Bi-directionally protective communication between neurons and astrocytes under ischemia

Abstract

The extensive existing knowledge on bi-directional communication between astrocytes and neurons led us to hypothesize that not only ischemia-preconditioned (IP) astrocytes can protect neurons but also IP neurons protect astrocytes from lethal ischemic injury. Here, we demonstrated for the first time that neurons have a significant role in protecting astrocytes from ischemic injury. The cultured medium from IP neurons (IPcNCM) induced a remarkable reduction in LDH and an increase in cell viability in ischemic astrocytes in vitro. Selective neuronal loss by kainic acid injection induced a significant increase in apoptotic astrocyte numbers in the brain of ischemic rats in vivo. Furthermore, TUNEL analysis, DNA ladder assay, and the measurements of ROS, GSH, pro- and anti-apoptotic factors, anti-oxidant enzymes and signal molecules in vitro and/or in vivo demonstrated that IP neurons protect astrocytes by an EPO-mediated inhibition of pro-apoptotic signals, activation of anti-apoptotic proteins via the P13K/ERK/STAT5 pathways and activation of anti-oxidant proteins via up-regulation of anti-oxidant enzymes. We demonstrated the existence of astro-protection by IP neurons under ischemia and proposed that the bi-directionally protective communications between cells might be a common activity in the brain or peripheral organs under most if not all pathological conditions.

Keywords: Anti-apoptosis and anti-oxidant; Astro-protection; Bi-directional communication.

Fig. 1

A–C: Effects of different durations of ischemia on cell viability and expression of hypoxia-inducible factor-1 alpha in neurons. Primary neurons were subjected to combined conditions of 1% O₂ and serum-free DMEM without glucose for 0, 0.5, 1, 2, 3 or 4-h, and then back to normoxia in DMEM containing 5% FBS for 24-h. The cell viability (A, n = 12) of neurons was then measured by MTT assay and HIF-1 alpha level in neuronal nuclei (B&C, n = 5) detected by western blot. Data were Mean ± SD. *p < 0.05, ***p < 0.001 versus the control (0-h). D-H: Ischemia-preconditioned neurons protected astrocytes against ischemic injury by inhibition of apoptosis. Astrocytes were pre-incubated with IPc-0 h, 0.5 h, 1 h or 2 h NCM for 48-h before undergoing ischemia (12-h). Cell viability (D) and LDH release (E) were measured, and the degree of apoptosis analyzed by TUNEL and DAPI staining. F. TUNEL (upper) and DAPI (lower) staining; G, apoptotic cells (% of total cells); H, DNA laddering analysis (M = marker). Data were Mean ± SD (n = 10). **P < 0.01, ***P < 0.001 versus ‘IPc-0h’ or ‘IPc-0h + Isch’ (Isch = Ischemia).

Fig. 2

Effects of EPO secreted by ischemia-preconditioned neurons on apoptosis of astrocytes in ischemia. Astrocytes were incubated with IPc-0 h or IPc-1 h NCM in the presence or absence of EPO antibody (aEPO) or recombinant human EPO (rhEPO, 75 pg/ml) followed by treatment with ischemia. The contents of cleaved/active caspase-3 (A), Bcl-2 (B), Bcl-xL (C), ¹³⁶p-Bad (D), ¹¹²p-Bad (E), cell viability (G), LDH release (H), TUNEL-positive cells (I, Representative photographs of TUNEL (upper) and DAPI (lower) staining; J, The percentage of apoptotic cells in total cells) were measured as described in ‘Methods’. F, EPO contents in IPcNCMs measured by ELISA. Data were Mean ± SD (A-E: n = 4; F: n = 6). *p < 0.05, **P < 0.01, ***P < 0.001 versus ‘IPc-0h’, ‘IPc-0h + Isch’ or ‘IPc-1h + Isch’ (Isch = Ischemia).

Fig. 3

Involvement of ERK, PI3K and STAT5 in EPO-mediated anti-apoptosis in astrocytes. In A (n = 10) and B (n = 6), astrocytes were incubated with IPc-0 h NCM (IPc-0h) with or without ischemia (Isch), or with IPc-1h in the presence of AG490 (AG), PD98059 (PD), LY294001 (LY), 573108 (STAT5 inhibitor), BAY11-7082 (BAY) and SB203850 (SB) for 1-h, followed with IPc-1h NCM for 48-h and exposure to ischemia for 12-h. Viabilities were then assayed. In C to F, astrocytes were incubated in IPc-1h NCM (IPc-1h) with or without anti-EPO antibody (aEPO) for 20 min, and p-Akt (C), p-Erk (D), p-STAT3 (E) and p-STAT5 (F) were then measured by Western blot (n = 3). G–J: Astrocytes were incubated in IPc-1h NCM with or without aEPO for 48-h, and Bcl-2 (G), ¹³⁶p-Bad (H), ¹¹²p-Bad (I) and Bcl-xL (J) were then measured by Western blot (n = 3). K-N: Astrocytes were pre-treated with or without PD98059, LY294002 or 573108 for 1-h before being incubated in IPc-1h NCM for 48-h, and Bcl-2 (K), ¹³⁶p-Bad (L), ¹¹²p-Bad (M) and Bcl-xL (N) were then measured by Western blot (n = 3). Data were Mean ±SD. *p < 0.05, **p < 0.01,***p < 0.001. versus the corresponding control (Isch = Ischemia).

