Evidence that the EphA2 receptor exacerbates ischemic brain injury

Abstract

Ephrin (Eph) signaling within the central nervous system is known to modulate axon guidance, synaptic plasticity, and to promote long-term potentiation. We investigated the potential involvement of EphA2 receptors in ischemic stroke-induced brain inflammation in a mouse model of focal stroke. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and EphA2-deficient (EphA2(-/-)) mice by middle cerebral artery occlusion (MCAO; 60 min), followed by reperfusion (24 or 72 h). Brain infarction was measured using triphenyltetrazolium chloride staining. Neurological deficit scores and brain infarct volumes were significantly less in EphA2(-/-) mice compared with WT controls. This protection by EphA2 deletion was associated with a comparative decrease in brain edema, blood-brain barrier damage, MMP-9 expression and leukocyte infiltration, and higher expression levels of the tight junction protein, zona occludens-1. Moreover, EphA2(-/-) brains had significantly lower levels of the pro-apoptotic proteins, cleaved caspase-3 and BAX, and higher levels of the anti-apoptotic protein, Bcl-2 as compared to WT group. We confirmed that isolated WT cortical neurons express the EphA2 receptor and its ligands (ephrin-A1-A3). Furthermore, expression of all four proteins was increased in WT primary cortical neurons following 24 h of glucose deprivation, and in the brains of WT mice following stroke. Glucose deprivation induced less cell death in primary neurons from EphA2(-/-) compared with WT mice. In conclusion, our data provide the first evidence that the EphA2 receptor directly contributes to blood-brain barrier damage and neuronal death following ischemic stroke.

Figure 1. EphA2^−/− mice have reduced brain damage and better functional outcome after focal cerebral ischemia.

Wild type and EphA2^−/− mice were subjected to 60 min MCAO, followed by 72 h reperfusion. (A) Quantified infarct volume was significantly smaller in EphA2^−/− compared with WT mice. (B) Representative ipsilateral stroke infarct volumes detected using TTC staining are shown. White (unstained) areas indicate infarction; red (stained) areas indicate normal tissue. (C) A five point neurological score was applied to the wild type and EphA2^−/− mice following ischemia and reperfusion. Data are means ± SEM from 10 to 12 animals. **p<0.01 relative to wild type. (D) Regional cerebral blood flow in WT animals was recorded during and after ischemia and expressed as a percentage of the pre-ischemic value.

Figure 2. EphA2^−/− mice have reduced post-stroke blood-brain barrier (BBB) leakage, edema and infiltrating immune cells.

(A) Quantitative measurement of brain edema in ipsilateral brain hemisphere by the wet-dry method. EphA2^−/− mice have lower levels of post-stroke brain edema in comparison with wild type mice. (B) Quantitative measurement of Evans Blue dye extravasation in ipsilateral brain hemisphere. Evans Blue dye content was significantly lower in brains from EphA2^−/− mice compared with wild type. (C) Flow cytometry analysis of the ipsilateral hemisphere following 72 h of reperfusion showed significantly fewer infiltrating (CD45^high) immune cells in EphA2^−/− mice compared to vehicle treated controls. Data are means ± SEM from 12 to 13 animals. **p<0.01 relative to wild type.

Figure 3. EphA2^−/− mice demonstrate less tight junction protein disruption following focal cerebral I/R.

Cerebral I/R-induced tight junction disruption and BBB damage were analyzed with zona occludens-1 (ZO-1) and matrix metalloproteinase-9 (MMP-9) antibodies respectively, by immunoblotting. (A and B) EphA2^−/− mice demonstrate significantly lower levels of MMP-9 in I/R samples compared to the wild type group, suggestive of lower I/R-induced BBB damage. (A and C) EphA2^−/− mice demonstrate significantly higher levels of ZO-1 in sham and I/R samples when compared to the wild type group, indicative of less I/R-induced tight junction disruption. Data are mean ± SEM, n  = 4–6. *p<0.05 or **p<0.01 relative to the wild type controls.

Figure 4. Apoptotic neuronal death is reduced in EphA2^−/− mice following focal cerebral I/R.

Ipsilateral cortical protein lysates from sham and I/R brains from both wild type and EphA2^−/− mice were analyzed for selected pro- and anti-apoptotic proteins using immunoblotting. (A–D) EphA2^−/− mice demonstrate significantly lower levels of apoptotic cell death (cleaved caspase-3; Cl. Cas3) and pro-apoptotic proteins (P-SAP/JNK, BAX) as compared to the wild type group following focal cerebral I/R. (A and E) EphA2^−/− mice demonstrate significantly higher levels of the anti-apoptotic protein, Bcl–2, compared to wild type following cerebral I/R. Data are mean ± SEM, n = 4–5. *p<0.05, ***p<0.0001 relative to wild type.

Figure 5. EphA2 expression is modulated following ischemic conditions in vitro and in vivo.

Wild type primary cortical neurons were used to show expression profiles of EphA2 and its ligands - Ephrins A1, 2 and 3 - using immunocytochemistry. (A–D) Cortical neurons showed expression of EphA2 and Ephrins A1–3 under normal and GD conditions. The neuronal protein lysates obtained from normal and GD conditions were probed for changes in the above-mentioned proteins using immunoblotting. (E–I) Expression of EphA2 and its ligands ephrin-A1, ephrin-A2, and ephrin-A3, was increased following GD for 12–24 h when compared to normal conditions. (J–N) A similar increase in expression was observed in vivo following cerebral I/R when compared to sham conditions. Cell-specific expression profile of EphA2 in the ischemic (24 I/R) brain sections was analysed in peri-infarct regions (200X) (O–P). EphA2 was primarily localized to neurons and blood vessels in the peri-infarct regions. Protein lysates of cortical neuronal cultures subjected to GD for 24 h were analyzed for the apoptotic cell death marker cleaved caspase-3 (Q–R). EphA2^−/− neuronal cultures showed significantly lower levels of cleaved caspase-3 when compared to wild type cultures. Data are mean ± SEM, n = 3–5. *p<0.05, **p<0.001, ***p<0.0001 relative to sham (in vivo) and normal controls (in vitro) for EphA2, Ephrin A1 or Ephrin A3 blots. Data are mean ± SEM, n = 3. *p<0.05 relative to the wild type controls in Cl.Cas3 blots.