Aziz Guellich

Elisa Negroni

Valérie Decostre

Alexandre Demoule

Catherine Coirault
email: catherine.coirault@inserm.fr

Ranganath Mamidi

Altered cross-bridge properties in skeletal muscle dystrophies

Keywords: myosin, cross-bridge kinetics, skeletal muscle, muscle dystrophy, myopathies www.frontiersin.org

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Muscular dystrophies (MDs) are a group of more than 30 clinical and molecular heterogeneous genetic disorders that cause progressive degeneration of the skeletal muscle fibers. They are characterized by severe muscle weakness that generally affects limb, axial, and/or facial muscles to a variable extent. The age of onset, severity and rate of progression greatly vary in the different forms of MD. The primary cause of various forms of MDs is an individual mutation in genes encoding a wide variety of proteins, including extracellular matrix (ECM) proteins, transmembrane, and membrane-associated proteins, cytoplasmic proteases and nuclear proteins. Detailed classification and list of causative genes in MD have been recently reviewed [START_REF] Cohn | Oxidative stress of myosin contributes to skeletal muscle dysfunction in rats with chronic heart failure[END_REF][START_REF] Flanigan | The muscular dystrophies[END_REF][START_REF] Kaplan | The 2013 version of the gene table of monogenic neuromuscular disorders (nuclear genome)[END_REF][START_REF] Mercuri | Muscular dystrophies[END_REF]. Although still incompletely understood, considerable progress has been now made to reveal the pathophysiological mechanisms in MDs. It has been shown that most MDs share Abbreviations: ADP, adenosine 5 -diphosphate; ATP, adenosine 5 -triphosphate; CB, cross-bridge; DMD, Duchenne muscular dystrophy; ECM, extracellular matrix; FSHD, fascioscapulohumeral muscular dystrophy; MD, muscular dystrophy; MsrA and MrB, methionine sulfoxyde reductase A and B; MyHC, myosin heavy chain; nNOS, neuronal NO synthase; NO, nitric oxide; Pi, inorganic phosphate; ROS, reactive oxygen species; Tm, tropomyosin. common pathologic features, such as altered Ca 2+ homeostasis, infiltration of muscle tissue by inflammatory immune cells, accretion of proinflammatory and profibrotic cytokines, activation of proteolytic enzymes, metabolic/mitochondrial alterations, intracellular accumulation of reactive oxygen species (ROS) production, and/or defective autophagy, which can contribute to muscle wasting. At the earliest stages of the disease, reduced myofibrillar protein content can occur in apparent uninjured fibers, secondary to an imbalance between protein synthesis and proteolysis [START_REF] Mckeran | Increased myofibrillar protein catabolism in Duchenne muscular dystrophy measured by 3-methylhistidine excretion in the urine[END_REF][START_REF] Warnes | Increased rates of myofibrillar protein breakdown in muscle-wasting diseases[END_REF]. Then, successive rounds of degeneration and regeneration lead to fibrosis and fatty replacement of muscle tissue and in turn reduce the number of potential cross-bridges (CBs) generating force (Figure 1). In addition, functional changes in the CB properties may contribute to muscle weakness in distinct types of muscle disease. It is well-established that shift in the relative myosin isoform expression is associated with modifications of the kinetics of actomyosin interactions [START_REF] Bar | Three fast myosin heavy chains in adult rat skeletal muscle[END_REF][START_REF] Schiaffino | Three myosin heavy chain isoforms in type 2 skeletal muscle fibres[END_REF][START_REF] Schiaffino | Molecular diversity of myofibrillar proteins: gene regulation and functional significance[END_REF]. However, there are also increasing evidences that CB properties can change with no change in myosin isoform content [START_REF] Coirault | Velocity of actomyosin sliding in vitro is reduced in dystrophic mouse diaphragm[END_REF][START_REF] Canepari | Actin sliding velocity on pure myosin isoforms from dystrophic mouse muscles[END_REF], suggesting that post-translational modifications of contractile proteins have significant role in muscle weakness (Figure 1). The present review focuses on the changes in the function of contractile proteins and in CB performance that may occur in the context of skeletal muscle dystrophies (MDs) and their contribution to the pathophysiological mechanisms of these diseases.

CYCLICAL INTERACTIONS OF ACTIN WITH MYOSIN

Contractile force of striated muscle is produced within the half-sarcomere-the functional contractile unit-by the cyclical interactions of actin filament with myosin molecule, the muscle molecular motor. These cyclical interactions are controlled by membrane-located mechanisms that trigger intracellular raise of Ca 2+ and mechanisms inside the sarcomere itself. In skeletal muscle, sarcomeric mechanisms mainly involve the Ca 2+ -controlled conformational change of the regulatory proteins troponins and tropomyosin (Tm), the functional properties of myosin, and strong cooperative interaction between neighboring troponin-Tm unit along the actin filament [START_REF] Gordon | Regulation of contraction in striated muscle[END_REF].

In resting muscle, intracellular Ca 2+ concentration ([Ca 2+ ] i ) is low and the C-terminal domain of troponin I immobilizes the Tm in a position that prevents the binding of myosin to actin [START_REF] Galinska-Rakoczy | Structural basis for the regulation of muscle contraction by troponin and tropomyosin[END_REF]. Upon membrane depolarization, Ca 2+ is released from the sarcoplasmic reticulum and binds to troponin C, initiating azimuthal movement of Tm around the actin-containing thin filament. This in turn exposes the sites on actin on which the myosin heads can attach [START_REF] Kress | Structural changes during activation of frog muscle studied by time-resolved X-ray diffraction[END_REF][START_REF] Geeves | Molecular mechanism of actomyosin-based motility[END_REF]. In the conventional view, myosin binding is solely determined by the Ca 2+ transient causing structural changes in the thin filament. However, numerous studies have reported Ca 2+ -induced structural change in the myosin-containing thick filaments before myosin binding to the thin filament [START_REF] Huxley | Time-resolved X-ray diffraction studies of the myosin layer-line reflections during muscle contraction[END_REF][START_REF] Lowy | Studies of the diffuse x-ray scattering from contracting frog skeletal muscles[END_REF][START_REF] Yagi | An x-ray diffraction study on early structural changes in skeletal muscle contraction[END_REF][START_REF] Reconditi | Motion of myosin head domains during activation and force development in skeletal muscle[END_REF]. This has led to the proposal of a modified model of muscle activation in which fast coordinated changes in the structures of both thick and thin filaments follow concomitantly upon the rise in intracellular free Ca 2+ concentration [START_REF] Reconditi | Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog[END_REF].

