ORIGINAL ARTICLE Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 Published online 20 July 2020 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.12599 Motoneuron deafferentation and gliosis occur in association with neuromuscular regressive changes during ageing in mice † † Alba Blasco1 , Sílvia Gras1 , Guillem Mòdol-Caballero2 , Olga Tarabal1 , Anna Casanovas1 , Lídia Piedrafita1 , Alejandro Barranco3 , Tapas Das4 , Suzette L. Pereira4 , Xavier Navarro2 , Ricardo Rueda3 , Josep E. Esquerda1 & Jordi Calderó1* 1 Unitat de Neurobiologia Cel·lular, Departament de Medicina Experimental, Facultat de Medicina, Universitat de Lleida, Institut de Recerca Biomèdica de Lleida (IRBLleida), Lleida, Spain, 2Grup de Neuroplasticitat i Regeneració, Institut de Neurociències, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, CIBERNED, Bellaterra, Spain, 3Abbott Nutrition Research and Development, Granada, Spain, 4Abbott Nutrition Research and Development, Columbus, OH, USA Abstract Background The cellular mechanisms underlying the age-associated loss of muscle mass and function (sarcopenia) are poorly understood, hampering the development of effective treatment strategies. Here, we performed a detailed characterization of age-related pathophysiological changes in the mouse neuromuscular system. Methods Young, adult, middle-aged, and old (1, 4, 14, and 24–30 months old, respectively) C57BL/6J mice were used. Motor be- havioural and electrophysiological tests and histological and immunocytochemical procedures were carried out to simultaneously analyse structural, molecular, and functional age-related changes in distinct cellular components of the neuromuscular system. Results Ageing was not accompanied by a significant loss of spinal motoneurons (MNs), although a proportion (~15%) of them in old mice exhibited an abnormally dark appearance. Dark MNs were also observed in adult (~9%) and young (~4%) animals, suggesting that during ageing, some MNs undergo early deleterious changes, which may not lead to MN death. Old MNs were depleted of cholinergic and glutamatergic inputs (~40% and ~45%, respectively, P < 0.01), suggestive of age-associated alter- ations in MN excitability. Prominent microgliosis and astrogliosis [~93% (P < 0.001) and ~100% (P < 0.0001) increase vs. adults, respectively] were found in old spinal cords, with increased density of pro-inflammatory M1 microglia and A1 astroglia (25-fold and 4-fold increase, respectively, P < 0.0001). Ageing resulted in significant reductions in the nerve conduction velocity and the compound muscle action potential amplitude (~30%, P < 0.05, vs. adults) in old distal plantar muscles. Compared with adult muscles, old muscles exhibited significantly higher numbers of both denervated and polyinnervated neuromuscular junctions, changes in fibre type composition, higher proportion of fibres showing central nuclei and lipofuscin aggregates, depletion of satellite cells, and augmented expression of different molecules related to development, plasticity, and maintenance of neuro- muscular junctions, including calcitonin gene-related peptide, growth associated protein 43, agrin, fibroblast growth factor binding protein 1, and transforming growth factor-β1. Overall, these alterations occurred at varying degrees in all the muscles analysed, with no correlation between the age-related changes observed and myofiber type composition or muscle topography. Conclusions Our data provide a global view of age-associated neuromuscular changes in a mouse model of ageing and help to advance understanding of contributing pathways leading to development of sarcopenia. Keywords Sarcopenia; Ageing; C57BL/6J mice; Motoneurons; Central synapses; Glia; Neuromuscular junction; Skeletal muscle Received: 3 April 2020; Revised: 5 June 2020; Accepted: 15 June 2020 *Correspondence to: Jordi Calderó, Unitat de Neurobiologia Cellular, Departament de Medicina Experimental, Facultat de Medicina, Universitat de Lleida–Institut de Recerca Biomèdica de Lleida, Av. Rovira Roure 80, Lleida 25198, Spain. Phone: +34-973-702440, Email: jordi.caldero@udl.cat † These authors contributed equally to this work. © 2020 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any me- dium, provided the original work is properly cited and