Introduction

Fibrosis is a pathological alteration observed in almost any tissue/organ and is characterized by increased deposition of extracellular matrix, predominantly composed by collagen¹. In skeletal muscle, diffuse endomysial fibrosis is commonly observed in muscle dystrophies, where chronic injury ultimately leads to replacement of muscle by connective and adipose tissues². While fat infiltration can be quantified with NMR, it remains unclear how and to which extent endomysial fibrosis alters NMR variables. In dystrophic muscle, fibrosis most frequently develops simultaneously with other pathological processes as cell membrane leakage, inflammation, necrosis, regeneration and fat infiltration. Such processes influence NMR variables to different and sometimes opposing extends. Aiming at characterizing how endomysial fibrosis affects NMR variables, we developed a non-dystrophic mouse model with increased muscle fibrosis, but minimized confounding factors such as inflammation, fat infiltration and membrane leakage. Muscle T₂, native T₁ (natT₁) and T₁ values after Gd injection were measured. ECV (or Gd-distribution volume) was estimated, and NMR variables were compared to fibrosis as quantified by histological analysis.

Materials and methods

Sixteen C57Bl (BL) and 15 DBA/2J (DBA) mice were included and fibrosis was induced locally in the left tibialis anterior (TA). Fibrosis was induced by combining acute and chronic injuries. Acute muscle injury was induced by electroporation (8 pulses, 100V, 20ms pulse duration, 2Hz pulse frequency, pulse train repeated twice turning the electrodes by 90°). Chronic injuries were obtained by intra-muscular injections (NaCl 0.9% in the left TA, 50µl, 3 times per week during 3 weeks). Subsequently, mice had 2 weeks without injury to allow tissue regeneration and reduction of muscle inflammation. This combination injury/regeneration was repeated twice, completing a 10 weeks injury protocol. During this time, mice were treated with anti-fibrotic drugs, either halofuginone (200µg/kg, 3 times per week, 5 DBA and 5 C57Bl), or Angiotensin 1-7 (Tarix, 1mg/kg, daily, 5 C57Bl mice). Remaining mice did not receive any anti-fibrotic treatment. The purpose of these treatments was to induce different levels of muscle fibrosis in order to assess correlations between different degrees of fibrosis and changes in NMR variables. Uninjured right TAs served as normal control muscles in pairwise comparisons. After fibrosis induction, mice were evaluated by NMR with T₂ maps (MSME, TR=3500ms, TE=n*5.15ms, n=1,…, 32, EPG fitting), natT₁ maps (saturation-recovery RARE, TE=6.66ms, 13 TR: 82.9, 100, 128, 171, 214, 602, 286, 359, 1010, 1694, 2842, 4769, 8000ms), and T₁-maps 1 hour after Gd injection (Dotarem, 2µmol Gd/g mouse weight, intraperitoneal injection saline:Dotarem 1.6:1). Acquisitions were done under isoflurane anesthesia in a 7T Bruker system, with a Bruker cryoprobe (spatial resolution: 0.1x0.1x1mm³). Immediately before the NMR acquisitions and just after the last T₁-map, blood samples were collected, centrifuged, and plasma T₁ was measured with the same pulse sequence as used for muscle T₁ mapping. ECV or Gd distribution volume was estimated³ (Figure 1). After imaging, mice were euthanized and left and right TAs were dissected for histological analysis. Samples were stained with Sirius Red (SR) for quantification of the collagen content and hematoxilin-eosin (HE), for assessment of the degree of inflammation, centronucleation, fat infiltration and necrotic calcifications.

Results

The injury protocol successfully induced endomysial fibrosis (Figure 2, A-E), without fat infiltration and only mild to moderate inflammation in the injured muscles (Figure 2-F). Centronucleated fibers were common features in both mouse strains. Alterations identified as mineralized necrotic fibers could be observed mainly in injured DBA muscles (Figure 2-G, H). Pairwise T-test revealed increased ECV after injury (injured: 7.3±2.0%, control: 6.4±1.1%, p<0.05). ECV correlated significantly with collagen content on histological sections (R=0.6, p<0.001, Figure 3-A). No differences between injured and control TAs were observed in pairwise T-test for natT₁, but a loose correlation between natT₁ and collagen content could be detected (R=0.3, p<0.05, Figure 3-B). Injured TA presented slightly lower T₂ values (injured: 21.4±0.6ms, control: 21.7±0.6ms, p<0.05), and a loose negative correlation could be detected between T₂ and collagen content (R=-0.32, p<0.05, Figure 3-C).

Discussion and conclusion

Our observations show that muscle fibrosis correlates with increased extracellular volume, similarly to what is observed in myocardial fibrosis³. Our model, unlike dystrophic muscle, showed predominant fibrosis, but was not able to disentangle completely fibrosis from other pathological processes like inflammation and regeneration. Even though, collagen content correlated also, but to a lesser extent, with natT₁ (positive correlation) and muscle T₂ (negative correlation). Despite these promising results, caution should be taken as confounding factors can still have a disturbing influence on correlations between fibrosis and these NMR variables.

Acknowledgements

No acknowledgement found.