Myosin head binding to actin occurs first in a low binding, pre-force-generating state (Figure 2, step a). At this state, the CB has already hydrolyzed the adenosine 5 -triphosphate (ATP), but the products adenosine 5 -diphosphate (ADP) and inorganic phosphate (Pi) are still bound to the myosin head [START_REF] Lymn | Mechanism of adenosine triphosphate hydrolysis by actomyosin[END_REF][START_REF] Pate | A model of crossbridge action: the effects of ATP, ADP and Pi[END_REF]. Then, the CB goes through the power stroke (Figure 2, step b), during which the myosin head can generate a force of several piconewtons or an axial displacement of the actin filament toward the center of the sarcomere of 5-10 nm in vitro [START_REF] Molloy | Movement and force produced by a single myosin head[END_REF][START_REF] Veigel | The stiffness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers transducer[END_REF][START_REF] Mehta | Single-molecule biomechanics with optical methods[END_REF][START_REF] Reconditi | Motion of myosin head domains during activation and force development in skeletal muscle[END_REF] or 8-13 nm in situ [START_REF] Reconditi | The myosin motor in muscle generates a smaller and slower working stroke at higher load[END_REF]. There are evidences that force generation precedes the Pi release [START_REF] Dantzig | Reversal of the cross-bridge force-generating transition by photogeneration of phosphate in rabbit psoas muscle fibres[END_REF][START_REF] Caremani | Effect of inorganic phosphate on the force and number of myosin cross-bridges Frontiers in Physiology | Striated Muscle Physiology October 2014 | Volume 5 | Article 393 | 6 during the isometric contraction of permeabilized muscle fibers from rabbit psoas[END_REF]. Subsequent steps involve the release of ADP (Figure 2, step c), and the binding of ATP that rapidly dissociates the actomyosin complex (Figure 2, step d). When unbound from actin, myosin hydrolyzes ATP and reverses the power stroke, thus returning to its original position and allowing a new cycle to start [START_REF] Eisenberg | The relation of muscle biochemistry to muscle physiology[END_REF][START_REF] Steffen | Repriming the actomyosin crossbridge cycle[END_REF]) (Figure 2, step e). Thus, at the molecular level, skeletal myosin is a molecular motor that transduces chemical energy produced by the hydrolysis of one ATP molecule into mechanical work [START_REF] Huxley | Muscle structure and theories of contraction[END_REF][START_REF] Huxley | Proposed mechanism of force generation in striated muscle[END_REF][START_REF] Lymn | Mechanism of adenosine triphosphate hydrolysis by actomyosin[END_REF]Eisenberg et al., 1980; [START_REF] Pate | A model of crossbridge action: the effects of ATP, ADP and Pi[END_REF][START_REF] Gordon | Regulation of contraction in striated muscle[END_REF]. Skeletal MDs may reduce the number of active CBs producing force but may also potentially affect various steps of the cyclical interactions of actin with myosin, thus reducing the unitary force produced by each CB and/or modifying the CB kinetics.

CHANGES IN CB KINETICS RELATED TO MD-INDUCED SHIFT IN MYOSIN ISOFORM

The events in the CB cycle are essentially the same for all muscle myosins, but the kinetics of acto-myosin interaction varies widely among myosin heavy chain (MyHC) isoforms. Although the physiological differences between fast and slow skeletal muscle fibers depend on more than just differences in MyHC isoforms, it is well established that MyHC isoforms are a major determinant of the large variability in contractile and energetic properties of muscle fibers [START_REF] Pette | Cellular and molecular diversities of mammalian skeletal muscle fibers[END_REF]Bottinelli et al., 1994a,b). The existence of several MyHC isoforms differentially distributed in various fibers makes MyHCs useful to study fiber heterogeneity and plasticity in mammalian muscles. In the adult skeletal muscle, four MyHC isoforms can be expressed, namely the slow MyHC -1 and the fast MyHC -2A, MyHC-2X, and MyHC-2B, coded respectively by the Myh7, Myh2, Myh1, and Myh4 genes [START_REF] Bar | Three fast myosin heavy chains in adult rat skeletal muscle[END_REF][START_REF] Schiaffino | Three myosin heavy chain isoforms in type 2 skeletal muscle fibres[END_REF][START_REF] Schiaffino | Molecular diversity of myofibrillar proteins: gene regulation and functional significance[END_REF].

Changes in the proportion of fast relative to slow fiber types are frequently observed in muscular dystrophy, and may result either from a reduced muscular activity in affected patients or from a physiopathological consequence of the gene mutation. The pattern of wasted muscle and affected fibers is highly variable among the types of MDs. Preferential involvement in fast type 2 muscle fibers is a common feature in Duchenne Muscular Dystrophy (DMD) [START_REF] Webster | Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy[END_REF], with early disappearance of muscle fibers expressing MyHC-2X transcripts [START_REF] Pedemonte | Early decrease of IIx myosin heavy chain transcripts in Duchenne muscular dystrophy[END_REF]. Likewise, slow type 1 muscle fiber predominance has been reported in patients with facioscapulohumeral muscular dystrophy (FSHD) [START_REF] Celegato | Parallel protein and transcript profiles of FSHD patient muscles correlate to the D4Z4 arrangement and reveal a common impairment of slow to fast fibre differentiation and a general deregulation of MyoD-dependent genes[END_REF], dysferlinopathy [START_REF] Ervasti | Membrane organization of the dystrophin-glycoprotein complex[END_REF] and congenital muscular dystrophy type Ullrich [START_REF] Schessl | Predominant fiber atrophy and fiber type disproportion in early ullrich disease[END_REF], suggesting a selective loss of fast-twitch/type 2 muscle fibers [START_REF] D'antona | Structural and functional alterations of muscle fibres in the novel mouse model of facioscapulohumeral muscular dystrophy[END_REF] or an active fiber typing conversion process [START_REF] Schessl | Predominant fiber atrophy and fiber type disproportion in early ullrich disease[END_REF][START_REF] De La Torre | Proteomics identification of differentially expressed proteins in the muscle of dysferlin myopathy patients[END_REF]. This preferential involvement of the fastest fiber type leads to a fast to slow shift among fast MyHC isoforms in the dystrophic muscles [START_REF] Stedman | The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy[END_REF][START_REF] Petrof | Adaptations in myosin heavy chain expression and contractile function in dystrophic mouse diaphragm[END_REF][START_REF] Coirault | Myosin molecular motor dysfunction in dystrophic mouse diaphragm[END_REF], which may represent an adaptive response that would tend to preserve the economy of force contraction. However, fiber-type composition has been shown to be differently affected in other MDs. Hypertrophy of type 2 fibers without type 1 fiber atrophy have been found in biopsies of patients with laminopathies [START_REF] Kajino | Congenital fiber type disproportion myopathy caused by LMNA mutations[END_REF], whereas MyHC-2a fibers are markedly atrophied in affected cricopharyngeal muscle of oculopharyngeal muscular dystrophy patients [START_REF] Gidaro | Atrophy, fibrosis, and increased PAX7-positive cells in pharyngeal muscles of oculopharyngeal muscular dystrophy patients[END_REF]. How mutations responsible for MDs differently affect myofiber-type specification and/or MyHC deserve further studies. In addition to adult myosin isoform remodeling, dystrophic fiber can express developmental embryonic and neonatal MyHCs, coded by Myh3 and Myh8, respectively [START_REF] Wieczorek | Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature[END_REF][START_REF] Sartore | Fibre types in extraocular muscles: a new myosin isoform in the fast fibres[END_REF]. Such developmental or neonatal MyHC-expressing fibers can either be regenerating fibers or fibers that expressed inappropriate and immature MyHC, as has been observed in some congenital myopathies [START_REF] Sewry | The role of immunocytochemistry in congenital myopathies[END_REF].

