Targeted over-expression of endothelin-1 in astrocytes leads to more severe brain damage and vasospasm after subarachnoid hemorrhage

Abstract

Background: Endothelin-1 (ET-1) is a potent vasoconstrictor, and astrocytic ET-1 is reported to play a role in the pathogenesis of cerebral ischemic injury and cytotoxic edema. However, it is still unknown whether astrocytic ET-1 also contributes to vasogenic edema and vasospasm during subarachnoid hemorrhage (SAH). In the present study, transgenic mice with astrocytic endothelin-1 over-expression (GET-1 mice) were used to investigate the pathophysiological role of ET-1 in SAH pathogenesis.

Results: The GET-1 mice experienced a higher mortality rate and significantly more severe neurological deficits, blood-brain barrier breakdown and vasogenic edema compared to the non-transgenic (Ntg) mice following SAH. Oral administration of vasopressin V1a receptor antagonist, SR 49059, significantly reduced the cerebral water content in the GET-1 mice. Furthermore, the GET-1 mice showed significantly more pronounced middle cerebral arterial (MCA) constriction after SAH. Immunocytochemical analysis showed that the calcium-activated potassium channels and the phospho-eNOS were significantly downregulated, whereas PKC-α expression was significantly upregulated in the MCA of the GET-1 mice when compared to Ntg mice after SAH. Administration of ABT-627 (ETA receptor antagonist) significantly down-regulated PKC-α expression in the MCA of the GET-1 mice following SAH.

Conclusions: The present study suggests that astrocytic ET-1 involves in SAH-induced cerebral injury, edema and vasospasm, through ETA receptor and PKC-mediated potassium channel dysfunction. Administration of ABT-627 (ETA receptor antagonist) and SR 49059 (vasopressin V1a receptor antagonist) resulted in amelioration of edema and vasospasm in mice following SAH. These data provide a strong rationale to investigate SR 49059 and ABT-627 as therapeutic drugs for the treatment of SAH patients.

Figure 1

Effects of subarachnoid hemorrhage induction in Ntg and GET-1 mice after subarachnoid hemorrhage. (A) Kaplan-Meier curves show the survival data of the Ntg (solid line, n = 10) and the GET-1 (broken line, n = 13) mice after subarachnoid hemorrhage. (B) Line graph shows the cerebral blood flow of Ntg and GET-1 mice during SAH. (C) Histogram comparing the neurological deficit scores for the Ntg (white bar) and GET-1 (black bar) mice after SAH for 3 days. (*P < 0.05, Mann-Whitey test; n = 10 for Ntg and GET-1 mice).

Figure 2

GET-1 mice showed more severe brain edema with upregulation of ET-1 and GFAP expressions after subarachnoid hemorrhage. (A) Analysis the integrity of the BBB with the EB extravasation in Ntg and GET-1 brain after SAH. (*P < 0.05, ANOVA followed by Bonferroni’s test; n = 4 for the sham groups and n = 8 for the SAH groups). (B) Histogram comparing the water content of the Ntg and GET-1 mice with or without SR 49059 treatment after SAH (*P < 0.05, ANOVA followed by Bonferroni’s test; n = 4 for each group of mice). (C) Immunocytochemical analysis of ET-1 expression in Ntg brain after SAH. Representative micrograph shows the expression of ET-1 in the hippocampal region of the Ntg brain after Sham (i) and SAH (ii) (n = 4 for each group). Histogram below shows the quantification results (relative value in percentage) of ET-1 Sham and SAH of Ntg brain sections using software ImageJ. (**P < 0.01, student t-test; n = 4 for each group of mice). (D) Immunocytochemical analysis of GFAP expression after SAH. Representative micrograph shows the localization and expression of GFAP in the astrocytes of the hippocampus of the Ntg and GET-1 brain at (i-iv) low and (v-viii) high magnification (n = 4 for each group). The GFAP signals in the astrocytes at the SAH groups of the Ntg and GET-1 brains is increased, and they have longer processes (arrowheads). Histogram below shows the quantification results (relative value in percentage) of GFAP Sham and SAH of Ntg and GET-1 brain sections using software ImageJ. (*P < 0.05, **P < 0.01, ***P < 0.005, ANOVA followed by Bonferroni’s test; n = 4 for each group of mice).

