Senescence-associated beta-galactosidase activity expression in aging hippocampal neurons

doi:10.1016/j.bbrc.2010.05.011

Biochemical and Biophysical Research Communications

Volume 396, Issue 4, 11 June 2010, Pages 866-869

https://doi.org/10.1016/j.bbrc.2010.05.011 Get rights and content

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 cultured 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 senescent neurons in hippocampal pyramidal cells of the CA3 region in advanced aging.

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 chromosomal 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 biomarker to identify senescent cells in situ is needed.

Recently, senescence biomarkers and related genes were applied 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 identification of senescent cells both in culture and in mammalian tissues [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 correlation between elevated SA-beta-GAL activity and aging in vivo remain to be determined [8].

Section snippets

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-controlled environment with a 12:12-hour light–dark cycle. All rats were fed standard lab chow. Water was freely available to all groups.

Tissue preparation

Rats were sacrificed under anesthesia administered by intraperitoneal 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

Hippocampal SA-beta-GAL expression increases with increasing age in rat brain

The activity of SA-beta-GAL in hippocampal pyramidal cells in CA3 region increased in 18 and 24 month age of rats (Table 1).

The staining of SA-beta-GAL

SA-beta-GAL activity increased in neurons of CA3 region of hippocampus tissue in 18 and 24 month age of rats (Fig. 1A–C).

No positive SA-beta-GAL was found in neurons of dentate gyrus of hippocampus in all groups (Fig. 2).

SA-beta-GAL in cultured primary hippocampus neurons

The variation of SA-beta-GAL positive was determined as a function of passage number and time spent in culture. SA-beta-GAL in cultured primary hippocampus

Discussion

SA-beta-GAL has become one of the most commonly used markers for cell-aging [9]. Cellular senescence is a tumor-suppressive process characterized by irreversible cell cycle exit, unique morphology, and expression of SA-beta-GAL [5]. SA-beta-GAL expression in human skin fibroblasts was shown to be a reliable indicator of the switch mechanism used by cells to enter senescence [10]. We show that the activity of SA-beta-GAL was increased in the CA3 region of rats by 18 months of age. These neurons

Acknowledgments

The authors are indebted to Dr. Huirong Lin for laboratory assistance in the Endocrine Department of the Second Affiliated Hospital of the Medical College of Shantou University. Professor Jianjun Zhang is thanked for statistical analyses in the Statistics Department of the Medical College of Shantou University. The authors would like to thank Dr. Stanley Lin for English language editing. This work was supported by Medical Scientific Research Foundation of Guangdong Province (B2008140) and

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Senescence-associated beta-galactosidase activity expression in aging hippocampal neurons

Abstract

Introduction

Section snippets

Subject

Tissue preparation

Hippocampal SA-beta-GAL expression increases with increasing age in rat brain

The staining of SA-beta-GAL

SA-beta-GAL in cultured primary hippocampus neurons

Discussion

Acknowledgments

J. Biol. Chem.

Arch. Gerontol. Geriatr.

Exp. Gerontol.

Exp. Cell Res.

J. Biomol. Screen

Exp. Gerontol.

Multiple longevity phenotypes and the transition from health to senescence

Ann. NY Acad. Sci.

Protein kinase CK2 regulates cytoskeletal reorganization during ionizing radiation-induced senescence of human mesenchymal stem cells

Cancer Res.