Importantly, the different MyHC isoforms display large functional differences regarding the actin-activated ATPase activity of myosin, the rate of ADP release from acto-myosin during the time of attachment, and the velocity with which they can move actin. This results in large variability in contractile, thermodynamic, and kinetic coupling according to the fiber type [START_REF] Barany | ATPase activity of myosin correlated with speed of muscle shortening[END_REF]. For instance, fibers containing MyHC-1 have nearly threefold slower ATPase and lower tension cost than fibers with MyHC-2X, while fibers with MyHC-2A are intermediate [START_REF] Stienen | Myofibrillar ATPase activity in skinned human skeletal muscle fibres: fibre type and temperature dependence[END_REF]. Therefore, an increased proportion of the slow MyHC isoforms would tend to improve muscle efficiency and may represent an adaptive response at least in some MDs. An opposite response, namely a slow-to-fast shift with preferential atrophy of slow fiber, accelerates the time cycle, reduces muscle efficiency [START_REF] Coirault | Myosin molecular motor dysfunction in dystrophic mouse diaphragm[END_REF] and thus may contribute to the exercise intolerance in DMD patients. MyHC shift may also impact on CB recruitment, given that attachment of fast myosin involves a CBmediated facilitation mechanism absent in slow MyHC isoforms [START_REF] Galler | Qualitatively different cross-bridge attachments in fast and slow muscle fiber types[END_REF]. Indeed, fast myosin heads already attached to actin substantially accelerate the further attachment of neighboring myosin heads, thereby enabling a rapid change in the rate of force development at high levels of activation [START_REF] Galler | Qualitatively different cross-bridge attachments in fast and slow muscle fiber types[END_REF].

In contrast, slow MyHC isoforms have moderate dependence on level of activation, enabling slow CB recruitment to become faster only gradually at higher levels of activation [START_REF] Galler | Qualitatively different cross-bridge attachments in fast and slow muscle fiber types[END_REF]. Thus, pathologic changes in myosin isoforms modify specific steps of the CB cycling as well as the overall kinetics of the CB cycle, with functional consequences on muscle performance and energy cost of contraction.

CB ALTERATIONS CAUSED BY MICRO-ARCHITECTURAL CHANGES IN MYOFILAMENTS

Striated muscles are characterized by a highly ordered structure that is critical for normal function and muscle homeostasis. Micro-architectural changes in myofilaments are likely contributors to muscle weakness in MDs, either directly by affecting the function of critical structural proteins, or indirectly, by increasing the activity of Ca 2+ -dependent proteases.

In striated muscle, the dystrophin glycoprotein complex is preferentially located at the costamere [START_REF] Ervasti | Membrane organization of the dystrophin-glycoprotein complex[END_REF], a protein network connecting the outermost myofibrils to the sarcolemma at each Z-disc [START_REF] Pardo | A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements ("costameres") mark sites of attachment between myofibrils and sarcolemma[END_REF][START_REF] Ervasti | Membrane organization of the dystrophin-glycoprotein complex[END_REF][START_REF] Bloch | Lateral force transmission across costameres in skeletal muscle[END_REF][START_REF] Ervasti | Costameres: the Achilles' heel of Herculean muscle[END_REF][START_REF] Michele | Dystrophin-glycoprotein complex: post-translational processing and dystroglycan function[END_REF]. These lateral linkages are critical in the maintenance of sarcomere stability and in the transmission of forces generated by the CBs (Figure 3) [START_REF] Rybakova | The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin[END_REF][START_REF] Bloch | Lateral force transmission across costameres in skeletal muscle[END_REF][START_REF] Ervasti | Costameres: the Achilles' heel of Herculean muscle[END_REF]. Indeed, while part of the forces generated in sarcomeres is longitudinally transmitted down myofibrils in muscle to the tendon, costameres are critical to transmit the forces laterally to the ECM and neighboring muscle fibers [START_REF] Street | Lateral transmission of tension in frog myofibers: a myofibrillar network and transverse cytoskeletal connections are possible transmitters[END_REF][START_REF] Ervasti | Costameres: the Achilles' heel of Herculean muscle[END_REF][START_REF] Bloch | Costameres mediate force transduction in healthy skeletal muscle and are altered in muscular dystrophies[END_REF][START_REF] Ramaswamy | Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats[END_REF]. Dystrophin deficiency reduces the lateral transfer of forces between activated fibers [START_REF] Ramaswamy | Lateral transmission of force is impaired in skeletal muscles of dystrophic mice and very old rats[END_REF], thus compromising the homogeneity of sarcomere contraction between adjacent fibers and impairing the efficacy of force transmission. As a result, each fiber tends to act as an independent longitudinal force generator, thus increasing the susceptibility of muscle membrane to contraction-induced muscle damage.

Skeletal muscle fiber contains the ubiquitously expressed and well-conserved family of Ca 2+ -dependent cysteine proteases calpains, μ-calpain, and m-calpain, as well as a muscle-specific calpain i.e., calpain-3 [START_REF] Goll | The calpain system[END_REF][START_REF] Bartoli | Calpains in muscle wasting[END_REF]. Elevated calpain amount, primarily due to a significant increase in m-calpain concentration is observed in dystrophic muscles from the mdx mice [START_REF] Spencer | Calpains are activated in necrotic fibers from mdx dystrophic mice[END_REF], a mouse model of DMD. Increased activity of calpains has been implicated in the progression of muscle wasting in dystrophic muscles [START_REF] Spencer | Calpains are activated in necrotic fibers from mdx dystrophic mice[END_REF][START_REF] Alderton | Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes[END_REF][START_REF] Tidball | Calpains and muscular dystrophies[END_REF][START_REF] Zhang | Role of the calcium-calpain pathway in cytoskeletal damage after eccentric contractions[END_REF]. The two ubiquitous μand m-calpains both cleave the same substrates in vitro, including the troponin complex (Troponin C, Troponin I, and Troponin T), Tm, α-actinin, titin, desmin, the Z-disk protein fodrin and the sarcolemmal associated spectrin complex of proteins [START_REF] Goll | The calpain system[END_REF]. Such substrates are consistent with a role of μand m-calpains in sarcomeric organization and/or dismantling. Interestingly, the two sarcomeric tropomodulin isoforms Tmod1 and Tmod4 have been recently identified as proteolytic targets of m-calpain in dystrophic muscle [START_REF] Gokhin | Calpain-mediated proteolysis of tropomodulin isoforms leads to thin filament elongation in dystrophic skeletal muscle[END_REF]. Tropomodulins are dynamic actin filament pointed-end-capping proteins [START_REF] Weber | Tropomodulin caps the pointed ends of actin filaments[END_REF], that localize in sarcomere to each side of the M-line [START_REF] Fowler | Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle[END_REF]. Absence of Tmod1 and its replacement by Tmod3 and Tmod4 in turn impair initial Tm movement over actin subunits during thin filament activation, thus reducing both the fraction of actomyosin CBs in the strongly bound state and fiber forcegenerating capacity [START_REF] Ochala | Pointed-end capping by tropomodulin modulates actomyosin crossbridge formation in skeletal muscle fibers[END_REF]. Calpain-3 binds to the N2A region of titin [START_REF] Sorimachi | Muscle-specific calpain, p94, responsible for limb girdle muscular dystrophy type 2A, associates with connectin through IS2, a p94specific sequence[END_REF], but has also been identified in the nucleus of muscle cells [START_REF] Baghdiguian | Calpain 3 deficiency is associated with myonuclear apoptosis and profound perturbation of the IkappaB alpha/NF-kappaB pathway in limbgirdle muscular dystrophy type 2A[END_REF]. Interestingly, it has been shown that calpain-3 binding to the myofibrillar structure in vitro is modulated by the presence of CBs locked in rigor [START_REF] Murphy | Ca2+ activation of diffusible and bound pools of mu-calpain in rat skeletal muscle[END_REF][START_REF] Murphy | Endogenous calpain-3 activation is primarily governed by small increases in resting cytoplasmic [Ca2+] and is not dependent on stretch[END_REF], thus supporting the hypothesis that CB interactions modulate calpain-3 activity. This may explain why in vivo, eccentric exercise is the only physiological circumstance shown to result in the activation of calpain-3 [START_REF] Murphy | Calpain-3 is autolyzed and hence activated in human skeletal muscle 24 h following a single bout of eccentric exercise[END_REF]. A number of myofibrillar proteins have been identified as potential calpain-3 substrates in vitro, but none have been confirmed as in vivo targets. These include titin (at the PEVK region, adjacent to the N2A region of titin), and myosin light chain [START_REF] Cohen | Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice[END_REF]. Future works are needed to determine the interrelation between calpain-3 and the CB interactions.

