Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis

C5a receptors are found in the central nervous system (CNS), on both neurons and glia. However, the origin of the C5a, which activates these receptors, is unclear. In the present study, we show that primary cultured mouse cortical neurons constitutively express C5, the precursor of C5a, and express the classical receptor for C5a, CD88. With cell ischemia caused by 12 h glucose deprivation, or oxygen-glucose deprivation (OGD), neurons demonstrated increased apoptosis, up-regulation of CD88, and increased levels of C5a in the media. Exogenous murine C5a (100 nM) added to the neuronal cultures resulted in apoptosis, without affecting cell necrosis. Pretreatment of the cells with the specific CD88 receptor antagonist PMX53 (100 nM) significantly blocked ischemia-induced apoptosis (∼50%), and neurons from CD88(-/-) mice were similarly protected. In a murine model of stroke, using middle cerebral artery occlusion (MCAO), we found that C5a levels in the brain increased; this also occurred in cerebral slice cultures exposed to OGD. CD88(-/-) mice subjected to MCAO had significantly reduced infarct volumes and improved neurological scores. Taken together, our results demonstrate that neurons in the CNS have the capability to generate C5a following ischemic stress, and this has the potential to activate their C5a receptors, with deleterious consequences

C5a receptor (CD88) and TLR2 inhibition improves hypothermia-induced neuroprotection in an in vitro ischemic model

The concept of 'salvageble penumbra' has prompted both scientists and physicians to explore various neuroprotective approaches that could be beneficial during stroke therapy. Unfortunately, most of them have proved ineffective in targeting multiple cellular death cascades incited within the ischemic penumbra. Hypothermia has been shown to be capable of addressing this problem to some extent. Although many studies have shown that hypothermia targets several cellular processes, its effects on innate immune receptor-mediated apoptotic death still remain unclear. Moreover, whether inhibiting the signaling of innate immune receptors like complement anaphylatoxin C5a receptor (CD88) plays a role in this hypothermic neuroprotection still need to be deciphered. Using various types of ischemic insults in different neuronal cells, we confirm that hypothermia does indeed attenuate apoptotic neuronal cell death in vitro and this effect can be further enhanced by pharmacologically blocking or knocking out CD88. Thus, our study raises a promising therapeutic possibility of adding CD88 antagonists along with hypothermia to improve stroke outcome

EphA2 expression is modulated following ischemic conditions <i>in vitro</i> and <i>in vivo.</i>

<p>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 <i>in vivo</i> 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&lt;0.05, **p&lt;0.001, ***p&lt;0.0001 relative to sham (<i>in vivo)</i> and normal controls (<i>in vitro)</i> for EphA2, Ephrin A1 or Ephrin A3 blots. Data are mean ± SEM, n = 3. *p&lt;0.05 relative to the wild type controls in Cl.Cas3 blots.</p

Evidence that the EphA2 receptor exacerbates ischemic brain injury

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