Anti-apoptotic effect of adipose tissue-derived stromal vascular fraction in denervated rat muscle

P. J. Kingham,L. J. Backman,R. El-Habta,G. Andersson

doi:10.1186/s13287-021-02230-y

Abstract

BackgroundRecovery of muscle function after peripheral nerve injury is often poor, and this can be attributed to muscle fiber atrophy and cell death. In the current study, we have investigated the effects of stromal vascular fraction (SVF) on muscle cell apoptosis and its potential to preserve muscle tissue following denervation.MethodsRat gastrocnemius muscle was denervated by sciatic nerve transection. At 2 and 4 weeks after injury, muscles were examined histologically and apoptosis was measured using TUNEL assay and PCR array for a range of apoptotic genes. Additionally, an in vitro TNF-α apoptosis model was established using SVF cells co-cultured indirectly with primary rat myoblasts. Annexin V and TUNEL were used together with Western blotting to investigate the signaling pathways.ResultsDenervated muscles showed significantly higher TUNEL reactivity at 2 and 4 weeks following nerve injury, and an increased expression of caspase family genes, mitochondria-related apoptotic genes, and tumor necrosis factor family genes. In cultured rat primary myoblasts, Annexin V labeling was significantly increased at 12 h after TNF-α treatment, and this was followed by a significant increase in TUNEL reactivity at 48 h. Western blotting showed that caspase-7 was activated/cleaved as well as the downstream substrate, poly (ADP-ribose) polymerase (PARP). Co-culture of myoblasts with SVF significantly reduced all these measures of apoptosis. Bax and Bcl-2 levels were not changed suggesting that the TNF-α-induced apoptosis occurred via mitochondria-independent pathways. The protective effect of SVF was also shown in vivo; injections of SVF cells into denervated muscle significantly improved the mean fiber area and diameter, as well as reduced the levels of TUNEL reactivity.ConclusionsThis study provides new insights into how adipose tissue-derived cells might provide therapeutic benefits by preserving muscle tissue.

Highlights

The incidence of peripheral nerve injuries is approximately 3% in trauma-injury patients [1]
We have previously shown that denervated muscle has increased expression of muscarinic acetylcholine (ACh) receptors and that Adipose tissue-derived stem cell (ASC) are capable of increasing the proliferation of myoblasts in vitro through paracrine secretion of ACh [8]
Anti-apoptotic effect of stromal vascular fraction (SVF) on Tumor necrosis factor alpha (TNF-α)-induced apoptosis Indirect co-culture of SVF and tumor necrosis factor (TNF)-α-treated myoblasts significantly reduced all measures of apoptosis, including cleavage of poly (ADP-ribose) polymerase (PARP) (Fig. 3a), and caspase 3/7 activity (Fig. 3b)

Summary

Introduction

The incidence of peripheral nerve injuries is approximately 3% in trauma-injury patients [1]. Schaakxs et al showed in an experimental animal model that injections of in vitro-stimulated adipose-derived stem cells (ASCs) into denervated muscle reduced the atrophy and enhanced hind limb functionality [7]. We have previously shown that denervated muscle has increased expression of muscarinic acetylcholine (ACh) receptors and that ASCs are capable of increasing the proliferation of myoblasts in vitro through paracrine secretion of ACh [8]. In this current study, we hypothesized that injections of freshly isolated adipose-tissue stromal vascular fraction (SVF) would maintain the integrity of the muscle fibers during the period of nerve regeneration. We have investigated the effects of stromal vascular fraction (SVF) on muscle cell apoptosis and its potential to preserve muscle tissue following denervation

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Results

Discussion

Conclusion