MODULATIONS AND DAMAGE OF CB KINETICS BY OXIDATIVE STRESS DEFINITION AND INTRACELLULAR SOURCES OF OXIDATIVE STRESS IN MDs

Oxidative stress has been reported as primum movens in both loss of cell viability and contractile dysfunction in MDs [START_REF] Ragusa | Oxidative stress as a potential pathogenic mechanism in an animal model of Duchenne muscular dystrophy[END_REF][START_REF] Rando | Muscle cells from mdx mice have an increased susceptibility to oxidative stress[END_REF][START_REF] Tidball | The role of free radicals in the pathophysiology of muscular dystrophy[END_REF][START_REF] Menazza | Oxidative stress by monoamine oxidases is causally involved in myofiber damage in muscular dystrophy[END_REF][START_REF] Lawler | Exacerbation of pathology by oxidative stress in respiratory and locomotor muscles with Duchenne muscular dystrophy[END_REF]. By definition, oxidative stress refers to a deregulation of the cellular redox-status due to an imbalance between reactive oxygen species (ROS) production and antioxidant capacities.

There are numerous potential intracellular sources of ROS in muscle tissue including the mitochondria [START_REF] Murphy | How mitochondria produce reactive oxygen species[END_REF][START_REF] Brand | The sites and topology of mitochondrial superoxide production[END_REF], the family of nicotinamide adenine dinucleotide phosphatate oxidases, now collectively known as NOX enzymes family, and a wide range of enzymes, such as xanthine oxidase, nitric oxide synthase, cyclo oxygenases, cytochrome P450 enzymes and lipoxygenases [START_REF] Cheng | Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, and Nox5[END_REF]. In addition, extracellular sources of ROS can arise from other non-muscle cell types, including activated neutrophils and macrophages [START_REF] Moylan | Oxidative stress, chronic disease, and muscle wasting[END_REF].

Because striated muscle cells have to adapt very rapidly and in a co-ordinated manner to changes in energy supply and oxygen flux, the control of ROS production and redox-status is tightly regulated during contraction. Importantly, the neuronal form of nitric oxide synthase (nNOS) is normally present in the sarcolemma of fast-twitch muscle fibers and associates with dystrophin [START_REF] Brenman | Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy[END_REF]. Although the reaction between nitric oxide (NO) and the superoxide anion is known to form peroxynitrite, NO can also act as a protective molecule against the damaging actions of ROS [START_REF] Wink | Nitric oxide (NO) protects against cellular damage by reactive oxygen species[END_REF]. Accordingly, the absence of nNOS and NO in DMD muscle removes this protective action, increasing the susceptibility of DMD muscle to the damaging action of ROS [START_REF] Brenman | Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy[END_REF][START_REF] Haycock | Differential susceptibility of human skeletal muscle proteins to free radical induced oxidative damage: a histochemical, immunocytochemical and electron microscopical study in vitro[END_REF][START_REF] Disatnik | Evidence of oxidative stress in mdx mouse muscle: studies of the prenecrotic state[END_REF].

PHYSIOLOGICAL STRESS INDUCES REVERSIBLE MODIFICATIONS OF PROTEINS THAT CONTRIBUTE TO NORMAL MUSCLE PHYSIOLOGY

There is increasing evidence that ROS reversibly modulate important intracellular pathways involved in muscle homeostasis [START_REF] Droge | Free radicals in the physiological control of cell function[END_REF][START_REF] Smith | Redox modulation of contractile function in respiratory and limb skeletal muscle[END_REF][START_REF] Jackson | Redox regulation of skeletal muscle[END_REF][START_REF] Musaro | Oxidative stress and muscle homeostasis[END_REF]. Small, compartmentalized, and transient increases in ROS regulate intracellular signals by reversible oxidation of specific protein residues [START_REF] Meng | Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo[END_REF][START_REF] Ghezzi | Oxidoreduction of protein thiols in redox regulation[END_REF][START_REF] Janssen-Heininger | Redox-based regulation of signal transduction: principles, pitfalls, and promises[END_REF][START_REF] Drazic | Methionine oxidation activates a transcription factor in response to oxidative stress[END_REF]. Hydrogen peroxide modifies protein function by oxidizing the thiol (-SH) groups of redox sensitive cysteine residues to form disulfide bonds with adjacent cysteine residues, glutathione (glutathionylation), or small protein thiols such as thioredoxin. Reduction of the disulfide bond can then be obtained through the action of enzymes such as glutaredoxin or the peroxiredoxins [START_REF] Rhee | Cell signaling. H2O2, a necessary evil for cell signaling[END_REF][START_REF] Hashemy | Oxidation and S-nitrosylation of cysteines in human cytosolic and mitochondrial glutaredoxins: effects on structure and activity[END_REF]. Accordingly, specific cysteine and methionine residues can function as redox-dependent switches, thereby modulating the function of myofibrillar proteins [START_REF] Nogueira | Myosin is reversibly inhibited by S-nitrosylation[END_REF][START_REF] Mollica | S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans[END_REF][START_REF] Gross | Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants[END_REF], phosphatase or transcriptional activities [START_REF] Xanthoudakis | Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme[END_REF][START_REF] Ji | Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle[END_REF][START_REF] Powers | Mechanisms of disuse muscle atrophy: role of oxidative stress[END_REF][START_REF] Powers | Reactive oxygen species: impact on skeletal muscle[END_REF][START_REF] Jackson | Redox regulation of skeletal muscle[END_REF][START_REF] Ugarte | Oxidized mitochondrial protein degradation and repair in aging and oxidative stress[END_REF]. Indeed, recent findings indicate that myosin, actin, troponin I and Tm from skeletal muscle have cysteines that are critical to the effects from oxidants [START_REF] Nogueira | Myosin is reversibly inhibited by S-nitrosylation[END_REF][START_REF] Mollica | S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans[END_REF][START_REF] Gross | Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants[END_REF], and that accessibility depends on the conformation of the CB [START_REF] Gross | Accessibility of myofilament cysteines and effects on ATPase depend on the activation state during exposure to oxidants[END_REF]. In addition, the function of myosin [START_REF] Nogueira | Myosin is reversibly inhibited by S-nitrosylation[END_REF] and of the ryanodine 1 receptor [START_REF] Bellinger | Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle[END_REF]) can be reversibly modified by S-nitrosylation, a redox-related modification of cysteine thiol by NO. Reversible oxidation of contractile proteins likely regulates muscle force production and actomyosin interactions, thereby contributing to the physiological adaptation of muscle to mechanical challenge [START_REF] Powers | Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production[END_REF]. Disturbance or removal of this redox sensitive modulations may limit the adaptability of the CB kinetics, with deleterious effects on muscle function and homeostasis [START_REF] Stone | Hydrogen peroxide: a signaling messenger[END_REF][START_REF] Linnane | The essential requirement for superoxide radical and nitric oxide formation for normal physiological function and healthy aging[END_REF]. Apart cysteine, methionine residue can be oxidized to its sulfoxyde, generating a mixture of two diastereoimers, denoted S and R. The S and the R forms are reduced back to methionine by methionine sulfoxyde reductase A (MsrA) and B (MsrB), respectively [START_REF] Stadtman | Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism[END_REF][START_REF] Petropoulos | Protein maintenance in aging and replicative senescence: a role for the peptide methionine sulfoxide reductases[END_REF][START_REF] Ugarte | Oxidized mitochondrial protein degradation and repair in aging and oxidative stress[END_REF]. Interestingly, recent studies have highlighted a new role for these Msr enzymes, identifying them as actors controlling the assembly/disassembly of actin filaments [START_REF] Hung | SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics[END_REF][START_REF] Lee | MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation[END_REF]. A specific oxidation-reduction (redox) enzyme, the Mical protein, selectively modifies two methionines on the conserved pointed-end in actin filaments. Oxidized methionines at these positions simultaneously disassemble and decrease the polymerization of actin filaments [START_REF] Hung | SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics[END_REF]. Recently, Hung et al. and Lee et al. showed in Drosophila bristle processes model and in mouse macrophages that MsrB1 stereospecifically reverses the oxidative modification of the methionines introduced by Mical, indicating that Msr system antagonizes the Mical-actin depolymerization dependent process and reversibly control actin polymerization [START_REF] Hung | SelR reverses Mical-mediated oxidation of actin to regulate F-actin dynamics[END_REF][START_REF] Lee | MsrB1 and MICALs regulate actin assembly and macrophage function via reversible stereoselective methionine oxidation[END_REF]. These data indicate new post-translational regulatory mechanisms involving oxidoreductase systems, but also open up new paths of investigation in the field of muscle diseases.

