Our previous studies and others have identified that

Our previous studies and others have identified that the inhalational anesthetic isoflurane induces neuronal apoptosis via [Ca2+]i overload through the opening of synaptic voltage-dependent calcium channels (VDCCs) and the excessive Ca2+ release from the endoplasmic reticulum (Zhao et al., 2011; Zhao et al., 2010). Isoflurane-induced [Ca2+]i overload not only activates mitochondrial caspase-dependent apoptosis pathway by decreasing the Bcl-2/Bax or Bcl-xl/Bad ratio, opening the mitochondrial permeability transition pore (mPTP), releasing of cytochrome c from the mitochondria and activating caspase-3 (Li et al., 2014; Wei et al., 2005; Yon et al., 2005; Zhang et al., 2010; Zhang et al., 2012), but also is linked to the activation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) pathways (Li et al., 2013; Liao et al., 2014), which leads to neuronal apoptosis and learning and memory impairment in young animals. Whether caspase-independent pathway contributes to isoflurane-induced neuroapoptosis remains underdetermined. The aim of the current study was therefore to investigate the role of AIF in the isoflurane-induced neuroapoptosis in the developing rat brain. Moreover the effects of calpain or JNK activation on isoflurane-induced AIF apoptosis pathway were also investigated.
Materials and methods
Results
Discussion Consistent with previous reports (Sanchez et al., 2011; Zhang et al., 2010; Zhang et al., 2012), our present data showed that isoflurane increased pro-apoptotic factor Bax expression in the mitochondria, decreased anti-apoptotic factor Bcl-2 levels, facilitated cytochrome c releases from the mitochondria to the cytosol, and induced activation of caspase-dependent apoptotic pathways in the developing rat brain. AIF-mediated neuronal cell death plays a causal role in a variety of pathological settings, such as traumatic Pyoluteorin receptor injury, hypoxia, ischemic brain injury or ethanol- mediated neuron damage (Cao et al., 2007; Cherian et al., 2008; Slemmer et al., 2008). Our experiments demonstrated that AIF-mediated apoptotic pathway was also involved in isoflurane-induced neurotoxicity by eliciting AIF release from the mitochondria and subsequent translocation into nucleus. The nuclear AIF induced neuroapoptosis which was reflected by a significant increase of cells positively stained for both tAIF and TUNEL in the cortices of neonatal rats after isoflurane exposure. It was noticed that the peak time of cytochrome c and caspase-3 expression was within 6 h; whereas the peak time of AIF translocation into nucleus was at 24 h, which suggest that the caspase-dependent apoptotic pathway is activated in the early period; whereas AIF apoptotic pathways is activated in the late period after isoflurane anesthesia. These findings are consistent with previous studies that report that the mitochondrial release of AIF proceeds more slowly than the release of cytochrome c (Cherian et al., 2008; Munoz-Pinedo et al., 2006; Ramachandran et al., 2003). This slower-rate kinetics has been associated with the requirement of a putative secondary event such as proteolytic cleavage necessary for the mitochondrial release of AIF (Otera et al., 2005). This cleavage of AIF is achieved through the activation of cysteine proteases cathepsins and calpains (Yuste et al., 2005). The majority of studies have reported that AIF release does not require caspase activation (Susin et al., 2000; Susin et al., 1999; Yu et al., 2002) and it occurs in a similar way independently of the presence or absence of caspase inhibitors (Munoz-Pinedo et al., 2006). Therefore, isoflurane activated two distinct pathways leading to nuclear apoptosis in the developing brain. There is abundant evidence supporting a connection between activation of calpain, an important calcium-dependent protease, and AIF-mediated apoptotic pathway (Lu et al., 2013; Yang et al., 2008). Prolonged increase in intracellular Ca2+ level is a prerequisite step for the activation of mitochondrial calpain to release AIF during cell death (Brambrink et al., 2010; Lu et al., 2013). Recent researches have shown that isoflurane induces cytosolic calcium overload in the developing neurons by activation GABAA receptor and opening of synaptic VDCCs. The GABA-induced [Ca2+]i increase further induces Ca2+ release from the endoplasmic reticulum via activation of IP3 receptors (Wei et al., 2008; Yang et al., 2008; Zhao et al., 2011). Mitochondria are able to buffer [Ca2+]i to certain extent under both physiological and pathophysiological conditions. Free mitochondrial Ca2+ ([Ca2+]m) will increase when the Ca2+ buffering capacity of mitochondrial matrix becomes nearly saturated, which activates mitochondrial calpains and subsequently trigger apoptotic cell death (Norberg et al., 2008). Our results showed that isoflurane enhanced the expression of μ-calpain and AIF truncation in the mitochondria within 6 h after exposure, the expression of mitochondrial m-calpain, tAIF release and subsequent nuclear translocation within 6 h to 24 h after isoflurane exposure, which are consistent with the studies of Ozaki et al. (Ozaki et al., 2009; Wang et al., 2008), suggesting that mitochondrial μ-calpain is an initiator of the AIF pathway by cleaving the mature AIF to tAIF and that the mitochondrial m-calpain plays a significant role in the release of tAIF into the cytosol. Pretreatment with calpain inhibitor MDL-28170 blocked isoflurane-induced AIF release and nuclear translocation, while MDL-28170 had no influence in isoflurane-induced expression of Bax, Bcl-2 and caspase-3. These findings is in agreement with the observations of Wang et al. (Wang et al., 2008), which confirms that the calpain inhibitor MDL-27180 blocks the release of AIF and cytochrome c from the mitochondria in rats after lithium-pilocarpine-induced status epilepticus. Actually, our results found MDL-27180 only slightly reduced isoflurane-induced release of cytochrome c and the expression of cytochrome c was still higher than normal level. Cytochrome c release is a direct consequence of the Bax/Bak-mediated mitochondrial permeabilization (Antonsson et al., 2000). Since MDL-27180 did not influence the isoflurane-induced Bax translocated to mitochondria, which suggest MDL-27180 may not change Bax/Bak-mediated mitochondrial permeabilization. This may be the reason that MDL-27180 only slightly reduced cytochrome c release. Our previous studies have verified JNK and P38 MAPK signal pathways are also involved in isoflurane-induced caspase-3 activation in addition to cytochrome c (Li et al., 2013; Liao et al., 2014), therefore inhibition calpain by MDL-27180 had no effects on isoflurane-induced caspase-3 activation although it slightly reduced cytochrome c level. On the other hand, Bax/Bak-mediated mitochondrial permeabilization does not directly induce AIF release (Norberg et al., 2008). AIF has to be cleaved to soluble tAIF by mitochondrial μ-calpain firstly and then released to the cytosol by VDAC-Bax pores that are promoted to be formed by the activation of mitochondrial m-calpain (Ozaki et al., 2009). Our results further emphasize the importance of a mitochondrial calpain for AIF processing, in isoflurane-induced neurotoxicity.