Figure 3

Vasospasm analysis and NOS expressions in MCA after subarachnoid hemorrhage. (A) Representative pictures show the histological analysis of MCA. Vasospasm is observed in both Ntg and GET-1 brain after SAH when compared to that of their sham group (n = 5). Histogram shows the arterial diameter measurement of MCA in Ntg and GET-1 after SAH. (*P < 0.05, Mann-Whitey test; n = 5 for Ntg and GET-1 mice). Expression of NOS in MCA after SAH. Representative photos shows the (B) nNOS, (C) eNOS and (D)p-eNOS expressions of sham and SAH of MCA in Ntg and GET-1 brain. Arrowheads show the expression of nNOS (tunica adventitia) and eNOS/p-eNOS (tunica intima), (n = 4 for each group of mice). Histograms show the quantification results (relative value in percentage) of immunocytochemistry of sham and SAH of Ntg and GET-1 MCA sections. (*P < 0.05, **P < 0.01, ANOVA followed by Bonferroni’s test; n = 4 for each group of mice).

Figure 4

Western blot analysis of PKC-α and p-PKC-α in Ntg and GET-1 brain after SAH. Representative blots show PKC-α and p-PKC-α expression in the sham and SAH groups of Ntg and GET-1 brain. Histogram below shows the quantification (relative percentage) of the Western blot. (*P < 0.05, **P < 0.01, Mann-Whitey test; n = 4 for Ntg and GET-1 mice).

Figure 5

Immunocytochemical analysis of Ca²⁺-activated K⁺ channel and ET_AR expressions in Ntg and GET-1 MCA after SAH. (A) Representative micrograph shows the localization and expression of Ca²⁺-activated K⁺ channel in the MCA of the Ntg and GET-1 after SAH. (n = 3 from each group of mice). Histogram shows the quantification results (relative value in percentage) of immunocytochemistry of sham and SAH of Ntg and GET-1 MCA sections. (*P < 0.05, **P < 0.01, ***P < 0.005, ANOVA followed by Bonferroni’s test; n = 3 for each group of mice). (B) Representative micrograph shows the localization and expression of ET_AR in the smooth muscle cells (arrows) of MCA in Ntg and GET-1 mice. Histogram shows the quantification results (relative value in percentage) of immunocytochemistry of sham and SAH of Ntg and GET-1 MCA sections. (*P < 0.05, **P < 0.01, ANOVA followed by Bonferroni’s test; n = 3 for each group of mice). (C) Representative micrograph shows the immunocytochemical expressions of PKC-α and the diameters of MCA after treating with ET_AR antagonist ABT-627. Histogram below shows the measurements of MCA diameter. (*P < 0.05, **P < 0.01, Mann-Whitey test; n = 3 for Ntg and GET-1 mice).

Figure 6

Proposed mechanism of astrocytic ET-1 mediated cerebral vasospasm after SAH. GET-1 mice with over-expressed in ET-1 showed more severe neurological deficits and vasospasm after SAH. Increased astrocytic ET-1 during SAH induces cerebral vasospasm through the ET_A receptor and mediated by PKC-α, which leads to dynfunction in the K⁺ channels. Administration of ET_A receptor antagonist ABT-627 ameliorates the SAH-induced vasospasm. The impairment of NO system also exaggerates the vasospasm effect. Astrocytic ET-1 leads to more severe cerebral edema and BBB breakdown that further contributes to cerebral vasoconstriction. Vasopressin V_1a receptor antagonist, SR 49059, significantly reduced the SAH-induced edema, suggesting that astrocytic ET-1 induces edema in SAH through vasopressin V_1a receptor.