Fig. 4

Effects of ischemia-preconditioned neurons on apoptosis of astrocytes in the cortex of rats after ischemia-reperfusion in vivo. Rats received 0.5 nmol kainic acid (KA, in 1.5 µl PBS) injections in the left cerebral cortex. After 24-h, the animals were treated with 4 min forebrain ischemia (IP) and then subjected to 20 min forebrain ischemia followed by reperfusion (I/R) for 24-h. A, Neurons and astrocytes in the peri-injected area (asterisk) were identified by immunostaining for MAP2 (green) and GFAP (red) respectively. In this area, selective loss of neurons but not astrocytes was observed. B, Astrocytic apoptosis under different conditions was analyzed by TUNEL (green) and GFAP (red) double staining. C, the number of TUNEL positive cells co-localized with GFAP staining was counted in the area 0.5–1.0 mm from the injection site (n = 12). D, Rats were injected with KA or PBS into the left or right cerebral cortex. After 24-h, the animals were treated with 4 min forebrain ischemia followed by a 24-h interval. The tissues around the injection site were cut out and used to measure the content of EPO (mIU/mg protein) by ELISA (n = 11). Data were presented as mean ± SD. * p < 0.05; **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 5

Ischemia-preconditioned neurons protect astrocytes against I/R injury by inhibiting oxidative stress. A–D: Astrocytes were pre-incubated with IPc-0h, IPc-0.5h, IPc-1h or IPc-2h NCM for 48-h before being exposed to ischemia (I) for 12-h followed by reperfusion (R) for 24-h. The ROS and GSH contents were determined, and fluorescence intensities were observed under confocal microscopy and quantified by using a fluorescent spectrophotometer as described in Methods. A, Representative confocal images indicating ROS levels in astrocyte which were measured by a fluorometric assay with 2′, 7′-dichlorofluorescin diacetate (DCFH-DA); B, DCF intensity (% IPc-0h, n = 10); C, 8-isoprostane contents (% IPc-0h, n = 10); D, GSH contents (n = 6). E and F: Astrocytes were incubated with IPc-0h or IPc-1h NCM in the presence or absence of EPO antibody (aEPO) followed by treatment with ischemia, and DCF intensity (% IPc-0h, n = 10) (E) and 8-isoprostane contents (%I Pc-0h, n = 10) (F) were then determined as described in Methods. G: Effects of ischemia-preconditioned neurons on expression of anti-oxidant enzymes in astrocytes. Astrocytes were incubated with IPc-0h or IPc-1h NCM for 48-h, and the contents of T-SOD, Mn-SOD, CuZn-SOD, CAT (catalase) and GSH-PX were then measured (n = 6). Data were Mean ± SD. *P < 0.05, **P < 0.01 vs. IPc-0h or IPc-0h + I/R.

Fig. 6

A hypothetical scheme for the mechanisms involved in the astro-protection by ischemia-preconditioned neurons. IP neurons have the ability to express and release sufficient amounts of EPO for paracrine astro-protection via the increased HIF-1 alpha induced by IP. The increased EPO binds to EPOR on the membrane of astrocytes, triggering dimerization of EPOR and inducing Jak2 activation, which in turn activates three different downstream-signaling pathways to prevent apoptosis: PI3K/Akt, ERK and STAT5. Activated PI3K/Akt can phosphorylate Bad at ser-136 and up-regulate Bcl-2 expression, and in turn preventing apoptosis. ERK signaling pathway-mediated anti-apoptosis is via enhanced Bcl-2 levels and phosphorylation of Bad at ser-112, while STAT5 suppresses astrocyte apotosis via the up-regulated expression of Bcl-xL. The anti-apoptotic effects of EPO from IP neurons on astrocytes require the combined activation of these three pathways. In addition, IP neurons were able to inhibit I/R induced-oxidative stress by up-regulation of anti-oxidant enzymes (SOD, CAT, GSH-PX) partly via EPO. Therefore. IP neurons protect astrocytes from ischemia-induced injury by an EPO-mediated anti-apoptosis and anti-oxidant effect.