IRREVERSIBLE MODIFICATIONS OF PROTEINS: OXIDIZED PROTEINS ARE DYSFUNCTIONAL AND ARE TARGETED TO REMOVAL

In contrast to reversible modifications caused by low oxidative stress, chronic disruption of the oxidative balance and/or high levels of free ROS radicals cause potential biological damage, termed oxidative stress. This is a complex process that probably depends on the type of oxidant, on the site and intensity of its production, on the composition and activity of various antioxidants and on the ability of skeletal muscle to repair damaged fibers.

Irreversible damage of myofibrillar proteins may affect the CB cycle, as observed after nitrosylation and carbonylation of myosin (Coirault et al., 2007;[START_REF] Guellich | Role of oxidative stress in cardiac dysfunction of PPARalpha-/mice[END_REF], and target them for catabolic proteolysis (Jung et al., 2007). Alternatively, oxidative stress can activate caspases that in turn cleave myofibrillar proteins and impair CB properties [START_REF] Du | Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions[END_REF].

FUNCTIONAL CONSEQUENCES OF ALTERED CB CYCLING ON MUSCLE CONTRACTION

The functional consequences of altered CB cycle in MD remain to be precisely determined. It has been reported that the reduced muscle strength per cross section in mdx diaphragm is associated with an accelerated cycle compared with control diaphragm muscle [START_REF] Coirault | Myosin molecular motor dysfunction in dystrophic mouse diaphragm[END_REF]. Assuming that one molecule of ATP is hydrolyzed per CB cycle [START_REF] Huxley | Muscle structure and theories of contraction[END_REF][START_REF] Huxley | Proposed mechanism of force generation in striated muscle[END_REF]Eisenberg et al., 1980), these data suggested that the overall cycle of ATP splitting takes place more rapidly in mdx than in control mouse diaphragm, thus reducing the efficiency of muscle contraction to sustain force. Overall, these data suggested that absence of the sarcolemmal dystrophin protein leads to damaged or dysfunctional myosin molecules that impair CB kinetics and may account for the contractile deficit in mdx diaphragm. However, while mechanical differences are observed between mdx and control diaphragms at the intact muscle strip level, these differences are not observed at the single permeabilized cell [START_REF] Bates | Molecular, cellular, and muscle strip mechanics of the mdx mouse diaphragm[END_REF]. In addition, it was reported that myosin extracted from bulk mdx mouse diaphragm muscle moves actin filaments in an "in vitro motility assay" at a lower velocity than myosin extracted from controls [START_REF] Coirault | Velocity of actomyosin sliding in vitro is reduced in dystrophic mouse diaphragm[END_REF]. Consistent reduction in actin sliding velocity has been found in pure MyHC-2 isoform from mdx gastrocnemius muscle, but not in type 1 myosin from wild-type and mdx muscle [START_REF] Canepari | Actin sliding velocity on pure myosin isoforms from dystrophic mouse muscles[END_REF]. Therefore, observed changes in myosin velocity have been related to a change in the intrinsic properties of the molecule, but was not attributed to a change in the proportion of different myosin isoforms in the sample. In addition, it has been shown that nebulin in skeletal muscle increases thin filament activation via increasing CB cycling kinetics leading to an increased force and efficiency of contraction [START_REF] Chandra | Nebulin alters cross-bridge cycling kinetics and increases thin filament activation: a novel mechanism for increasing tension and reducing tension cost[END_REF]. Results from that study provide novel insights regarding nebulin-based nemaline myopathy. Thus, complex mechanisms other than translational changes in myosin isoform may contribute to CB modifications in muscular disorders. Future studies are needed to precisely determine the contribution of post-translational modifications of contractile proteins to muscle weakness in MDs.

CONCLUSIONS AND FUTURE DIRECTIONS

The mechanisms of muscle weakness have been a fundamental question of the physiopathology of muscle dystrophy for more than 50 years. It is now clear that the molecular causes responsible for the impaired performance in MDs involve both structural and functional modifications of the acto-myosin interactions. The precise mechanisms are complex with probably some specificities related to each individual mutation responsible for the MD, the severity of the translational and post-translational alterations in myofibrillar proteins, and on the ability of skeletal muscle to adapt to these changes. Whereas the functional consequences of translational changes in contractile proteins have been extensively studied, the impact of post-translational changes on the function of various contractile proteins still remains to be precisely assessed in the context of MDs. This is a particularly exciting time to study this question as recent findings strongly suggest that the loss of transient structural modifications of the contractile proteins such as reversible oxidation of specific protein residues impair the ability of CBs to adapt very rapidly to changes in workload and energy supply. Future works will assess the impact of non-permanent structural modifications of actin, myosin, and regulatory proteins on muscle performance and determine their potential contribution to muscle weakness in MDs.

FIGURE 1 |

 1 FIGURE 1 | Schematic representation of the main mechanisms leading to muscle weakness in MDs. Altered CB cycle in muscular dystrophy can arise from multiples causes including progressive loss of muscle tissue due to necrosis, fibrosis, atrophy, or oxidative damage, micro-architectural

FIGURE 2 |

 2 FIGURE 2 | The CB cycle.Step a: attachment of myosin to actin. This step begins with the "weak binding" of actin (in magenta) to the myosin head (in brown). A stereospecific interaction between actin and myosin leads to strong binding of actin to myosin. Step b: power stroke. The loss of inorganic phosphate (Pi) is assumed to trigger the onset of the power stroke and a reversal of the previous conformational bent. Step c: ADP release step. The power stroke induces reopening of the nucleotide binding pocket and ADP release. Step d: ATP binding. ATP can then rapidly bind to myosin which dissociates from actin. Step e: ATP hydrolysis. ATP is then rapidly hydrolyzed to ADP and Pi.

