Opioids prevent regeneration in adult mammals through inhibition of ROS production

Abstract

Inhibition of regeneration and induction of tissue fibrosis are classic outcomes of tissue repair in adult mammals. Here, using a newly developed model of regeneration in adult mammals i.e. regeneration after massive resection of an inguinal fat pad, we demonstrate that both endogenous and exogenous opioids prevent tissue regeneration in adults, by inhibiting the early production of reactive oxygen species (ROS) that generally occurs after lesion and is required for regeneration. These effects can be overcome and regeneration induced by the use of an opioid antagonist. The results obtained in both our new model and the gold standard adult zebrafish demonstrate that this mechanism can be considered as a general paradigm in vertebrates. This work clearly demonstrates that ROS is required for tissue regeneration in adult mammals and shows the deleterious effect of opioids on tissue regeneration through the control of this ROS production. It thus raises questions about opioid-based analgesia in perioperative care.

Figure 1

MRL but not C57BL/6 mice can regenerate inguinal fat pad (IFP). (a) Macroscopic view of IFP in adult mouse. Dotted line: ablation plane. The lymph node appears as a dark spot (location indicated by a star on the picture) at the crossing of the three main blood vessels (arrows). Scale bar: 0.5 cm. Cartoon; IFP in situ localization. (b) Macroscopic view of uninjured IFP and IFP at 0 and 8 weeks post-resection in MRL and C57BL/6 mice. Scale bars: 0.5 cm. (c) Imaging of uninjured IFP and IFP 8 weeks post-resection, showing adipocytes (BODIPY staining, grey), vascularization (lectin staining, red), sympathetic innervation (tyrosine hydroxylase staining, green) and collagen deposition (second harmonic generation, blue) in MRL and C57BL/6 mice. Scale bars: 100 µm. (d) Contralateral IFP weight from sham (○) versus resected C57BL/6 mice (⊗) 8 weeks post-resection. (e) Quantification of IFP regeneration in MRL (black) and C57BL/6 (white) mice 0, 2, 4 and 8 weeks post-resection, using the weight ratio (regeneration index) between the resected and the uninjured contralateral IFP. n = 7 to 25 animals per group. Data are represented as mean ± SEM. (ns; not significant, **p < 0.005, ***p < 0.0001). IFP: inguinal fat pad.

Figure 2

Opioid signalling controls tissue regeneration. (a) Quantification of IFP regeneration 4 weeks post resection in MRL mice treated (⊠) or not (■) with TRAM. (b) Quantification of IFP regeneration 4 weeks post resection in C57BL/6 mice treated (●) or not (○) with NAL-M. (c) Representative images of C57BL/6 mice IFP 4 weeks post resection and NAL-M treatment. (d) PENK mRNA expression in MRL (black) and C57BL/6 (white) mice IFP. Data are represented as mean ± SEM. (*p < 0.05, ***p < 0.0005 in a, ***p < 0.0001 in b). NAL-M: naloxone methiodide, TRAM: Tramadol.

Figure 3

Opioid signalling prevents regeneration through control of ROS production in zebrafish. (a) Scheme of the experiment. Caudal fins of adult fish were amputated and then allowed to regenerate for 16 or 72 hours. (b) Quantification of the size of the regenerated tissue at 72 hpa (hours post-amputation) in the control (H₂O) (○) and in fish treated with NAL-M (●) or TRAM (⦻). (c) Representative images 72 hpa of caudal fins challenged to regenerate in the presence of NAL-M or TRAM. (d) ROS detection (representative images) at the level of the amputation plane at 16 hpa. (e) ROS quantification in the control (H₂O) (white) and in fish treated with NAL-M (black) or TRAM (white checkered). Data are represented as mean ± SEM. (**p < 0.01; ***p < 0.0001). NAL-M: naloxone methiodide, TRAM: Tramadol.

Figure 4

ROS production is required for IFP regeneration. (a) Representative in vivo imaging of ROS production at 6 hours after surgery in resected MRL mice treated or not with TRAM. (b) In vivo quantification of ROS production at 0, 3, 6, 12, 24, 48 and 72 hours post-resection in MRL mice treated (⊠) or not (■) with TRAM. n = 8 per group. A.U: arbitrary units. A.U.C: Quantification of ROS production in vivo from 0 to 72 hours post-resection in MRL mice treated (white checkered) or not (black) with TRAM. (c) Quantification of IFP regeneration in MRL mice 2 weeks post-resection without (■) or with (⊡) APO treatment. (d) Representative in vivo imaging of ROS production at 6 hours after surgery in resected C57BL/6 mice treated or not with NAL-M or NAL-M and APO. (e) In vivo quantification of ROS production at 0, 3, 6, 12, 24, 48 and 72 hours post-resection in C57BL/6 mice treated (●) or not (○) with NAL-M or with NAL-M and APO (⨀). n = 8 per group. A.U: arbitrary units. A.U.C: Quantification of ROS production in vivo from 0 to 72 hours post-resection in C57BL/6 mice treated (black) or not (white) with NAL-M or with NAL-M and APO (dotted).(f) Quantification of IFP regeneration 2 weeks post-resection in C57BL/6 mice treated (●) or not (○) with NAL-M or with NAL-M and APO (⨀). Data are represented as mean ± SEM. (*p < 0.05, **p < 0.005, ***p < 0.0005). APO: apocynin, NAL-M: naloxone methiodide, TRAM: Tramadol.