Massive resection of IFP induces tissue regeneration or fibrosis in MRL and C57BL/6 adult mice respectively

To investigate both tissue regeneration and fibrotic healing in mammals, we developed a robust and quantifiable model relying on the massive resection (around 35% of the whole tissue) of the inguinal fat pad (IFP) in adult mice. Using the specific anatomy of the IFP, the resection was systematically performed adjacent to the lymph node, which was used as a visual reference allowing the reproducibility of the resection (Fig. 1a). Macroscopic and microscopic observations as well as IFP weight quantification were performed 8 weeks after surgery. As expected, spontaneous macroscopic regeneration was observed in MRL mice (Fig. 1b upper panel) in contrast to C57BL/6 mice, which did not regenerate (Fig. 1b lower panel). Regenerated IFP exhibited adipocytes, blood vessels and nerves organized in a typical shape and structure similar to the ones observed in the contralateral IFP used as an internal control (Fig. 1c upper panel). In contrast, non-regenerated IFP was characterized by the absence of adipocytes and exhibited fibrotic high collagen deposition (Fig. 1c lower panel). Regeneration was then quantified by the regeneration index (RI), i.e. the weight ratio between the resected IFP and the uninjured contralateral IFP, after a previous check that uninjured IFP weight did not change over the same time following unilateral IFP resection (Fig. 1d). Consistently with macroscopic and microscopic observations, RI was significantly higher in regenerative mice than in non-regenerative mice as early as 2 weeks after resection and the difference was enhanced 8 weeks after resection (0.94 ± 0.037 in MRL vs 0.69 ± 0.017 in C57BL/6 mice) (Fig. 1e). No further regeneration was observed in C57BL/6 mice, even one year after resection. According to these results, this newly developed tissue lesion can be used to decipher regeneration and scarring regulatory processes.

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. Full size image

Opioid signalling controls IFP regeneration

To investigate whether opioids inhibited regeneration and/or induced fibrosis, spontaneously regenerative MRL mice and non-regenerative C57BL/6 mice were treated, just after IFP resection, with an opioid receptor agonist (Tramadol, TRAM) or antagonist (naloxone methiodide, NAL-M), respectively. TRAM treatment induced a significant decrease in RI 4 weeks after resection (Fig. 2a; 0.79 ± 0.02 without TRAM vs 0.65 ± 0.030 with TRAM). In contrast, RI was significantly higher in NAL-M treated mice than in untreated mice 4 weeks after resection (RI of 0.84 ± 0.009 with NAL-M vs 0.69 ± 0.013 without NAL-M) (Fig. 2b,c). No change in food intake or body weight was observed, so an indirect effect on feeding behaviour and fat intake regulation could be excluded (Supplementary Figure S1). Altogether, these results demonstrate that opioids inhibit spontaneous tissue regeneration and favour fibrotic tissue formation.

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. Full size image

We thus postulated that the absence of spontaneous regeneration in C57BL/6 compared to MRL mice could be associated with a higher synthesis of endogenous opioids in the IFP of C57BL/6 mice. Consistently with this hypothesis, the IFP-expression of proenkephalin (PENK, enkephalin poly-peptide precursor) was significantly higher in IFP of C56BL/6 than in MRL mice (Fig. 2d). In contrast, neither prodynorphin nor pro-opio-melanocortin mRNA was detected in IFP in either mouse strain. These results suggest that inhibition of regeneration in adult mammals could be mediated by endogenous PENK.

Opioid signalling prevents regeneration through control of ROS levels in zebrafish

To demonstrate that the anti-regenerative effects of opioids were not tissue and/or model dependent, we used the gold standard adult zebrafish caudal fin regeneration model28. Fish were incubated in NAL-M or TRAM from the time of amputation to analysis, and the size of the regenerate was quantified after amputation (Fig. 3a). As in our mouse model, NAL-M enhanced the regenerate size (Fig. 3b,c, 137.7 ± 26%), while TRAM inhibited regeneration (Fig. 3b,c, 70 ± 13%). These results demonstrate that the inhibitory effect of opioids on tissue regeneration is a prevailing process in adult vertebrates.

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 (H 2 O) (○) 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 (H 2 O) (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. Full size image

In zebrafish, regeneration has been widely demonstrated to be controlled by ROS4. We thus postulated that opioids controlled caudal fin regeneration through regulation of ROS production. NAL-M treatment enhanced ROS levels at the tip of the amputated fin, the major site of ROS production after amputation (Fig. 3d,e), while TRAM reduced the overall ROS production, even when the area of ROS detection was extended compared to the control (Fig. 3d,e). These results obtained in the caudal fin of zebrafish demonstrate that opioids inhibit tissue regeneration by abolishing the transient peak of ROS.

Opioid signalling prevents regeneration through control of ROS levels in adult mammals

We therefore investigated whether regeneration in adult mammals was associated with ROS production. In line with this hypothesis, IFP resection induced a robust and transient peak of ROS in the injured tissue of regenerative MRL mice (Fig. 4a,b). ROS production in MRL mice reached maximum values at 12 hours after resection and returned to normal values 72 hours following surgery (Fig. 4b). The treatment of MRL mice with apocynin (APO, inhibitor of NADPH p47 phox subunit translocation29) after IFP resection induced a severe decrease in RI two weeks after surgery (Fig. 4c; 0.85 ± 0.027 in untreated mice vs 0.62 ± 0.019 in APO treated mice). These results demonstrate that a robust and transient peak of ROS is required for proper tissue regeneration in adult MRL mice.

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. Full size image

The effects of opioids on ROS production were then investigated in vivo by treatments with antagonists or agonists of opioid receptors. In vivo imaging revealed that the ROS peak observed after resection in IFP of MRL mice was strongly inhibited within 96 hours after TRAM treatment (Fig. 4a,b). In contrast, whereas no peak of ROS was observed in control C57BL/6 mice, NAL-M treatment induced a significant increase in ROS production (Fig. 4d,e). This NAL-M effect on ROS production was abolished after APO treatment (Fig. 4d,e), leading to abolition of a NAL-M effect on RI two weeks post-resection (Fig. 4f). Similar results were obtained using the antioxidant alpha-tocopherol (Supplementary Figure S2). In vitro experiments revealed that ROS production under NAL M treatment was specifically increased in immune cells (CD45+) and not in non-immune (CD45−) cells isolated from IFP stromal vascular fraction (Supplementary Figure S3).

Taken together, these data demonstrate that ROS production is required for proper tissue regeneration in adult mammals and that opioids inhibit this regeneration through the control of ROS production.