FIGURE 3 |

 3 FIGURE 3 | Transmission of the forces generated in sarcomere. The dystrophin-glycoprotein complex is preferentially located at the costamere, a protein network that reside at the sarcolemma membrane in register with the Z-lines of sarcomeres and that is critical in the lateral transmission of forces generated by the CB to the ECM and neighboring muscle fibers. Dystrophin deficiency at the lateral membrane alters costamere assembly and thus may impair the efficacy of lateral force transmission.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Marie-Hélène Errera

Marthe Laguarrigue

Florence Rossant

Edouard Koch

Céline Chaumette

Christine Fardeau

Mark Westcott

José-Alain Sahel

Bahram Bodaghi

Jonathan Benesty

Michel Paques

High resolution imaging of retinal vasculitis by flood illumination adaptive optics ophthalmoscopy: a follow-up study

Keywords: retina, vasculitis, flood-illumination adaptive optics ophthalmoscopy adaptive optics, posterior uveitis, retina, uveitis, retinal vasculitis

Purpose: To monitor perivascular sheathing during the course of retinal vasculitis by flood illumination adaptive optics ophthalmoscopy (AOO

Retinal vasculitis may occur either as isolated or in association with various inflammatory diseases affecting the eye. [START_REF] Rosenbaum | Retinal vasculitis[END_REF] Ophthalmoscopic presentations of retinal vasculitis combine vascular sheathing and focal fluorescein leakage. It affects more frequently veins than arteries. It may be associated with inflammatory diseases involving the central nervous system, most notably Behcet's disease, sarcoidosis, systemic lupus erythematosus ('vasoocclusive retinopathy') or multiple sclerosis.

Retinal vasculitis may lead to severe visual loss, either because of the complications of vascular obstruction which can lead to capillary non perfusion and development of pre-retinal new vessels and/or tractional detachment, or macular edema due to blood-retinal barrier rupture. Much remains to be known about the clinical and pathologic features of retinal vasculitis and the mechanisms leading to visual loss. In experimental retinal vasculitis, paravascular cellular infiltrates consist of a variable association of macrophages, T cells and CD45+ leucocytes. [START_REF] Copland | The clinical time-course of experimental autoimmune uveoretinitis using topical endoscopic fundal imaging with histologic and cellular infiltrate correlation[END_REF] They may co-localize with areas of leakage on fluorescein angiography (FA) [START_REF] Chu | Assessment and in vivo scoring of murine experimental autoimmune uveoretinitis using optical coherence tomography[END_REF][START_REF] Stanford | Retinal vasculitis: correlation of animal and human disease[END_REF] suggesting a causal relationship. In humans, segmental perivenous lymphoplasmocytic infiltration has been documented in Behcet's disease, multiple sclerosis and idiopathic retinal vasculitis. [START_REF] Charteris | Behcet's disease: activated T lymphocytes in retinal perivasculitis[END_REF][START_REF] George | Ocular immunopathology of Behcet's disease[END_REF][START_REF] Hughes | The pathology and pathogenesis of retinal vasculitis[END_REF][START_REF] Eichenbaum | A clinical and histopathological review of intermediate uveitis ("pars planitis")[END_REF][START_REF] Engell | Periphlebitis retinae in multiple sclerosis. A histopathological study of two cases[END_REF] However, the specific mediators involved in perivascular infiltration remain elusive and a detailed time-course of vascular infiltrates has not been reported.

Given the diagnostic and prognostic value of retinal perivascular sheathing, it would be of clinical interest to better identify, quantify and monitor retinal vasculitis. In experimental uveitis, grading systems based on fundus imaging 2 or optical coherence tomography (OCT) [START_REF] Chu | Assessment and in vivo scoring of murine experimental autoimmune uveoretinitis using optical coherence tomography[END_REF] have been proposed but have not been applied yet in humans. As a result, there is no validated procedure for the grading of human vasculitis.

Adaptive optics is an optoelectronic technology which improves the lateral resolution of fundus images, allowing quantitative analysis of microvascular structures. [START_REF] Koch | Morphometric analysis of small arteries in the human retina using adaptive optics imaging: relationship with blood pressure and focal vascular changes[END_REF][START_REF] Chui | Imaging of vascular wall fine structure in the human retina using adaptive optics scanning laser ophthalmoscopy[END_REF] We recently reported that flood-illumination adaptive optics (AOO) imaging allows highly sensitive detection of paravascular infiltration characteristic of retinal vasculitis. [START_REF] Errera | Retinal vasculitis imaging by adaptive optics[END_REF] By AOO, vascular sheathing appears as fusiform or linear opacities on both sides of vessels, often co-localizing with focal vascular narrowing. We also reported a case of regression of sheathing under therapy, suggesting that AOO may be useful for monitoring treatment. [START_REF] Errera | Retinal vasculitis imaging by adaptive optics[END_REF] Here, we quantified the extent of vascular sheathing and monitored its evolution using a semi-automated segmentation software. Our objective was to quantitatively explore the changes over time of vasculitis.

Material and Methods

This retrospective study adhered to the tenets set forth in the Declaration of Helsinki, and approval of the local Ethics Committee (St Antoine hospital, Paris, France) was obtained.

Inclusion was proposed to treatment-naive patients with retinal vasculitis of different etiologies and idiopathic and clear ocular media. Retinal vasculitis was identified by the presence of typical vascular sheathing and/or by foci of fluorescein leakage. All cases were reviewed by a senior uveitis specialist (MHE). Each subject received full oral and written information and gave consent prior to inclusion. Part of the data from two patients (cases 6 and 9) has been previously reported. [START_REF] Koch | Morphometric analysis of small arteries in the human retina using adaptive optics imaging: relationship with blood pressure and focal vascular changes[END_REF] Follow-up was customized according to each case's needs.

Color fundus photographs, scanning laser ophthalmoscopy and FA were performed at the Quinze-Vingts Hospital using standard procedures. AOO imaging was done with a commercially available AOO camera (rtx1™ camera; Imagine Eyes, Orsay, France; illumination wavelength 850 nm; field of view 4° x 4°) using a previously described procedure. [START_REF] Koch | Morphometric analysis of small arteries in the human retina using adaptive optics imaging: relationship with blood pressure and focal vascular changes[END_REF] Briefly, en face AOO fundus images were obtained through dilated pupils (1% tropicamide; Novartis, France). Because of the asymmetric configuration of the camera, only the right eye could undergo such exploration. An external target guiding the fellow eye allowed navigating along large vessels up to approximately 25° from the disc. Retinal vessels (veins and arteries) up to 25 o from the disc were screened by AOO. No photographs of the macula were obtained. Perivascular cuffs were systematically searched for by moving the external target in order to navigate along the temporal vessels. When a vascular abnormality was identified on the live fundus display, a stack of images (40 images in 2 seconds) was acquired and automatically processed using the built-in software of the AOO camera (AO image 2.0). On average, one to three averaged images per minute could be taken.

