KEY POINTS: Spinal treatment can restore diaphragm function in all animals 1 month following C2 hemisection induced paralysis. Greater recovery occurs the longer after injury the treatment is applied. Through advanced assessment of muscle mechanics, innovative histology and oxygen tension modelling, we have comprehensively characterized in vivo diaphragm function and phenotype. Muscle work loops reveal a significant deficit in diaphragm functional properties following chronic injury and paralysis, which are normalized following restored muscle activity caused by plasticity-induced spinal reconnection. Injury causes global and local alterations in diaphragm muscle vascular supply, limiting oxygen diffusion and disturbing function. Restoration of muscle activity reverses these alterations, restoring oxygen supply to the tissue and enabling recovery of muscle functional properties. There remain metabolic deficits following restoration of diaphragm activity, probably explaining only partial functional recovery. We hypothesize that these deficits need to be resolved to restore complete respiratory motor function. ABSTRACT: Months after spinal cord injury (SCI), respiratory deficits remain the primary cause of morbidity and mortality for patients. It is possible to induce partial respiratory motor functional recovery in chronic SCI following 2 weeks of spinal neuroplasticity. However, the peripheral mechanisms underpinning this recovery are largely unknown, limiting development of new clinical treatments with potential for complete functional restoration. Utilizing a rat hemisection model, diaphragm function and paralysis was assessed and recovered at chronic time points following trauma through chondroitinase ABC induced neuroplasticity. We simulated the diaphragm's in vivo cyclical length change and activity patterns using the work loop technique at the same time as assessing global and local measures of the muscles histology to quantify changes in muscle phenotype, microvascular composition, and oxidative capacity following injury and recovery. These data were fed into a physiologically informed model of tissue oxygen transport. We demonstrate that hemidiaphragm paralysis causes muscle fibre hypertrophy, maintaining global oxygen supply, although it alters isolated muscle kinetics, limiting respiratory function. Treatment induced recovery of respiratory activity normalized these effects, increasing oxygen supply, restoring optimal diaphragm functional properties. However, metabolic demands of the diaphragm were significantly reduced following both injury and recovery, potentially limiting restoration of normal muscle performance. The mechanism of rapid respiratory muscle recovery following spinal trauma occurs through oxygen transport, metabolic demand and functional dynamics of striated muscle. Overall, these data support a systems-wide approach to the treatment of SCI, and identify new targets to mediate complete respiratory recovery.

Institute of Experimental Medicine Czech Academy of Sciences Prague Czech Republic

Institute of Life Course and Medical Sciences University of Liverpool Liverpool UK

School of Biomedical Sciences Faculty of Biological Sciences University of Leeds Leeds UK

The Wolfson Centre for Age Related Diseases Guy's Campus King's College London London UK

Komentář v

J Physiol. 2021 Feb;599(4):1009-1010. doi: 10.1113/JP281042. PubMed

Zobrazit více v PubMed

Al‐Shammari AA, Kissane RWP, Holbek S, Mackey AL, Andersen TR, Gaffney EA, Kjaer M & Egginton S (2019). Integrated method for quantitative morphometry and oxygen transport modeling in striated muscle. J Appl Physiol 126, 544–557. PubMed

Alilain WJ & Goshgarian HG (2008). Glutamate receptor plasticity and activity‐regulated cytoskeletal associated protein regulation in the phrenic motor nucleus may mediate spontaneous recovery of the hemidiaphragm following chronic cervical spinal cord injury. Exp Neurol 212, 348–357. PubMed PMC

Altringham JD & Young IS (1991). Power output and the frequency of oscillatory work in mammalian diaphragm muscle: the effects of animal size. J Exp Biol 157, 381–389. PubMed

Ameredes BT, Zhan WZ, Prakash YS, Vandenboom R & Sieck GC (2000). Power fatigue of the rat diaphragm muscle. J Appl Physiol 89, 2215–2219. PubMed

Askew GN & Marsh RL (1997). The effects of length trajectory on the mechanical power output of mouse skeletal muscles. J Exp Biol 200, 3119–3131. PubMed

Askew GN, Young IS & Altringham JD (1997). Fatigue of mouse soleus muscle, using the work loop technique. J Exp Biol 200, 2907–2912. PubMed

Barnouin Y, McPhee JS, Butler‐Browne G, Bosutti A, De Vito G, Jones DA, Narici M, Behin A, Hogrel J‐Y & Degens H (2017). Coupling between skeletal muscle fiber size and capillarization is maintained during healthy aging. J Cachexia Sarcopenia Muscle 8, 647–659. PubMed PMC

Bezdudnaya T, Hormigo KM, Marchenko V & Lane MA (2018). Spontaneous respiratory plasticity following unilateral high cervical spinal cord injury in behaving rats. Exp Neurol 305, 56–65. PubMed PMC

Biering‐Sorensen B, Kristensen IB, Kjaer M & Biering‐Sorensen F (2009). Muscle after spinal cord injury. Muscle Nerve 40, 499–519. PubMed

Bowen TS, Brauer D, Rolim NPL, Baekkerud FH, Kricke A, Ormbostad Berre A‐M, Fischer T, Linke A, da Silva GJ, Wisloff U & Adams V (2017). Exercise training reveals inflexibility of the diaphragm in an animal model of patients with obesity‐driven heart failure with a preserved ejection fraction. J Am Heart Assoc 6, e006416. PubMed PMC

Buttry JL & Goshgarian HG (2014). Injection of WGA‐Alexa 488 into the ipsilateral hemidiaphragm of acutely and chronically C2 hemisected rats reveals activity‐dependent synaptic plasticity in the respiratory motor pathways. Exp Neurol 261, 440–450. PubMed

Corpeno R, Dworkin B, Cacciani N, Salah H, Bergman HM, Ravara B, Vitadello M, Gorza L, Gustafson AM, Hedström Y, Petersson J, Feng HZ, Jin JP, Iwamoto H, Yagi N, Artemenko K, Bergquist J & Larsson L (2014). Time course analysis of mechanical ventilation‐induced diaphragm contractile muscle dysfunction in the rat. J Physiol 592, 3859–3880. PubMed PMC

Cregg JM, Chu KA, Hager LE, Maggard RSJ, Stoltz DR, Edmond M, Alilain WJ, Philippidou P, Landmesser LT & Silver J (2017). A latent propriospinal network can restore diaphragm function after high cervical spinal cord injury. Cell Rep 21, 654–665. PubMed PMC

Del Negro CA, Funk GD & Feldman JL (2018). Breathing matters. Nat Rev Neurosci 19, 351–367. PubMed PMC

Dietz V (2010). Behavior of spinal neurons deprived of supraspinal input. Sci Rep 6, 167–174. PubMed

Dunford EC, Leclair E, Aiken J, Mandel ER, Haas TL, Birot O & Riddell MC (2017). The effects of voluntary exercise and prazosin on capillary rarefaction and metabolism in streptozotocin‐induced diabetic male rats. J Appl Physiol 122, 492–502. PubMed PMC

Egginton S (1990). Morphometric analysis of tissue capillary supply In Vertebrate Gas Exchange: From Environment to Cell, ed. Boutilier RG, pp. 73–141. Springer, Berlin, Heidelberg.

Egginton S, Badr I, Williams J, Hauton D, Baan GC & Jaspers RT (2011). Physiological angiogenesis is a graded, not threshold, response. J Physiol 589, 195–206. PubMed PMC

Egginton S & Gaffney E (2010). Tissue capillary supply – it's quality not quantity that counts! Exp Physiol 95, 971–979. PubMed

Elliott JE, Omar TS, Mantilla CB & Sieck GC (2016). Diaphragm muscle sarcopenia in Fischer 344 and Brown Norway rats. Exp Physiol 101, 883–894. PubMed PMC

Felix MS, Bauer S, Darlot F, Muscatelli F, Kastner A, Gauthier P & Matarazzo V (2014). Activation of Akt/FKHR in the medulla oblongata contributes to spontaneous respiratory recovery after incomplete spinal cord injury in adult rats. Neurobiol of Dis 69, 93–107. PubMed

Fowler ED, Hauton D, Boyle J, Egginton S, Steele DS & White E (2019). Energy metabolism in the failing right ventricle: limitations of oxygen delivery and the creatine kinase system. Int J Mol Sci 20, 1805. PubMed PMC

Gill LC, Ross HH, Lee KZ, Gonzalez‐Rothi EJ, Dougherty BJ, Judge AR & Fuller DD (2014). Rapid diaphragm atrophy following cervical spinal cord hemisection. Respir Physiol Neurobiol 192, 66–73. PubMed PMC

Golder FJ & Mitchell GS (2005). Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury. J Neurosci 25, 2925–2932. PubMed PMC

Graham ZA, Siedlik JA, Harlow L, Sahbani K, Bauman WA, Tawfeek HA & Cardozo CP (2019). Key glycolytic metabolites in paralyzed skeletal muscle are altered seven days after spinal cord injury in mice. J Neurotrauma 36, 2722–2731. PubMed PMC

Greising SM, Mantilla CB, Medina‐Martínez JS, Stowe JM & Sieck GC (2015). Functional impact of diaphragm muscle sarcopenia in both male and female mice. Am J Physiol Lung Cell Mol Physiol 309, L46–L52. PubMed PMC

Grundy D (2015). Principles and standards for reporting animal experiments in The Journal of Physiology and Experimental Physiology. J Physiol 593, 2547–2549. PubMed PMC

Hauton D, Al‐Shammari A, Gaffney EA & Egginton S (2015a). Maternal hypoxia decreases capillary supply and increases metabolic inefficiency leading to divergence in myocardial oxygen supply and demand. PLoS One 10, e0127424. PubMed PMC

Hauton D, Winter J, Al‐Shammari AA, Gaffney EA, Evans RD & Egginton S (2015b). Changes to both cardiac metabolism and performance accompany acute reductions in functional capillary supply. Biochim Biophys Acta 1850, 681–690. PubMed

Hellsten Y, Rufener N, Nielsen JJ, Høier B, Krustrup P & Bangsbo J (2008). Passive leg movement enhances interstitial VEGF protein, endothelial cell proliferation, and eNOS mRNA content in human skeletal muscle. Am J Physiol Regul Integr Comp Physiol 294, R975–R982. PubMed

Høier B, Rufener N, Bojsen‐Møller J, Bangsbo J & Hellsten Y (2010). The effect of passive movement training on angiogenic factors and capillary growth in human skeletal muscle. J Physiol 588, 3833–3845. PubMed PMC

Høier B, Walker M, Passos M, Walker PJ, Green A, Bangsbo J, Askew CD & Hellsten Y (2013). Angiogenic response to passive movement and active exercise in individuals with peripheral arterial disease. J Appl Physiol 115, 1777–1787. PubMed PMC

Huijing PA, Nieberg SM, vander Veen EA & Ettema GJ (1994). A comparison of rat extensor digitorum longus and gastrocnemius medialis muscle architecture and length‐force characteristics. Acta anatomica 149, 111–120. PubMed

Jeong M‐a, Plunet W, Streijger F, Lee JHT, Plemel JR, Park S, Lam CK, Liu J & Tetzlaff W (2011). Intermittent fasting improves functional recovery after rat thoracic contusion spinal cord injury. J Neurotrauma 28, 479–492. PubMed PMC

Josephson RK (1985). Mechanical power output from striated muscle during cyclic contraction. J Exp Biol 114, 493.

Khurram OU, Fogarty MJ, Sarrafian TL, Bhatt A, Mantilla CB & Sieck GC (2018). Impact of aging on diaphragm muscle function in male and female Fischer 344 rats. Physiol Rep 6, e13786. PubMed PMC

Khurram OU, Sieck GC & Mantilla CB (2017). Compensatory effects following unilateral diaphragm paralysis. Respir Physiol Neurobiol 246, 39–46. PubMed PMC

Kissane RWP, Egginton S & Askew GN (2018a). Regional variation in the mechanical properties and fibre‐type composition of the rat extensor digitorum longus muscle. Exp Physiol 103, 111–124. PubMed

Kissane RWP, Wright O, Al'Joboori YD, Marczak P, Ichiyama RM & Egginton S (2018b). Effects of treadmill training on microvascular remodeling in the rat following spinal cord injury. Muscle Nerve 59, 370–379. PubMed

Lane MA, Fuller DD, White TE & Reier PJ (2008a). Respiratory neuroplasticity and cervical spinal cord injury: translational perspectives. Trends Neurosci 31, 538–547. PubMed PMC

Lane MA, White TE, Coutts MA, Jones AL, Sandhu MS, Bloom DC, Bolser DC, Yates BJ, Fuller DD & Reier PJ (2008b). Cervical prephrenic interneurons in the normal and lesioned spinal cord of the adult rat. J Comp Neurol 511, 692–709. PubMed PMC

Lang BT, Cregg JM, DePaul MA, Tran AP, Xu K, Dyck SM, Madalena KM, Brown BP, Weng Y‐L, Li S, Karimi‐Abdolrezaee S, Busch SA, Shen Y & Silver J (2015). Modulation of the proteoglycan receptor PTPsigma promotes recovery after spinal cord injury. Nature 518, 404–408. PubMed PMC

Liet J‐M, Pelletier V, Robinson BH, Laryea MD, Wendel U, Morneau S, Morin C, Mitchell G & Lacroix J (2003). The effect of short‐term dimethylglycine treatment on oxygen consumption in cytochrome oxidase deficiency: a double‐blind randomized crossover clinical trial. J Pediatr 142, 62–66. PubMed

Llano‐Diez M, Renaud G, Andersson M, Marrero HG, Cacciani N, Engquist H, Corpeno R, Artemenko K, Bergquist J & Larsson L (2012). Mechanisms underlying ICU muscle wasting and effects of passive mechanical loading. Crit Care 16, R209–216. PubMed PMC

López‐Vales R, Forés J, Navarro X & Verdú E (2007). Chronic transplantation of olfactory ensheathing cells promotes partial recovery after complete spinal cord transection in the rat. Glia 55, 303–311. PubMed

Lynn R & Morgan DL (1994). Decline running produces more sarcomeres in rat vastus intermedius muscle fibers than does incline running. J Appl Physiol 77, 1439–1444. PubMed

Mantilla CB, Bailey JP, Zhan W‐Z & Sieck GC (2012). Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury. Exp Neurol 234, 191–199. PubMed PMC

Mantilla CB, Gransee HM, Zhan W‐Z & Sieck GC (2013a). Motoneuron BDNF/TrkB signaling enhances functional recovery after cervical spinal cord injury. Exp Neurol 247, 101–109. PubMed PMC

Mantilla CB, Greising SM, Zhan W‐Z, Seven YB & Sieck GC (2013b). Prolonged C2 spinal hemisection‐induced inactivity reduces diaphragm muscle specific force with modest, selective atrophy of type IIx and/or IIb fibers. J Appl Physiol 114, 380–386. PubMed PMC

Mantilla CB, Rowley KL, Zhan WZ, Fahim MA & Sieck GC (2007). Synaptic vesicle pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon terminal, inactivity. Neuroscience 146, 178–189. PubMed

Mantilla CB, Seven YB, Hurtado‐Palomino JN, Zhan W‐Z & Sieck GC (2011). Chronic assessment of diaphragm muscle EMG activity across motor behaviors. Respir Physiol Neurobiol 177, 176–182. PubMed PMC

Mantilla CB, Seven YB, Zhan W‐Z & Sieck GC (2010). Diaphragm motor unit recruitment in rats. Respir Physiol Neurobiol 173, 101–106. PubMed PMC

Marsh RL & Bennett AF (1986). Thermal dependence of contractile properties of skeletal muscle from the lizard Sceloporus occidentalis with comments on methods for fitting and comparing force‐velocity curves. J Exp Biol 126, 63–77. PubMed

Metzger JM, Scheidt KB & Fitts RH (1985). Histochemical and physiological characteristics of the rat diaphragm. J Appl Physiol 58, 1085–1091. PubMed

Miyata H, Zhan WZ, Prakash YS & Sieck GC (1995). Myoneural interactions affect diaphragm muscle adaptations to inactivity. J Appl Physiol 79, 1640–1649. PubMed

Nantwi KD, El‐Bohy AA, Schrimsher GW, Reier PJ & Goshgarian HG (1999). Spontaneous functional recovery in a paralyzed hemidiaphragm following upper cervical spinal cord injury in adult rats. Neurorehabil Neural Repair 13, 225–234.

Nantwi KD & Goshgarian HG (1998). Effects of chronic systemic theophylline injections on recovery of hemidiaphragmatic function after cervical spinal cord injury in adult rats. Brain Res 789, 126–129. PubMed

Olfert IM, Baum O, Hellsten Y & Egginton S (2016). Advances and challenges in skeletal muscle angiogenesis. Am J Physiol Heart Circ Physiol 310, H326–H336. PubMed PMC

Porter WT (1895). The path of the respiratory impulse from the bulb to the phrenic nuclei. J Physiol 17, 455–485. PubMed PMC

Rayegani SM, Shojaee H, Sedighipour L, Soroush MR, Baghbani M & Amirani OaoB (2011). The effect of electrical passive cycling on spasticity in war veterans with spinal cord injury. Front Neurol 2, 39. PubMed PMC

Shumsky JS, Tobias CA, Tumolo M, Long WD, Giszter SF & Murray M (2003). Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT‐3 into a spinal cord injury site is associated with limited recovery of function. Exp Neurol 184, 114–130. PubMed

Sieck DC, Zhan W‐Z, Fang Y‐H, Ermilov LG, Sieck GC & Mantilla CB (2012). Structure‐activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions. Respir Physiol Neurobiol 180, 88–96. PubMed PMC

Sonjak V, Jacob K, Morais JA, Rivera‐Zengotita M, Spendiff S, Spake C, Taivassalo T, Chevalier S & Hepple RT (2019). Fidelity of muscle fibre reinnervation modulates ageing muscle impact in elderly women. J Physiol 597, 5009–5023. PubMed

Stevens ED & Syme DA (1993). Effect of stimulus duty cycle and cycle frequency on power output during fatigue in rat diaphragm muscle doing oscillatory work. Can J Physiol Pharmacol 71, 910–916. PubMed

Syme DA (1990). Passive viscoelastic work of isolated rat, Rattus norvegicus, diaphragm muscle. J Physiol 424, 301–315. PubMed PMC

Syme DA & Stevens ED (1989). Effect of cycle frequency and excursion amplitude on work done by rat diaphragm muscle. Can J Physiol Pharmacol 67, 1294–1299. PubMed

Tallis J, Higgins MF, Seebacher F, Cox VM, Duncan MJ & James RS (2017). The effects of 8 weeks voluntary wheel running on the contractile performance of isolated locomotory (soleus) and respiratory (diaphragm) skeletal muscle during early ageing. J Exp Biol 220, 3733–3741. PubMed

Tallis J, James RS, Little AG, Cox VM, Duncan MJ & Seebacher F (2014). Early effects of ageing on the mechanical performance of isolated locomotory (EDL) and respiratory (diaphragm) skeletal muscle using the work‐loop technique. Am J Physiol Regul Integr Comp Physiol 307, R670–R684. PubMed

Tickle PG, Hendrickse PW, Degens H & Egginton S (2020). Impaired skeletal muscle performance as a consequence of random functional capillary rarefaction can be restored with overload‐dependent angiogenesis. J Physiol 598, 1187–1203. PubMed PMC

Tran AP, Warren PM & Silver J (2018). The biology of regeneration failure and success after spinal cord injury. Physiol Rev 98, 881–917. PubMed PMC

Wang K & Ramirez‐Mitchell R (1983). A network of transverse and longitudinal intermediate filaments is associated with sarcomeres of adult vertebrate skeletal muscle. J Cell Biol 96, 562–570. PubMed PMC

Warren PM & Alilian W (2018). Plasticity induced recovery of breathing occurs at chronic stages after cervical contusion. J Neurotrauma 36, 1985–1999. PubMed PMC

Warren PM, Campanaro C, Jacono FJ & Alilain WJ (2018a). Mid‐cervical spinal cord contusion causes robust deficits in respiratory parameters and pattern variability. Exp Neurol 306, 122–131. PubMed PMC

Warren PM, Steiger SC, Dick TE, MacFarlane PM, Alilain WJ & Silver J (2018b). Rapid and robust restoration of breathing long after spinal cord injury. Nat Commun 9, 4843. PubMed PMC

Zholudeva LV, Iyer NR, Qiang L, Spruance VM, Randelman ML, White NW, Bezdudnaya T, Fischer I, Sakiyama‐Elbert SE & Lane MA (2018). Transplantation of neural progenitors and v2a interneurons after spinal cord injury. J Neurotrauma 35, 2883–2903. PubMed PMC

Recovery of Forearm and Fine Digit Function After Chronic Spinal Cord Injury by Simultaneous Blockade of Inhibitory Matrix Chondroitin Sulfate Proteoglycan Production and the Receptor PTPσ