EVALUATION OF THE POTENTIAL USE OF MICRO-COMPUTED TOMOGRAPHY IN THE STUDY OF MUSCLES FROM MURINE MODELS FOR MUSCLE DYSTROPHIES

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Microvascular network organisation in three dimensions

Young and old Flk-1GFP/+ and mdx-4Cv:Flk-1GFP/+ mice were anesthetised with isoflurane inhalation (Forene, Abbott, Rungis, France) and killed by cervical dislocation. Gastrocnemius muscles were removed and imaging of vascular network was carried out in two conditions: thick cryo-sections or whole muscle. Gastrocnemius muscle was snap frozen in liquid nitrogen-cooled isopentane before cryosectionning (100μm-thick sections). Confocal acquisitions were performed using a spinning disk microscope (Leica, Wetzlar, Germany), laser femto-second was used: Chameleon Ultra, 20×/0.7 and 40x/0.75 objectives and a CoolSnap HQ2 camera. Optical slices were taken every 0.5 or 0.3μm interval along the z-axis (80μm). For whole muscle conditions, images of Gastrocnemius blood vessels were obtained from the entire muscle using multi-photon scanner resonant confocal Leica TCS-SP5 with 20x/0.95 objective. Optical slices were taken every 0.5μm along the z-axis.

Histology

Gastrocnemius muscles were collected from mice after NMR experiments, snap frozen in liquid nitrogen-cooled isopentane and kept at –80°C. Six different levels of 7μm-thick sections were cut and stained with hematoxylin-eosin (HE) to describe histopathological modifications of muscle tissue, and Sirius red for visualisation of collagen. For immunohistochemistry analyses, muscle cryosections were incubated with anti-CD31 (Pharmingen) and anti-laminin (Sigma) antibodies, overnight at 4°C, revealed by cy3- or TRITC-labeled secondary antibodies (Jackson ImmunoResearch Laboratories).

Morphometric analysis

Two-dimension analysis was performed to evaluate distribution of muscle fiber diameter, percentage of centro- or peri-nucleated fibers, capillary count and distribution around each myofiber using ImageJ (NIH, Bethesda, USA) and NIS-Element (Nikon) softwares. At least 200 fibers were considered for each muscle.
Three-dimensional analysis was performed to evaluate organisation of vascular network. For each muscle, 10z-stack image reconstructions were achieved on 80 to 150μm-thick frozen sections. Analysis was carried out using IMARIS (ImarisBitplane, Zurich, Switzerland) software (quantification of vessel density, tortuosity, volume, anastomose count and distance between capillaries).

Nuclear Magnetic Resonance analysis

NMR experiments were performed on: 3 month-old mdx-4Cv (n=6) and control C57Bl/6J (n=9) and on 12 month-old mdx-4Cv (n=5) and control C57Bl/6J (n=7).

Hyperaemic response paradigm

To highlight differences between normal and altered muscles we classically applied a stress to increase the global need for perfusion. Ischemia-reperfusion stress was applied to the mouse left hindlimb which provokes maximal vasodilatation and limited resistance of arteries/arterioles (Bertoldi et al., 2008) just after tourniquet release.
In practice, anaesthesia was induced with 4% isoflurane delivered in 1.5L/min air and maintained with 1.75% isoflurane. During experiments, a water heating pad ensured a constant temperature of 37°C and breathing was monitored. After a 24min NMR acquisition at rest (baseline), ischemia of the leg was induced by occlusion of femoral artery by two surgical threads placed around the thigh and pulled tight by application of a weight (Bertoldi et al., 2008). After 30min of ischemia, the weight was instantly removed, inducing a hyperaemic response, which was monitored over the next 30min. During whole protocol, dynamic acquisitions of NMR scans of interleaved perfusion imaging and 31P-spectroscopy (31P-NMRS) were collected.

Multi parametric functional NMR (mpf-NMR) acquisitions

In vivo NMR experiments were conducted in a 4Tesla Biospec system equipped with a 20cm diameter 200mT.m-1 gradient insert (BrukerBioSpin MRI GmbH, Ettlingen, Germany). Mice were placed supine in a 6cm diameter, 12cm length volume transmitter 1H coil for whole-body signal excitation. An actively decoupled 2cm diameter surface 1H coil, positioned below the left calf, was used for image signal reception. Muscle metabolites were probed by a 10mm 31P saddle-shaped coil placed around the left leg.
As described in detail elsewhere (Baligand et al., 2009; Bertoldi et al., 2008), Arterial Spin Labelling (ASL)-NMR imaging and 31P-NMRS acquisitions were interleaved using the dedicated Bruker MultiScanControl software (BrukerBioSpin GmbH) in order to follow simultaneously and non-invasively: (i) muscle perfusion signal by SATuration-Inversion Recovery (SATIR) (time resolution: 10sec), and (ii) mitochondrial activity by dynamic 31P-NMRS (time resolution: 2.5sec). In brief, ASL imaging is based on non-invasive alternate magnetic tagging of blood water spins to provide endogenous markers of muscle perfusion, measured in regions of interest (ROI) drawn in posterior compartment of the leg. Muscle bioenergetics and pH were assessed from ratios of energetic phosphates measurable by 31P-NMRS at rest, in vivo mitochondrial oxidative capacity was directly assessed from the rate of creatine rephosphorylation at the end of ischemia, and intramuscular pH was calculated from chemical shift between phosphocreatine (PCr) and inorganic phosphate (Pi). A minimum of 50% PCr depletion at the end of ischemia was necessary to reliably measure dynamics for PCr recovery, and examinations which did not reach this threshold were rejected.

Muscle blood perfusion is modified in young-adult mdx mice.

Despite similar microvascular organisation, profiles of reactive hyperaemia were significantly different in mdx (n=6) and wild-type (wt) (n=9) mice (Figure 3.3 A, p<10-6 with ANOVA). The release of ischemia provoked an instantaneous increase of perfusion, which was lower in wt mice (mdx: 78.7±27.1 ml/min/100g; wt: 41.3±32.3 ml/min/100g, 20s post-release). In wt mice, this first perfusion peak was followed by a drop to reach a plateau around a value of 26.6±9.2 ml/min/100g peaking at 270s post-ischemia. In contrast, mdx muscle perfusion slightly increased to a mean perfusion value of 84.8±24.8 ml/min/100g at 300s post-ischemia and reduced to 26.3±25.9 ml/min/100g only 850s after stress release (Figure 3.3 A). Moreover, the global volume repaid after ischemia was significantly higher in mdx mice (wt: 474.3±216.3 ml/100g; mdx: 1017±369.2 ml/100g, p<0.05). The response to ischemic stress was therefore different and enhanced in young-adult mdx mice while no morphological modification of microvessels was detected.

Alteration of microvascular network organisation in old Flk1GFP/+::mdx mouse

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Old mdx mice displayed marked histological lesions; some already observed in young-adult as anisocytosis or centrally nucleated myofibers, others included persistence of chronic inflammation, and presence of endomysial/perimysial fibrosis (Figure 3.5 A-D). The microvascular network was as well organised in old Flk1GFP/+ as in young-adult mice (Figure 3.5 E, G). In contrast, Flk1GFP/+::mdx mice displayed significant alterations, characterised by a marked increase in tortuosity and irregular scattering of capillaries (Figure 3.5 F, H). Capillary diameter was similar in both groups (12 μm, Figure 3.5 I), but we identified a higher anastomose count, from more than 50,000 anastomoses/mm3 for Flk1GFP/+::mdx mice to less than 1,000 anastomoses/mm3 for control Flk1GFP/+ (p<0.01) (Figure 3.5 J). Collectively, these results pointed to an anarchic blood vessel organisation in this context of dystrophinopathy.

Alteration of muscle perfusion in old mdx mice

Despite severe alterations in mdx muscle microvascular network organization, no difference in muscle perfusion was observed between mdx and wt mice at rest (mdx: 12.09±5.90 ml/min/100g; wt: 8.19±2.19 ml/min/100g). After tourniquet release, rapid increase of perfusion was detected in muscles of the posterior hindlimb compartment; this increase was significantly lower in old mdx mice (mdx perfusion maximal value at 380s post-ischemia: 60.5±39.3 ml/min/100g; wt perfusion maximal value at 400s post-ischemia: 106.1±38.1 ml/min/100g, p<0.05), in contrast to what was seen in young mice.
Analysis of variance of perfusion time-courses demonstrated differing profiles between WT and mdx (p<10-4), with specific differences in the early phase of reperfusion. A similar initial peak of perfusion, as the one observed in young-adult wt mice, was detected in the old wt group, 20s post-ischemia, but was absent in mdx mice (Figure 3.3 B).
Thus at 12 months, both mdx and wt showed different profiles from young-adult animals (ANOVA, p<10-6), and in contrast to wt and young-adult mdx, old mdx mice displayed a decrease in muscle perfusion and a modified perfusion profile after an ischemic stress.

Muscle bioenergetics in 12 month-old mice (Table 3.2)

At rest, no difference in pH was observed between wt (n=7) and mdx (n=5) mice but hypoxic stress induced a significant acidosis in both groups (p<0.005), more pronounced in mdx (wt: ΔpH = 0.22±0.06; mdx: ΔpH = 0.30±0.04; p<0.05). Ischemia was associated with a significant increase in PCr depletion in old dystrophic mice compared to wt, though the difference in Pi/PCr ratio between the two groups was not significant. Unlike in young mice, the rephosphorylation rate was comparable in both groups. Indeed τPCr was shorter in the old compared to the young wt mice (p<0.01), but it was unchanged with age in the mdx mice.Thus, no alteration of oxidative capacities was observed in old mdx mice in response to hypoxic stress compared to age-matched control mice, despite reduced perfusion. In older mice (wt and mdx), no correlation was found between τPCr and perfusion variables.

