and occludin was observed by immunohistochemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, First Bethune Hospital of Jilin University, Changchun, China.

ABSTRACT

Background and purpose: Complications due to brain edema and breakdown of blood brain barrier are an important factor affecting the treatment effects of patients with severe carotid stenosis. In this study, we investigated the protective effects of ischemic postconditioning on brain edema and disruption of blood brain barrier via establishing rat model of hypoperfusion due to severe carotid stenosis.

Methods: Wistar rat model of hypoperfusion due to severe carotid stenosis was established by binding a stainless microtube to both carotid arteries. Ischemic postconditioning procedure consisted of three cycles of 30 seconds ischemia and 30 seconds reperfusion. Brain edema was evaluated by measuring cerebral water content, and blood brain barrier permeability was assayed by examining cerebral concentration of Evans' Blue (EB) and fluorescein sodium (NaF). ELISA was used to analyze the expression of MMP-9, claudin-5 and occludin. The activity and location of MMP-9 was analyzed by gelatin zymography and in situ zymography, respectively. The distribution of tight junction proteins claudin-5 and occludin was observed by immunohistochemistry.

Results: The increased brain water content and cerebral concentration of EB and NaF were suppressed by administration of ischemic postconditioning prior to relief of carotid stenosis. Zymographic studies showed that MMP-9 was mainly located in the cortex and its activity was significantly improved by relief of carotid stenosis and, but the elevated MMP-9 activity was inhibited markedly by ischemic postconditioning. Immunohistochemistry revealed that ischemic postconditioning improved the discontinuous distribution of claudin-5 and occludin. ELISA detected that the expression of up-regulated MMP-9 and down-regulated claudin-5 and occludin caused by carotid relief were all attenuated by ischemic postconditioning.

Conclusions: Ischemic postconditioning is an effective method to prevent brain edema and improve BBB permeability and could be used during relief of severe carotid stenosis.

Show MeSH
Related in: MedlinePlus