Selected images were automatically segmented using a custom software running under Matlab (Mathworks, Natick, Massachussetts, USA). [START_REF] Lermé | A fully automatic method for segmenting retinal artery walls in adaptive optics images[END_REF] Briefly, after detection of the axial reflex of vessels, the external and internal walls of the perivascular cuff were simultaneously detected using an original tracking procedure to cope with deformation along vessels. Then, their positioning was refined using a deformable model embedding a parallelism constraint in order to control the distance of the curves to their reference line and improve the robustness to image noise. [START_REF] Lermé | A fully automatic method for segmenting retinal artery walls in adaptive optics images[END_REF] From this segmentation, the lumen and the sum of the width of the perivascular sheathing on both sides were extracted along a vascular segment up to 1000µm long (Fig 1). At sites of vascular narrowing, the relative narrowing was calculated as the ratio of minimal diameter over the average of upstream and downstream diameters. The whole procedure was done under human supervision; inadequate segmentations were discarded.

Results

We reviewed twenty cases of uveitis with retinal vasculitis seen in our department who underwent AOO imaging between June 2014 and March 2016. Images were then examined by three of us (JB, MHE and MP). The presence of perivascular sheathing was assessed.

Images showing acceptable contrast were selected for morphometric analysis. Nine cases were rejected because of a poor contrast of vascular sheathing. Twelve eyes of 12 consecutive subjects (8 women, 4 men; mean age, 42 years) were included in final evaluation. Diagnoses included birdshot chorioretinopathy (n=3), toxoplasmic retinochoroiditis (n=2), ocular tuberculosis (n=2), MS, idiopathic retinal vasculitis, aneurysm and neuroretinitis (IRVAN), ocular Lyme borreliosis, syphilitic chorioretinitis and idiopathic retinal vasculitis (one case each). Their mean duration of visual symptoms was 7.8 months (range, 1-20) and the mean follow-up was 6.5 months (± 5.6). None was treated by antiinflammatory treatment at time of first examination in our department. Their clinical characteristics are summarized in Table 1.

Mean (± SD) width of perivascular opacification was 101.0 (± 54. More than one area of sheathing was observed in 6 eyes. A relationship was found between leakage of vessels on fluorescein angiography and vascular sheathing of the same area on AOO in 8 out of 12 eyes (66%). Seven eyes (58%) showed perivenous sheathing in additional areas with no detectable abnormalities on fundus photographs or on FA (Fig 4).

On the opposite, all areas showing fluorescein leakage showed perivascular sheathing (Figs 5 and6).

Corticosteroid treatment was initiated in ten patients. Four were also treated with immunomodulator agents and six received specific antibiotic treatment. Patient 8, which had IRVAN, was not treated. Intervals between follow-up exams varied according to the treatment regimen, clinical evolution and availability of the patient. The range of intervals between AOO imaging sessions varied between 2 weeks and 2 years (median, 2.5 months).

Clinical evolution showed a progressive resolution of inflammatory signs in all but two cases (cases with IRVAN and ocular Lyme borreliosis). At last examination, on average the thinning had decreased from 101.0 µm ± 54.3 to 23.6µm ± 15.5 (p=0.003). Two eyes showed persistent venous sheathing at last examination. No vein occlusion occurred. Recurrence of perivenous sheathing was observed in 4 cases (example in Fig 6). These recurrences occurred at the same site as the initial one.

Changes of vessel diameters did not show a clear correlation with the overall level of ocular inflammation. Among the 10 eyes with focal vascular narrowing at presentation, 5 eyes showed an increase in vein diameter. Figure 7 shows morphograms of the long-term evolution of perivenous sheathing and venous diameters.

In an attempt to clarify the relationship between vascular sheathing and venous narrowing, we analyzed a subgroup of four eyes that had monthly AOO imaging. This showed a regular decrease of perivenous sheathing in all cases, which contrasted with the apparent stability of the diameter of the underlying veins (Fig 8). Note that none of these cases showed venous narrowing initially. However, in another subgroup of 5 eyes, it could be seen that the reduction of perivascular sheathing was accompanied by displacement of adjacent nerve fiber bundles (figure 9 and supplementary figures-videos), suggesting that expansion of perivenous infiltration displaced adjacent nerve fiber bundles (cases 1,3,7,8,9 and 11).

In areas of sheathing showing breakdown of the blood-retinal barrier, it could be observed that resolution of the fluorescein leakage occurred with the disappearance of sheathing (figure 3b).

Periarterial sheathing was present in two eyes (cases 8 and 11). One of them showed also focal reduction of diameter (figure 5). During follow-up, patient 8 (IRVAN syndrome) showed enlargement of the macroaneurysm but no change in the width of periarterial sheathing (figure 10). Although, patient 11 (idiopathic vasculitis) showed a disappearance of perivascular opacification and of vessels narrowing (figure 5).

Discussion

We show here that thanks to its capabilities to document at a microscopic level both the width of perivascular sheathing and the vascular narrowing, AOO brings new opportunities for the evaluation of retinal vasculitis. This may not only reveal the severity of the disease but also indicate the risk of local complications. Albeit AOO imaging is of limited interest in case of media opacities such as vitritis, it offers the unique possibility to detail fine features of paravascular opacities. We demonstrated for instance that IRVAN was associated with perivenous opacities. We also observed in many cases the presence of arteriolar narrowing associated with arteritis. The contrast was highly variable between the infiltrate and the surrounding retina, which may be due to the specific nature of the infiltrate or to the background pigmentation. Infiltrates associated with blood-retinal barrier rupture were more often located along the major vessels of the temporal arcades in an area within 10° from the optic disc. We also documented here the time-course of resolution of sheathing, showing that the width of the sheathing was halved in three months for one patient who underwent serial AOO imaging. Recurrence was often observed in the same site as initially, suggesting that cell infiltrates do not occur randomly but that there are preferential sites. Recently, using another software, Mahendradas et al, have also shown a complete resolution of the sheathing by AO on follow-up. 14 Venous narrowing and occlusion are common complications of vasculitis. By AOO imaging, minute venous narrowing could be documented. For few patients, there was a limited yet clear correlation between the width of sheathing and venous narrowing. The mechanisms of vascular occlusion during vasculitis are uncertain. Pathologic studies have suggested a thrombotic or obliterative mechanism. [START_REF] Hughes | The pathology and pathogenesis of retinal vasculitis[END_REF] In our patients, morphometric analyzes showed that in most eyes venous narrowing temporally and spatially correlate with the width of the perivascular cuff. Furthermore, in 5 cases, it could be documented that adjacent inner retinal structures were displaced by the perivascular cuffs rather than infiltrated. This strongly suggests that local compression by the infiltrate itself may contribute to vascular occlusions. This suggests that there is compression rather than infiltration. This was not observed in all eye, hence one should be cautious about such interpretation. Relationship with blood-retinal barrier also remains uncertain.

Periarterial infiltrates are rare. We documented here two cases with periarteritis, one case of IRVAN syndrome and one case of idiopathic vasculitis. The arteriolar wall remained visible through the infiltration, suggesting that the infiltrate was extra-parietal. AOO imaging demonstrated a similar appearance of both periarterial infiltrates and perivenous infiltrates, i.e, perivascular opacifications. There was also a reduction of arteriolar caliber.