is not used for commercial purposes. Regressive neuromuscular changes in aged mice 1629 Introduction death could account for the age-associated loss of motor units, particularly of those innervating fast-twitch muscles, The rise of human life expectancy is a contributing factor of and the decrease in muscle power.8,18–20 However, the as- frailty, a common clinical syndrome in the elderly, which is sumption that MN degeneration is the cause of age-related characterized by a state of increased vulnerability to adverse alterations in NMJs is controversial. Thus, some studies per- outcomes with subsequent disability and morbidity. Frailty is formed in murine models have not reported a significant loss considered a consequence of a collective age-related decline of MNs with age.21,22 Whatever the case, the fact that in the function and reserve of multiple organs, with particular age-related alterations in motor nerve terminals are a conse- involvement of the neuromuscular, endocrine, and immuno- quence of an intrinsic skeletal muscle pathology rather than a logical systems.1,2 Sarcopenia, defined as the progressive loss primary defect in MNs cannot be excluded. In fact, it is well of muscle mass, strength, and function, is a key component known that muscle fibres play an important role in the main- of physical frailty. Sarcopenia leads to mobility impairments tenance of NMJs.23–27 Therefore, a retrograde process (‘dy- and falls in older adults, negatively affecting their quality of ing-back’) leading to progressive NMJ disruption and MN life.3 The prevalence of sarcopenia in people over age 60 years dysfunction could result from a defect inherent to muscle fi- has been reported to be 8–40% depending on the sarcopenia bres with the subsequent decline in muscle-derived trophic definition used; starting about the age 50, humans lose 1–2% signals necessary to maintain the integrity and stability of of muscle mass per year, with an annual decline in muscle the neuromuscular presynaptic components. This theory is, strength of 1.5% between ages 50 and 60 and of 3% thereaf- in part, strengthened by findings indicating that NMJs in ter.4,5 Sarcopenia in the elderly has been proposed to have a humans are remarkably stable throughout the entire adult multifactorial aetiology, which includes chronic inflammation lifespan,28 suggesting that the structural and functional neu- (‘inflammageing’), metabolic and endocrine alterations, poor romuscular alterations occurring in the elderly have their nutrition, mitochondrial dysfunction, oxidative damage, and main cause in intrinsic muscle deficiencies. neurogenic factors.6–8 Although notable progress has been To elucidate the mechanisms leading to age-associated made in the understanding of causative factors leading to age- neuromuscular alterations, we undertook a detailed charac- ing sarcopenia, the exact cellular and molecular mechanisms terization of the pathophysiological changes occurring in the responsible for this process still remain elusive. Moreover, mouse neuromuscular system over the course of ageing. there is a remarkable divergence in reported results on the Here, we examined, as a whole, the various distinct cellular age-related changes observed in distinct components of the components, which shape the neuromuscular system and neuromuscular system and their contribution to progressive are essential for proper motor function. We simultaneously muscle wasting in the elderly. These differences are particu- analysed structural and molecular changes occurring with larly noticeable when comparing data from different ageing age in spinal cord MNs and glia, dorsal root ganglion (DRG) animal models and, even more, when compared with neuro- sensory neurons, motor nerves, NMJs, and skeletal muscles muscular changes reported in human studies. These differ- of C57BL/6JRj mice. To assess whether age-related changes ences could be due to the fact that many studies focus on in muscles are influenced by their regional location (distal specific components of the neuromuscular system rather than or proximal) and specific function, different hindlimb muscles a more holistic analysis of the neuromuscular system in its with a distinct position and myofiber type composition were entirety. studied. Moreover, to examine in vivo the potential Some studies have provided information indicating that an age-related changes in muscle innervation, an electrophysio- impaired neuromuscular innervation could account for the logical analysis using quantitative electromyography was per- loss of muscle mass in advanced ages9; in line with this, struc- formed. A better understanding of the neuromuscular tural changes in both the presynaptic and postsynaptic por- changes occurring with ageing and the pathogenic mecha- tions of the neuromuscular junction (NMJ) have been nisms leading to sarcopenia is crucial towards development reported to occur with age in both animals and humans.10– of effective interventions aimed at prevention and treatment. 12 Other studies in aged rodents have reported morphological alterations in motor nerve terminals with increased extent and complexity of their arborization, accompanied by frag- mentation of postsynaptic endplates. All these changes ap- Materials and methods pear to reflect continuous cycles of muscle denervation and reinnervation with ageing.13 Overall, this process of NMJ re- Animals modelling is considered to be a consequence of motoneuron (MN) degeneration with age. Some investigations in humans All animal handling and experimentation procedures were have reported a significant decrease in the number of MNs conducted in accordance with the ethical standards laid down in people over 60 years.14 Similarly, loss of MNs with advanc- in the 1964 Declaration of Helsinki and its later amendments, ing age has been reported in rodents as well.9,15–17 This MN the European Communities Council Directive 2010/63/EU for Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1630 A. Blasco et al. the care and use of laboratory animals, and the norms 25 μs duration delivered through a pair of needle electrodes established by the Generalitat de Catalunya [published in placed at the sciatic notch. The compound muscle action po- the Diari Oficial de la Generalitat de Catalunya (DOGC) tential (CMAP, M wave) and the H-reflex wave were recorded 2073, 1995]. All experiments were previously evaluated and from tibialis anterior (TA) and plantar interossei (PL) muscles approved by the Committee for Animal Care and Use of the with microneedle electrodes.33 The H/M ratio was calculated Universitat de Lleida and the Committee for Ethics on Exper- as the quotient of the maximal H wave and maximal M wave imental Animal and Human Research of the Universitat amplitude for each recorded muscle. For sensory nerve con- Autònoma de Barcelona. All efforts were made to minimize duction tests, the recording electrodes are placed near the suffering and reduce the number of animals in agreement digital nerves of the fourth toe to record the compound sen- with the European Communities Council Directive (24 No- sory nerve action potential following stimulation of the sciatic vember 1986; 86/609/EEC). nerve as mentioned previously. All potentials were amplified C57BL/6JRj mice were supplied by Janvier Labs (Saint and displayed on a digital oscilloscope (Tektronix 450S, Berthevin, France) and were included in four groups of age Tektronix, Beaverton, OR, USA) at appropriate settings to (young, adult, middle-aged, and old) according to previously measure the amplitude from baseline to the maximal nega- defined criteria.29 Briefly, young age was considered at 1– tive peak and the latency from stimulus to the maximal neg- 2 months, adult age at 4–10 months, middle age at 10– ative peak. To ensure reproducibility, the recording needles 18 months, and old age at ~19 months and older. The animals were placed under microscope to secure the same placement used in the present study had the following ages: 1 month on all animals guided by anatomical landmarks. During the (young), 4 months (adult), 14 months (middle-aged), and tests, the mice body temperature was maintained constant 24–30 months (old). To avoid potential bias due to gender, between 34°C and 36°C by means of a thermostat heating only male animals were used in this study. Mice were housed pad. (3 per cage) in the animal care facility of the Universitat de We also performed a motor unit number estimation Lleida on a 12-h light/dark cycle, having ad libitum access to (MUNE) test in the TA