All trans retinoic acid modulates peripheral nerve fibroblasts viability and apoptosis
Introduction
Axons of the peripheral nervous system (PNS) have the ability to grow beyond the lesion area (Yiu and He, 2006). Compelling evidence suggests the presence of permissive environment allowing axonal regeneration in PNS (Chen et al., 2007, Vargas and Barres, 2007). Indeed, a complex of cellular and molecular changes referred to Wallerian degeneration takes place within the distal stump of the damaged nerve as the natural mechanism for healing (Dubovy, 2011). Following loss of contact with axon, Schwann cells initiate proliferation and migration (Rotshenker, 2011) and participate actively with accumulated fibroblasts at the injury site, establishing guidance channel (i.e. Bands of Bungner) in a specialized cell sorting manner (Parrinello et al., 2010). The accomplishment or failure of peripheral nerve regeneration, to the most part, depends on the integrity of connective tissue which surrounds the nerve unit (Dreesmann et al., 2009). Disruption of nerve structure reduces the functional recovery since the regenerating axons gets stuck within a scar tissue (i.e. neuroma) made of fibroblasts originating from surrounding connective tissues and Schwann cells as well (Parrinello et al., 2010). Post-traumatic neuroma not only produces neurite growth repellent factors (Tannemaat et al., 2007), such as semaphorin3A produced by fibroblasts (Berger et al., 2011) but also plays a major role in chronic neuropathic pain development (Kotulska et al., 2006). On the other hand, it has been shown that fibroblastic cells may be as a contaminator agent in the Schwann cell cultures. This predominantly happens once implemented peripheral nerves do not lapse the duration of Wallerian degeneration or elapse over 10 days. Thus, it is recommended implementing Wallerian degeneration with one-week duration for attaining more and activated Schwann cells in culture (Kraus et al., 2010).
All trans retinoic acid (ATRA) is a functional metabolite of vitamin A that participates in multiple biological processes from embryogenesis, reproduction, inflammation, and proliferation to differentiation and apoptosis (Noy, 2010, Wolf, 2008). Interestingly, retinoic acid signaling involves in response to peripheral nerve injury (Zhelyaznik and Mey, 2006). It has been shown that retinaldehyde dehydrogenase II enzyme (RALDH II), which synthesizes retinoic acid, attains its maximum activity in seven days of peripheral nerve insult (Maden, 2007). Moreover, retinoic acid exert an influence on initiation and progression of fibrotic disease (Zhou et al., 2012). In this regard, Dong and colleagues displayed that ATRA could ameliorate bleomycin-induced pulmonary fibrosis through inhibition of interleukin-6 (IL-6) and transforming growth factor β (TGF-β) gene expression which both are considered as fibroblasts growth enhancers (Dong et al., 2012). The same mechanism was verified in case of ATRA administration for epidural fibrosis relief after laminectomy (Zhang et al., 2013). Therefore, the aim of the current study was to evaluate the possible effects of ATRA on peripheral nerve fibroblasts in culture condition.
Section snippets
Cell culture
Human gingival fibroblast cell line (HGF2, NCBI: C166) was purchased from National Cell Bank of Iran, Pasteur institute in Tehran, Iran (Khosravi et al., 2004). Peripheral nerve fibroblasts were isolated from rats sciatic nerve. Adult Sprague-Dawley rats (150–200 g; Pasteur Institute, Tehran, Iran) were deeply anesthetized with intraperitoneal injection of ketamine (30 mg/kg) and xylazine (3 mg/kg). Animal experiments were carried out in strict compliance with the approval of Institutional Animal
Results
The effect of different doses of ATRA on the viability rate of C166 cell line and peripheral nerve fibroblasts was shown in Fig. 1A. The 0.05 nM of ATRA significantly decreased mean peripheral nerve fibroblasts viability (19.4%) and continued up to 42.8% at 10 nM (P < 0.001), while no significant change in nerve fibroblasts viability was observed from 1 nM to 1 μM using MTT assay. Moreover, cumulative amount of peripheral nerve fibroblasts in successive days of counting demonstrated the growth rate
Discussion
Retinoic acid signaling elements including its synthesizing enzymes, cytosolic binding protein and nuclear receptors are expressed in PNS (Latasa and Cosgaya, 2011). The amplification and activation of retinoic acid signaling imply a potential role for retinoic acid during PNS regeneration and repair (Latasa and Cosgaya, 2011). It is well known that retinoic acid helps axonal regeneration in nervous system, at least, through three mechanisms; (a) directly stimulation of axonal elongation (Wong
Conclusion
Taken together, although more in vivo studies need to be clarified, our findings provide a preliminary in vitro base that retinoic acid may modify nerve fibroblasts viability following peripheral nerve damage and modulate scare tissue (i.e. neuroma) formation.
Acknowledgments
We thank Dr. Naser Jafari (Department of Biochemistry, School of Medicine, Ardabil University of Medical Sciences) for helpful comments on acridine orange and ethidium bromide staining. This work was financially supported (Grant no. 91393) by the Vice Chancellor for Research of the Ardabil University of Medical Sciences, Ardabil, Iran.
References (31)
- et al.
Cellular and molecular insights into neuropathy-induced pain hypersensitivity for mechanism-based treatment approaches
Brain Res. Rev.
(2011) - et al.
Retinoid effects on fibroblast proliferation collagen synthesis in vitro and on fibrotic disease in vivo
JAAD
(1986) - et al.
Nerve fibroblast impact on Schwann cell behavior
Eur. J. Cell Biol.
(2009) Wallerian degeneration and peripheral nerve conditions for both axonal regeneration and neuropathic pain induction
Ann. Anat.
(2011)- et al.
A reliable method to reduce collagen scar formation in the lesioned rat spinal cord
J. Neurosci. Methods
(2001) - et al.
Apoptosis and cell cycle arrest of human colorectal cancer cell line HT-29 induced by vanillin
Cancer Epidemiol.
(2009) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
J. Immunol. Methods
(1983)- et al.
EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting
Cell
(2010) - et al.
Schwann cell migration and neurite outgrowth are influenced by media conditioned by epineural fibroblasts
Neuroscience
(2013) - et al.
Regulation of retinoic acid receptors α, β and retinoid X receptor α after sciatic nerve injury
Neuroscience
(2006)