Evaluation of senescent cells in intervertebral discs by lipofuscin staining

Highlights

• Lipofuscin staining proved to be a robust marker of senescent IVD cells

• GL13 staining can be used in FFPE intervertebral discs

• GL13 staining confirms the increased number of senescent cells in aged IVDs

Abstract

Intervertebral disc (IVD) degeneration is considered an important contributor of low back pain, a major age-related disease. Interestingly, an unprecedented high number of senescent cells has been reported in aged and degenerated IVDs, most probably affecting tissue homeostasis. In previous studies classical markers of cellular senescence have been used, such as SA-β-gal staining or p16^Ink4a expression. Aim of the presented study was a re-evaluation of the number of senescent IVD cells by using a newly established staining procedure for lipofuscin, based on a Sudan Black-B analogue (GL13), which can be used in fresh, as well as in fixed and embedded tissues. In cultures of senescent rat and human IVD cells both SA-β-gal and GL13 gave similar percentages of senescent cells. Similarly, in fresh tissues from old rats the ratios of senescent cells were high with both detection procedures. Finally, in formalin-fixed and paraffin-embedded tissues from humans, a significant increased number of GL13-positive cells was found in herniated tissues, as compared to apparently normal ones, while similar numbers of p16^Ink4a-positive cells were observed. These data confirm the significantly enhanced number of senescent cells in aged and degenerated IVDs, most probably contributing to the degeneration of this tissue.

Introduction

Low back pain (LBP) is one of the major age-related chronic diseases, affecting a large part of the population worldwide, thus representing an important medical and socioeconomic problem (Taylor et al., 1994; Hart et al., 1995; Maniadakis and Gray, 2000; Vos et al., 2012; Stewart Williams et al., 2015). Intervertebral disc (IVD) degeneration, a process that starts early in life, is considered a crucial parameter in the development of LBP. This degeneration is associated with biochemical changes, such as the expression of several inflammatory mediators and catabolic enzymes, i.e. inflammatory cytokines, matrix metalloproteinases (MMPs) and aggrecanases (Wuertz et al., 2012). These alterations lead to the loss of the predominant extracellular matrix components, such as collagen type I and II and proteoglycans (mainly aggrecan), and eventually to the deterioration of IVD structure and function (Urban et al., 1982; Urban and Roberts 1995; Ishihara et al., 1996; Roberts et al., 1996; Ishihara et al., 1997; Urban and Roberts 2003).

One of the striking characteristics of the intervertebral disc is its extremely low cellularity, dropping abruptly from the periphery of the tissue (Annulus Fibrosus, AF) towards its core (Nucleus Pulposus, NP) (Maroudas et al., 1975; Trout et al., 1982; Urban et al., 1982; Urban and Roberts 1995, Ishihara et al., 1996; Roberts et al., 1996; Ishihara et al., 1997; Urban and Roberts 2003; Liebscher et al., 2011). However, these cells are crucial for the homeostasis of the tissue, as they control the synthesis and degradation of extracellular matrix, which is surrounding them. Under normal conditions, these cells express a minute proliferative rate (Pratsinis and Kletsas 2007; Pratsinis and Kletsas, 2008; Pratsinis et al., 2012; Pratsinis and Kletsas 2015), which increases with the degeneration of the tissue, leading to the formation of characteristic cell clusters (Johnson et al., 2001; Urban and Roberts 2003). In addition, we have observed that with increasing age and degeneration of the IVD a significant number of senescent cells (Urban and Roberts 1995; Roberts et al., 2006), most probably being the outcome of the intense stresses prevailing in the tissue, such as nutritional, mechanical, oxidative or osmotic (Bibby et al., 2005; Dimozi et al., 2015; Li et al., 2017a; Zhang and Yang, 2019; Yin et al., 2019; Zhang et al., 2019).

Normal cells can become senescent either due to continuous proliferation leading to telomere attrition (replicative senescence) or due to various extrinsic or intrinsic stimuli, such as irradiation, oxidative stress, genotoxic drugs, oncogenic stress, metabolic and epigenetic changes, etc. (collectively termed stress-induced premature senescence); in both cases senescent cells are characterized by their inability for proliferation, due to the combined action of cell cycle inhibitors, e.g. p21^WAF1 and p16^Ink4a (Gorgoulis et al., 2019). In addition, these cells express a specific phenotype (termed senescence associated secretory phenotype, SASP), marked by the increased secretion of inflammatory molecules, MMPs and other catabolic molecules, thus affecting locally tissue homeostasis (Rodier et al., 2009; Freund et al., 2010; Tchkonia et al., 2013; Vamvakas et al., 2017; Kouroumalis et al., 2019). Interestingly, similar changes in inflammatory and catabolic molecules are observed in the degenerated IVD (Le Maitre et al., 2007; Wuertz et al., 2012; Mavrogonatou et al., 2019).

The majority of the studies that evaluate the presence of senescent cells in intervertebral discs in vivo have been based on the widely used biomarker of cellular senescence Senescence-Associated β-galactosidase assay (SA-β-gal) (Roberts et al., 2006; Gruber et al., 2007). Interestingly, the percentages of the senescent cells, even within the same study, vary tremendously and in some cases the numbers of senescent cells are extremely high, as compared with other aged or pathological tissues (Roberts et al., 2006; Gruber et al., 2009; Kim et al., 2009; Kletsas 2009). Here, it must be mentioned that SA‐β‐gal assay has been shown to produce false‐positive results, under certain cell culture conditions (cell density or serum starvation), while certain cells that fully undergo senescence do not exhibit SA‐β‐gal activity (Georgakopoulou et al., 2013; Munoz-Espin and Serrano 2014). Most important, SA-β-gal staining, as it is based on an enzymatic reaction, can be used solely in fresh cells and tissues (Roberts et al., 2006).

We recently reported a specific recognition of senescent cells in biological material, including cultured cells, fresh/frozen and, most importantly, archival (formalin‐fixed and paraffin‐embedded, FFPE) tissues, by applying a specific staining procedure using the Sudan Black B (SBB) analogue GL13, which effectively detects lipofuscin (Georgakopoulou et al., 2013; Evangelou et al., 2017). Lipofuscin is a non-degradable aggregate of oxidized proteins, lipids, and metals (Jung et al., 2007), which accumulates in senescent cells and is considered as a robust senescent marker (Kohli et al., 2021). Accordingly, aim of this work was to compare GL13 and other classical senescence markers for the evaluation of the number of senescent cells in rat and human IVD cells and tissues.