Brain Research Bulletin 101 (2014) 18–25 Contents lists available at ScienceDirect Brain Research Bulletin jo ur nal home p age: www.elsevier.com/locate/brainresbull Research report Increased expression of phosphorylated c-Jun and phosphorylated c-Jun N-terminal kinase associated with neuronal cell death in diabetic and high glucose exposed rat retinas Toshiyuki Oshitari ∗ , Guzel Bikbova, Shuichi Yamamoto Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba, Japan a r t i c l e i n f o Article history: Received 14 August 2013 Received in revised form 1 December 2013 Accepted 2 December 2013 Available online 12 December 2013 Keywords: c-Jun c-Jun N-terminal kinase Diabetic retinas High glucose Endoplasmic reticulum stress Neurotrophin-4 a b s t r a c t The aim of this study is to examine whether the increased expression of phosphorylated c-Jun (p-c-Jun) and phosphorylated c-Jun N-terminal kinase (p-JNK) are significantly associated with neuronal cell death in diabetic rat retinas and retinas exposed to high glucose. Retinas isolated from six adult male Sprague- Dawley rats and six streptozotocin-induced diabetic rats (DM) were cultured in serum-free medium. The explants from non-diabetic controls were cultured in normal-glucose (N) or high-glucose (HG) medium. Furthermore, neurotrophin-4 (NT-4) and Taurine-conjugated ursodeoxycholic acid (TUDCA) were incu- bated in HG medium. After 7 days, the numbers of regenerating neurites were counted per explant. After counting, the explants were fixed, cryosectioned, and stained by TUNEL, and also immunostained for p-c-Jun and p-JNK. The numbers of TUNEL-positive, p-c-Jun- and p-JNK-immunopositive cells in the GCL were significantly higher and the numbers of regenerating neurites were significantly lower in the HG and the DM groups than in the N groups. In the HG groups supplemented with NT-4 and TUDCA, the numbers of TUNEL-positive, p-c-Jun- and p-JNK-immunopositive cells were significantly lower and the numbers of neurites were significantly higher than in the HG group without NT-4 and TUDCA. Increased expression of p-c-Jun and p-JNK is associated with neuronal cell death in diabetic rat retinas and retinas exposed to high glucose. Neuroprotective effect of TUDCA and NT-4 is correlated with the suppression of p-c-Jun and p-JNK expression. These results provide a better understanding of the neurodegenerative process underlying DR. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Diabetic retinopathy (DR), one of the major complications of diabetic patients, is the leading causes of blindness in the adult population (Oshitari and Roy, 2007; Oshitari et al., 2008a; Oshitari and Mitamura, 2010). Despite extensive studies the precise mech- anisms for the onset and the progression of DR have not been elucidated (Oshitari and Roy, 2007; Oshitari et al., 2008a; Oshitari and Mitamura, 2010). Several studies including ours have indicated that not only vascular abnormalities but also neuronal abnormal- ities are associated with the pathogenesis of the early phase DR (Barber et al., 1998; Takano et al., 1999; Oshitari and Roy, 2005; Abbreviations: DR, diabetic retinopathy; ER, endoplasmic reticulum; PERK, PKR- like ER kinase; CHOP, C/EBP homologus protein; JNK, c-Jun N-terminal kinase; AP-1, activator protein-1; NT-4, neurotrophin-4; TUDCA, Taurine-conjugated ursodeoxy- cholic acid; STZ, streptozotocin; PFA, paraformaldehyde; GCL, ganglion cell layer. ∗ Corresponding author at: Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Inohana 1-8-1, Chuo-ku, Chiba 260- 8670, Chiba, Japan. Tel.: +81 43 226 2124; fax: +81 43 224 4162. E-mail addresses: Tarii@aol.com, oshitari@faculty.chiba-u.jp (T. Oshitari). Oshitari et al., 2008b, 2009, 2010, 2011; Jackson et al., 2011). Although neuronal abnormalities such as retinal neuronal cell death directly irreversibly affect the visual function in diabetic patients, the precise mechanisms of neuronal cell death induced by diabetic stress are still unclear (Oshitari and Roy, 2007; Oshitari et al., 2008a; Oshitari and Mitamura, 2010). Elucidation of the pre- cise mechanisms of neuronal cell death in early phase DR is needed to establish the neuroprotective therapies for preventing the devel- opment and progression of DR (Oshitari and Roy, 2007; Oshitari et al., 2008a; Oshitari and Mitamura, 2010). Endoplasmic reticulum (ER) stress and its involvement with retinal cell death in several models including diabetic retinas is being extensively studied by investigators (Awai et al., 2006; Li et al., 2009, 2011, 2012; Doh et al., 2010; Oshitari et al., 2011; Yang et al., 2011). Our recent study indicates that PKR-like ER kinase (PERK) and C/EBP homologus protein (CHOP) expression are related to neuronal cell death under diabetic stress (Oshitari et al., 2011). We have already demonstrated that the mitochondria- and caspase-dependent cell death pathway is associated with neu- ronal cell death induced by diabetic stress in rat and human retinas (Oshitari and Roy, 2005; Oshitari et al., 2008b, 2010). Growing 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.12.002