Senescence-associated beta-galactosidase activity expression in aging hippocampal neurons Yi-Qun Geng a , Ji-Tian Guan b , Xiao-Hu Xu c , Yu-Cai Fu d, * a Central Laboratory, Joint Shantou International Eye Center of Shantou University and The Chinese University of Hong Kong, Shantou University Medica College, Dongxia Bei Road, Shantou, 515041 Guangdong, China b Department of Radiology, The Second Affiliated Hospital of Shantou, University Medical College, Dongxia Bei Road, Shantou, 515041 Guangdong, China c Department of Forensic Medicine, Shantou University Medical College, No. 22 Xinling Road, Shantou, 515041 Guangdong, China d Laboratory of Cell Senescence, Department of Etiology, Shantou University Medical College, No. 22 Xinling Road, Shantou, 515041 Guangdong, China article info Article history: Received 24 April 2010 Available online 10 May 2010 Keywords: Senescence-associated beta-galactosidase (SA-beta-GAL) Hippocampus Rat Biomarker abstract To investigate the activity of senescence-associated beta-galactosidase (SA-beta-GAL) in the hippocampus of aging rats. Hippocampi of 6-, 18-, and 24-month-old rats were observed by histochemical staining for SA- beta-GAL and cytochemical staining for SA-beta-GAL in cultured hippocampal neurons. The activity of SA- beta-GAL doubled in hippocampal pyramidal cells of the CA3 region in rats between 6 and 18 months (14.57 ± 2.74% vs. 31.66 ± 14.12% SA-beta-GAL-positive, respectively), and reached 50.76 ± 14.41% positive at 24 months. The activity of SA-beta-GAL also increased as a function of time upon prolonged culture of cul- tured hippocampal neurons with 95% of cells being SA-beta-GAL-positive at 20 days in vitro. Interestingly, no SA-beta-GAL-positive cells were found in neurons of the hippocampal dentate gyrus, a neurogenic region of the brain, at any age examined. SA-beta-GAL can be used as a senescence biomarker in determining senes- cent neurons in hippocampal pyramidal cells of the CA3 region in advanced aging. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Studies of cell senescence have been largely derived from cell culture systems, in which cells undergo replication senescence characterized by irreversible replicative exhaustion, enlarged and flattened cellular morphology, and critical shortening of chromo- somal telomeres [1]. However, in vivo, senescent characteristics of cells are obscure and difficult to distinguish from quiescent or terminally differentiated cells. In brain research on aging, a bio- marker to identify senescent cells in situ is needed. Recently, senescence biomarkers and related genes were ap- plied to make the identification of senescent cells feasible [2,3]. SA-beta-GAL is a widely used marker for cellular senescence [4]. SA-beta-GAL activity is detectable at pH 6.0 and permits the iden- tification of senescent cells both in culture and in mammalian tis- sues [5]. In cultured cells, SA-beta-GAL activity has been observed in senescent human diploid fibroblasts (HDFs), and in skin, liver, muscle, and endothelial cells [6]. In addition, SA-beta-GAL activity increases upon prolonged culture of mouse neurons [7]. However, the types of neurons that display SA-beta-GAL activity and the cor- relation between elevated SA-beta-GAL activity and aging in vivo remain to be determined [8]. 2. Materials and methods 2.1. Subject Male Sprague–Dawley rats at 6 (n = 10), 18 (n = 10) and 24 (n = 10) months were housed at a temperature of 22–24 °C in a light-con- trolled environment with a 12:12-hour light–dark cycle. All rats were fed standard lab chow. Water was freely available to all groups. 2.2. Tissue preparation Rats were sacrificed under anesthesia administered by intraperito- neal injection of pentobarbital sodium (50 mg/kg body wt.). The brain was sagitally cut into two halves. One side of cerebral hemisphere was prepared for paraffin sections immunohistochemical staining. For SA- beta-GAL histochemistry, the other side was frozen in liquid nitrogen, and embedded in Optimal Cutting Temperature (OCT) compound (Laica, Wetzlar, German). Coronal sections were cut to 6 lm thickness and mounted onto glass slides. After air drying for 30 min, sections were fixed in 1% formalin for 1 min, washed in PBS, dried, and stored at À80 °C until use used for SA-beta-GAL staining. 2.3. Histochemical staining for SA-beta-GAL Frozen sections were rehydrated three times, 5 min each, with rinse buffer (100 mM sodium phosphate, 2 mM MgCl 2 , 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.011 * Corresponding author. E-mail addresses: yqgeng@126.com (Y.-Q. Geng), jtguan@126.com (J.-T. Guan), xhxu@stu.edu.cn (X.-H. Xu), ycfu@stu.edu.cn (Y.-C. Fu). Biochemical and Biophysical Research Communications 396 (2010) 866–869 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc