Endogenous CNTF mediates stroke-induced adult CNS neurogenesis in mice

Abstract

Focal brain ischemia in adult rats rapidly and robustly induces neurogenesis in the subventricular zone (SVZ) but there are few and inconsistent reports in mice, presenting a hurdle to genetically investigate the endogenous neurogenic regulators such as ciliary neurotrophic factor (CNTF). Here, we first provide a platform for further studies by showing that middle cerebral artery occlusion in adult male C57BL/6 mice robustly enhances neurogenesis in the SVZ only under very specific conditions, i.e., 14 days after a 30 min occlusion. CNTF expression paralleled changes in the number of proliferated, BrdU-positive, SVZ cells. Stroke-induced proliferation was absent in CNTF −/− mice, suggesting that it is mediated by CNTF. MCAO-increased CNTF appears to act on C cell proliferation and by inducing FGF2 expression but not via EGF expression or Notch1 signaling of neural stem cells in the SVZ. CNTF is unique, as expression of other gp130 ligands, IL-6 and LIF, did not predict SVZ proliferation or showed no or only small compensatory increases in CNTF −/− mice. Expression of tumor necrosis factor-α, which can inhibit neurogenesis, and the presence of leukocytes in the SVZ were inversely correlated with neurogenesis, but pro-inflammatory cytokines did not affect CNTF expression in cultured astrocytes. These results suggest that slowly up-regulated CNTF in the SVZ mediates stroke-induced neurogenesis and is counteracted by inflammation. Further pharmacological stimulation of endogenous CNTF might be a good therapeutic strategy for cell replacement after stroke as CNTF regulates normal patterns of neurogenesis and is expressed almost exclusively in the nervous system.

Introduction

It is well-known that focal cerebral and striatal ischemia enhances neurogenesis in the SVZ and subgranular zone of the dentate gyrus (Burns et al., 2009, Kernie and Parent, 2010, Zhang and Chopp, 2009). Many studies have shown that ischemic injury induced by MCAO in adult rats increases the number of proliferating SVZ cells (Arvidsson et al., 2002, Jin et al., 2001, Parent et al., 2002, Zhang et al., 2001). Stroke-induced neurogenesis also seems to occur in the human brain (Jin et al., 2006, Macas et al., 2006). These results have raised the possibility that new neurons formed by endogenous neural stem cells can be used to replace cells as a treatment for stroke. A better understanding of the molecular regulators of stroke-induced neurogenesis and identification of endogenous drug targets to promote neurogenesis would be facilitated by the use of transgenic and knockout mice. However, in contrast to rats, it is not clear under which conditions stroke induced by MCAO in mice enhances SVZ neurogenesis. Only a few papers have reported changes in the SVZ of mice with one showing decreases at 3 days after MCAO and a return to baseline at 7 days (Tsai et al., 2006) and another an approximately 50% increase at 14 days after a permanent cortically restricted stroke (Wang et al., 2007). Some of the reasons for these apparent discrepancies and the differences between rats and mice might include mouse-specific requirements for the occlusion and post-injury times, or technical issues in the much smaller mouse.

Injured cells release many cytokines and growth factors after ischemia, which may be responsible for the neurogenic responses to stroke. Treatment with exogenous growth factors such as FGF2, EGF, BDNF, VEGF, EPO, CNTF and IGF-1 have been shown to promote ischemia-induced neurogenesis, predominantly in rats (Chmielnicki et al., 2004, Esneault et al., 2008, Jin et al., 2004, Schabitz et al., 2007, Sun et al., 2003, Tureyen et al., 2005, Wang et al., 2007, Yan et al., 2006, Yang et al., 2008). Of these, IGF-1 (Yan et al., 2006) and CNTF have been confirmed to regulate a proportion of endogenous neurogenesis under normal conditions using knockout mice or antibodies (Yang et al., 2008). However, only IGF-1 has been shown, by antibody knock-down, to be involved in the response after stroke (Yan et al., 2006), reiterating the need for using more genetic mouse models. CNTF is of particular interest as it is up‐regulated in the ischemic cortex nearly 20 fold by 2 weeks after stroke (Lin et al., 1998), enhances normal adult SVZ neurogenesis in mice and can be increased through systemic D2 dopamine agonist injections (Emsley and Hagg, 2003, Yang et al., 2008). Thus, CNTF might be a good therapeutic target for stroke but its role in stroke-induced neurogenesis remains to be investigated. Some of the cytokines produced during the inflammatory response after stroke, such as IL-6, TNFα, and IFNγ, have anti-proliferative effects in the SVZ and hippocampal subgranular zone (Ben-Hur et al., 2003, Iosif et al., 2006, Iosif et al., 2008, Monje et al., 2003, Vallieres et al., 2002). The interactions between pro- and anti-neurogenic cytokines remain to be investigated and would be facilitated by using genetically altered mice.

The present study was aimed at investigating under which conditions reproducible MCAO in mice would enhance SVZ neurogenesis, providing a platform to determine whether and how endogenous CNTF regulates this stroke-induced neurogenesis.