There are some limits to our study. It is unable to image the retina in presence of vitreous haze or edema surrounding the vessel. It is significant to note that vitreous haze, retinal edema and/or cataract limited AOO fundus imaging in 8 eyes among the twenty cases initially selected. AOO imaging cannot provide images of the peripheral vessels more than 25° away from the disc. It is also uncertain to which extent the foci that were measured actually are representative of the whole inflammatory process. Our sample size was too small to differentiate the effect of the various anti-inflammatory drugs.

The possibility to monitor vascular inflammation offers interesting fundamental and clinical perspectives. For instance, our data suggest that perivascular infiltrates may contribute to venous occlusion through a compressive effect. The effect of therapy can be quantitatively evaluated. Additional studies are needed to further assess the correlation between clinical severity and AOO features and to document the time-course of sheathing.

Those will be helpful in determining the relationship between narrowing and obstruction, between sheathing and blood-retinal barrier rupture, and between AOO features and etiology. AOO may also be of interest for the screening of vascular inflammation in patients affected by cerebral diseases.

  3) µm. The length of perivascular opacification ranged from 250µm to several millimeters. At sites of sheathing, focal narrowing of veins was observed in 10 eyes; the degree of venous narrowing relative to adjacent areas ranged from 4 to 28% (mean, 14%) in 10 eyes. Focal narrowing of arteries was also present in one eye (25% of narrowing) diagnosed with idiopathic retinal vasculitis and arterial dilatation at the site of macroaneurysm in the IRVAN case. Examples of perivascular opacification and vein narrowing imaged by AOO is shown in Fig 2. Representative examples of segmented AOO images are shown in Fig 3.

Figure 1 .

 1 Figure 1. Patient 8 (IRVAN syndrome). Top left, retinophotography at baseline and bottom left, fluorescein angiography showing no detectable sheathing or leakage (in squares). Middle: Segmented AOO image of periphlebitis. On the right, the morphogram shows the variations of the width of the lumen diameter and perivascular cuff.

Figure 2 :

 2 Figure 2: Patient 3 (birdshot chorioretinopathy). Left, FA at baseline: note the fluorescein leakage in the area enclosed by the square. Right, AOO imaging showing perivenous sheathing (arrowheads) and focal narrowing (asterisk).

Figure 3 :

 3 Figure 3: Patient 12 (presumed ocular tuberculosis). (a) FA and AOO at baseline, AOO images of periphlebitis at baseline (arrows) in area enclosed in the square showing fluorescein leakage at baseline. (b) FA and AOO at one month, AOO images of periphlebitis diseappearance (asterisk) in area enclosed in the square showing resolution of fluorescein leakage. Bottom, segmented AOO image. Right, morphogram showing the variations of lumen diameter (red curve) and perivascular cuff width (violet curve).

Figure 4 .

 4 Figure 4. Patient 9 (ocular Lyme borreliosis). Top left, Retinophotography at baseline showing no detectable sheathing. Top right, FA at baseline showing no detectable fluorescein leakage in the area enclosed by the square. AOO imaging: follow-up of venous sheathing (arrowheads) and narrowing (arrows) along the superotemporal vein in a case of Lyme's disease. Note the fluctuations over time of focal narrowings (arrows).

Figure 5 :

 5 Figure 5: Patient 11 (idiopathic retinal vasculitis). FA at baseline: diffuse leakage of veins and retinal arteries (area enclosed by the square). AOO imaging showing loss of transparency around the vein and the artery suggestive of inflammatory infiltration. The peak venous sheathing was measured at 225µm for the vein and 66µm for the artery. The maximal vascular narrowing was measured at 28%for the vein and 25% for the artery. After 3 months of oral corticosteroids, AOO imaging shows regression of perivascular infiltrates. Note also the widening of the artery.

Figure 6 .

 6 Figure 6. Patient 1 (birdshot retinochoroidopathy). Top: FA at baseline. The square indicates the area magnified in AOO images. Bottom: Successive AOO images. Note the fluctuations over time of perivenous sheathing (arrowheads) and of venous narrowing (asterisk). The sheathing recurred at month 24.

Figure 7 :

 7 Figure 7: Illustration of the changes of perivenous sheathing and venous diameters (curves in pale grey colour) between the first and last examination in patient 7. Perivenous sheathing (curves in grey colour) and venous diameters (curves in pale grey colour).

Figure 8 .

 8 Figure8. Short-term evolution of the width of perivenous sheathing and of venous diameters normalized to initial values in a subgroup of four eyes (cases 3,4,5 and 7). For each case, a single area was followed-up. Note that the decrease of perivenous sheathing is not correlated with venous diameter changes.

Figure 9 .

 9 Figure 9. Patient 7. (a) FA at baseline, (b, c and d) AOO images showing perivenous sheathing with and no detectable fluorescein leakage in the area of sheathing. Arrowheads in c and d show axons fascicles that come closer to the vein following the resolution of sheathing.

Figure 10 .

 10 Figure 10. Case 8 (IRVAN). AOO follow-up images of periarterial sheathing (macroaneurysm).

Table 1 .

 1 Clinical characteristics of patients (IRVAN: Idiopathic retinal arteritis, vasculitis, aneurysms and neuroretinitis). NA: not applicable. *arterial microaneurysm.

	Case Age/sex	Diagnosis	Duration	Visual	Peak	treatment	evolution	Maximal	Peak arterial	Maximal
				(months)	acuity	venous			venous	sheathing	arterial
						sheathing			narrowing	(diameter)	narrowing
						(diameter			(%)		(%)
						µm)
	1	65/F	birdshot	8	20/30	+ (90)	PO prednisone	relapse	+ (15)	none	none
							+
							mycophenolate
							mofetil
	2	26/F	birdshot	4.5	20/25	+ (35)	PO prednisone	resolution	+ (5)	none	none
							+ azathioprine
	3	56/F	birdshot	8	20/40	+ (80)	PO prednisone	resolution	+ (20)	none	none
							+
							mycophenolate
							mofetil
	4	28/F	toxoplasmosis	1	20/32	+ (88)	PO prednisone	resolution	+ (4)	none	none
							+ PO
							antibiotics
	5	25/F	toxoplasmosis	2.5	20/32	+ (66)	PO prednisone	resolution	+ (4)	none	none
							+ PO
							antibiotics
	6	58/M	tuberculosis	1	20/25	+ (90)	PO prednisone	resolution	NA	none	none
							+ RIPE
	12	35/M	tuberculosis	3	20/32	+ (211)	PO prednisone	resolution	+ (15)	none	none
							+ RIPE
	9	52/M	Lyme	12	20/40	+ (110)	PO prednisone	chronic	+ (20)	none	none
			borreliosis				+
							mycophenolate
							mofetil
	10	35/M	syphilis	5	20/50	+ (45)	IV Penicillin	resolution	NA	none	none
	7	35/F	multiple	11	20/40	+ (99)	PO prednisone	resolution	+ (9)	none	none
			sclerosis
	8	44/F	IRVAN	4	20/20	+ (111)	None	chronic	+ (24)	+ (124) *	NA
	11	44/F	idiopathic	20	20/25	+ (225)	PO prednisone	resolution	+ (28)	+	+ (25)
										(unavalaible)

Supported by the Institut National de la Santé et de la Recherche Médicale (Contrat d'Interface 2011) and the Agence Nationale de la Recherche (ANR-09-TECS-009 and ANR-12-TECS-0015-03). The funding organizations had no role in the design or conduct of this research. Disclosure of interest: Professor Michel Paques is consultant for Imagine Eyes. ).