Table of contents :

ABSTRACT
RESUMO
RESUME
GENERAL INTRODUCTION
LIST OF PUBLICATIONS
CHAPTER 1. BIBLIOGRAPHIC REVIEW: NON-INVASIVE STUDY OF GENETIC MUSCLE DISORDERS 
MUSCULAR DYSTROPHIES
Vascular alterations in Duchenne muscle dystrophy
CONGENITAL MYOPATHIES
ANIMAL MODELS FOR GENETIC MUSCLE DISEASES
Animal models for Muscular Dystrophies
Animal models for congenital myopathies
NON-INVASIVE EVALUATION OF GENETIC MUSCLE DISEASES
NMR IN THE STUDY OF ANIMAL MODELS FOR GENETIC MUSCLE DISEASES
CHAPTER 2. QUANTITATIVE T2 COMBINED WITH TEXTURE ANALYSIS OF NUCLEAR MAGNETIC RESONANCE IMAGES IDENTIFY DIFFERENT DEGREES OF MUSCLE INVOLVEMENT IN THREE MOUSE MODELS OF MUSCLE DYSTROPHY: MDX, LARGEMYD AND MDX/LARGEMYD
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Ethics Statement
Animals
Magnetic Resonance Imaging acquisition and analysis
Histological analysis
Statistic analysis
RESULTS
Muscle T2
Muscle texture analysis
Histological analysis
DISCUSSION
CONCLUSIONS
SUPPORTING INFORMATION
S2.1. T2 calculation from two images at different echo times
S2.2. Features selected for Texture Analysis
COMPLEMENTS TO THE MANUSCRIPT
Validation of the 2 points T2 measurements with a multiecho sequence
Post-mortem changes in the T2 values
CHAPTER 3. STRUCTURAL AND FUNCTIONAL ALTERATIONS OF SKELETAL MUSCLE MICROVASCULAR NETWORK IN DYSTROPHIN-DEFICIENT MDX MICE
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
Mice
Microvascular network organisation in three dimensions
Histology
Morphometric analysis
Nuclear Magnetic Resonance analysis
Statistics
RESULTS
Normal microvascular network organisation in young-adult Flk1GFP/+::mdx mouse
Muscle blood perfusion is modified in young-adult mdx mice.
Muscle bioenergetics in young-adult mice (Table 3.1)
Alteration of microvascular network organisation in old Flk1GFP/+::mdx mouse
Alteration of muscle perfusion in old mdx mice
Muscle bioenergetics in 12 month-old mice (Table 3.2)
DISCUSSION
CONCLUSION
SUPPLEMENTARY INFORMATION
S3.1. Nuclear Magnetic Resonance analysis
CHAPTER 4. NON-INVASIVE NMR STUDY OF THE MOUSE MODEL FOR CENTRONUCLEAR MYOPATHY WITH MUTATION IN THE DYNAMIN-2 GENE
INTRODUCTION
MATERIALS AND METHODS
Animals
Nuclear Magnetic Resonance (NMR)
Data analysis
Histological analysis
Statistical analysis
RESULTS
Morphometrical evaluation
T1 measurements
T2 measurements
Histological analysis
DISCUSSION
CHAPTER 5. PILOT FUNCTIONAL AND METABOLIC EVALUATION OF THE KI-DNM2R465W MICE 
INTRODUCTION
PILOT STUDY 1. EXERCISE AS THE PARADIGM OF MUSCLE STRESS IN KI-DNM2R465W MICE
Materials and methods
Results
Discussion
PILOT STUDY 2. PROLONGED ISCHEMIA AS THE PARADIGM OF MUSCLE STRESS
Materials and Methods
Results
Discusssion
PILOT STUDY 3. REGENERATION IN THE DNM2 MICE: T1, T2 AND FUNCTIONAL ANALYSIS AFTER INJURY
Materials and Methods
Results
Discussion
CHAPTER 6. EVALUATION OF THE POTENTIAL USE OF MICRO-COMPUTED TOMOGRAPHY IN THE STUDY OF MUSCLES FROM MURINE MODELS FOR MUSCLE DYSTROPHIES
INTRODUCTION
MATERIALS AND METHODS
Animals
Muscle injury with electroporation
Micro-CT
Data Analysis
Statistical Analysis
RESULTS
Phenotypical characterization of the dystrophic muscle with micro-CT
Evaluation of injured muscle with micro-CT
DISCUSSION
GENERAL CONCLUSIONS AND PERSPECTIVES
BIBLIOGRAPHY

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