muscle, using the same setting as for standard laboratory chow and water. According to previous the motor nerve conduction test. The protocol used was defined criteria,30,31 mice displaying tumours, physical abnor- based on the incremental technique.34 Starting from sub- malities, and/or evidence of disease were excluded from the threshold intensity, the sciatic nerve was stimulated with sin- study and euthanized by an overdose of pentobarbital gle pulses of gradually increased intensity until the first CMAP (30 mg, intraperitoneally). response appeared, representing the first motor unit re- cruited. With the next stimuli, quantal increases in the re- sponse were recorded. Increments >50 μV were considered Motor behaviour tests as the recruitment of an additional motor unit. The amplitude of a single motor unit was calculated as the mean of more To evaluate the impact of age progression on motor skills, than 15 consistent increases. The estimated number of motor mice were periodically weighed and carefully examined. units resulted from the equation: MUNE = CMAP maximal Open-field (for assessing locomotor activity) and rotarod amplitude/mean amplitude of single motor unit action and grip strength (for assessing motor coordination and bal- potentials. ance) tests were subsequently performed every 4 weeks ac- cording to previously described guidelines.32 All tests were conducted by the same investigator. Except for open-field Tissue sample preparation, histological analysis, test, every mouse was evaluated 3 times with a 15 min recov- and motoneuron counts ery period between them; the value obtained in the best test performance was used as the final score. Open-field test was At chosen time points, mice were anaesthetized and performed by the automated recording of mouse movements transcardially perfused with 4% paraformaldehyde (PFA) in using Smart Video Tracking software (v2.5.21, Panlab Harvard 0.1 M phosphate buffer (PB), pH 7.4. After animal perfusion, Apparatus, Holliston, MA, USA); different parameters such as spinal cords, L4 DRGs and ventral nerve roots (VRs), and en- time, distance, entries in zones, and average speed were tire hindlimb skeletal muscles were rapidly dissected. Nerve measured. roots were then immersed in 1% PFA and 1% glutaraldehyde in 0.1 M PB at pH 7.4 for 24 h. Muscle samples were proc- essed for histology and further morphometry according to Electrophysiology tests previously described procedures.35 Muscles examined include the extensor digitorum longus (EDL), a fast-twitch muscle in- Nerve conduction tests were performed in adult and old volved in the dorsiflexion of the foot and extension of toes mice. For motor nerve conduction tests, the sciatic nerve during locomotion; the TA, a fast-twitch muscle, which helps was stimulated percutaneously by means of single pulses of with dorsiflexion and inversion of the foot; the soleus (Sol), a Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1631 relative slow-twitch muscle in mice, which plays an important antibody. The primary antibodies used are indicated in Table function in maintaining the standing posture and is also uti- 1. lized for plantarflexion during walking; and the gracilis (Gra), After incubation with the primary antibody, sections were a fast-twitch, more proximal muscle, located in the medial washed with phosphate-buffered saline and incubated for 1 h side of the thigh, which is mainly involved in adduction of at room temperature with the appropriate secondary fluores- the limb and also assists knee flexion.36 cent antibodies labelled with one of the following fluoro- For histology and MN counts, the spinal cords surrounded chromes (1:500): Alexa Fluor 488, Alexa Fluor 546 by their vertebrae were maintained in Bouin’s solution for (Molecular Probes, Eugene, OR, USA), Cy3, or Cy5 (Jackson 2 months and embedded in paraffin. Serial transverse sec- ImmunoResearch Laboratories, West Grove, PA, USA). The tions (14 μm thick), obtained throughout the entire lumbar spinal cord sections were finally counterstained with blue (L1–L5) segment, were stained with haematoxylin and eosin fluorescent NeuroTrace Nissl staining (1:100; Molecular (H&E). α-MNs, located in the ventral horn, were identified Probes). Muscle sections were incubated with Alexa Fluor by their size (soma diameter > 20 μm), morphology (multipo- 555-conjugated α-bungarotoxin (α-Bgtx, 1:500; Molecular lar appearance, prominent nucleolus, and abundant Nissl Probes) to identify postsynaptic acetylcholine receptors. granules in cytoplasm), and topography in Lamina IX of grey Some sections were also stained with 4′,6-diamidino-2- matter. MNs were counted blindly on one side of every phenylindole dihydrochloride (DAPI, 50 ng/mL; Molecular 10th section, according to previously described criteria.37,38 Probes) for DNA staining. To avoid lipofuscin-like autofluores- Briefly, only MNs with a large nucleus and a visible clump of cence, slides with sections of spinal cord from old mice were nuclear material and a substantial intense basophilic cyto- treated with the Autofluorescence Eliminator Reagent plasm were included in the counts. These stringent criteria (Merck, Madrid, Spain), following incubation with the second- make not necessary the use of a correction factor for double ary antibodies. Some slides with DRG were stained with fluo- counting.37 The number of cells counted was multiplied by 10 rescein isothiocyanate (FITC)-conjugated Bandeiraea to obtain the total number of MNs per ventral horn. simplicifolia lectin [isolectin B4 (IB4), 1:25; Sigma-Aldrich, St. Ventral nerve roots were postfixed in 1% osmium tetroxide Louis, MO, USA]. and embedded in Embed 812 (Electron Microscopy Sciences, After washing, slides were coverslipped using an Fort Washington, PA, USA) epoxy resin, following standard anti-fading medium containing 0.1 M Tris–HCl buffer procedures. Semithin transversal sections (1 μm thick) were (pH 8.5), 20% glycerol, 10% Mowiol, and 0.1% 1,4- stained with methylene blue and imaged using an Olympus diazabicyclo[2,2,2]octane. Slides were examined with and 60x/1.4NA PlanApo oil immersion objective (Olympus) and a Olympus BX51 epifluorescence microscope (Olympus, Ham- DMX 1200 Nikon (Tokyo, Japan) digital camera. burg, Germany) equipped with a DP30BW camera or Muscles were cleaned, cleared of the excess of connective FluoView 500 or FluoView 1000 Olympus laser scanning con- tissue, blotted dry, and weighed. After this, muscles were focal microscopes. For comparisons, slides from different an- postfixed in 4% PFA in 0.1 M PB (pH 7.4), cryoprotected with imals and experimental conditions were processed in parallel 30% sucrose in 0.1 M PB, embedded in tissue freezing me- for immunocytochemistry and subsequent imaging. The same dium (Triangle Biomedical Sciences, Durham, NC, USA), and scanning parameters were used for the acquisition of images frozen. Cryostat transverse sections (16 μm thick), obtained corresponding to different experimental groups. For spinal from the mid-belly of the muscle, were stained with H&E. cord immunocytochemical studies, digital images of the en- tire lumbar region were obtained from every 30th section. Immunocytochemistry and imaging Electron microscopy For immunocytochemical analysis, lumbar spinal cords, L4 DRGs, and skeletal muscles were postfixed by immersion in Ultrathin sections of L4 VRs embedded in Embed812 were 4% PFA in 0.1 M PB (pH 7.4), either overnight (for spinal cords obtained and counterstained with uranyl acetate and lead cit- and DRGs) or for 2 h (for muscles), and cryoprotected. Tissue rate. Sections were observed in a JEOL JEM 1400 (JEOL, To- samples were embedded in tissue freezing medium and fro- kyo, Japan) transmission electron microscope. zen. Longitudinal (16 μm thick for muscles) and transverse (16 μm thick for muscles and 14 μm thick for spinal cords and DRGs) serial cryostat sections were obtained and stored Image analysis and morphometry at 80°C. Tissue sections were sequentially rinsed in Image and morphometric examinations were performed on phosphate-buffered saline containing 0.1% Triton X-100 for blinded images by two independent investigators. Digital im- 30 min, blocked in normal goat serum or normal horse se- ages were analysed with ImageJ software (US National Insti- rum, and subsequently incubated with the chosen primary tutes of Health, Bethesda, MD, USA). Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1632 A. Blasco et al. Table 1 Primary antibodies used for immunocytochemistry Target Host species Source/catalogue no. Dilution 14-3-3 Mouse monoclonal BD Transduction Laboratories (San Jose, CA)/F46820 1:100 Agrin Mouse monoclonal Millipore (Temecula, CA)/MAB5204 1:100 Calcitonin gene-related peptide Rabbit polyclonal Sigma-Aldrich (St. Louis, MO)/C8198 1:1000 CD206 Goat polyclonal R&D Systems (Minneapolis, MN)/AF2535 1:100 Choline acetyltransferase Goat polyclonal Millipore/AB144 1:250 Fibroblast growth factor Rabbit polyclonal Bioss Antibodies, 1:80 binding protein 1 (Boston, MA)/bs-1768R Glial fibrillary acidic protein Chicken polyclonal Abcam (Cambridge, UK)/ab4674 1:1000 Growth associated Rabbit polyclonal Santa Cruz Biotechnology 1:50 protein 43 (H-100) (Dallas, TX)/sc-10786 Ionized calcium-binding Rabbit polyclonal Wako Pure Chemical Industries Ltd. 1:500 adaptor molecule 1 (Osaka, Japan)/019-19,741 Ionized calcium-binding Goat polyclonal Abcam/ab5076 1:500 adaptor molecule 1 Laminin Rabbit polyclonal Abcam/ab30320 1:1000 Mac-2 Rat monoclonal Cedarlane (Burlington, Canada)/CL8942AP 1:800 Matrix metalloproteinase-9 Goat polyclonal Sigma-Aldrich/M9570 1:10 Myosin heavy chain I Mouse monoclonal Developmental Studies Hybridoma Bank 1:5 [DSHB] (Iowa City, IA, USA)/A4-840 Myosin heavy chain IIA Mouse monoclonal DSHB/SC-71 1:20 Myosin heavy chain IIB Mouse monoclonal DSHB/BF-F3 1:20 Neurofilament 68 KDa Chicken polyclonal Abcam/ab72997 1:1000 Paired box protein 7 Mouse monoclonal R&D Systems/MAB1675 1:50 Parvalbumin Mouse monoclonal Swant (Marly, Switzerland)/PV235 1:2000 S100β Rabbit polyclonal Abcam/ab41548 1:3000 Synaptic vesicle protein 2 Mouse monoclonal DSHB/SV2 1:1000 Transforming growth factor-β1 Rabbit polyclonal Abcam/ab92486 1:100 Vesicular acetylcholine transporter Guinea pig polyclonal Synaptic Systems 1:500 (Gottingen, Germany)/#139105 Vesicular GABA transporter Guinea pig polyclonal Synaptic Systems/#131004 1:200 Vesicular glutamate transporter 1 Rabbit polyclonal Synaptic Systems/#135302 1:1000 Vesicular glutamate transporter 2 Guinea pig polyclonal Synaptic Systems/#135404 1:200 Myofiber cells counts and size measurements were per- postsynaptic site. Full or partial denervation is considered formed in a single random image per muscle taken from its when the postsynaptic site was not completely apposed by mid-belly and immunostained for laminin. Myofiber type com- the nerve terminal immunostained for both neurofilament position of different muscles was examined in transversal sec- 68 KDa (NF) and synaptic vesicle protein 2, as presynaptic tions immunostained with different isoform-specific anti- marker. Single or polyinnervation was estimated counting myosin heavy chain (I, IIA, and IIB) antibodies (for types 1, the number of preterminal axons, stained with anti-NF anti- 2A, and 2B myofibers, respectively). Images were captured body, entering a single postsynaptic site: an NMJ was defined with a digital camera (Olympus DP30BW), and the number of as polyinnervated when it was occupied by two or more types 1 (slow-twitch), 2A (fast-twitch fatigue-resistant), and axons. Terminal sprouting was quantified by counting the 2B (fast-twitch fast-fatigable) myofibers was counted and number of NF-stained axonal processes coming from a nerve expressed as a percentage of total myofiber number. The pro- terminal that escaped from an α-Bgtx-labelled postsynaptic portion of myofibers showing central nuclei was also counted site; the number of sprouts per NMJ counted was referred on images from DAPI-stained sections. The percentage of as a percentage of the total number of NMJs examined per myofibers showing lipofuscin aggregates and the number and muscle. On the basis of the morphological appearance of the the area of these aggregates were also measured using ImageJ. postsynaptic site, the degree of NMJ maturity was classified The cytoarchitecture of the NMJs was assessed in longitudi- as plaque, folds, perforations, or pretzel-like (secondary) nal immunostained sections of muscles. Z-stack optical sec- structure ranging from immature to mature, according to pre- tions (1 μm thick) were obtained with the confocal viously described criteria.39 An NMJ was defined as microscope and projected to reconstruct NMJs; maximum in- fragmented when its postsynaptic site displayed a tensity projections of stacks were created using the micro- discontinued appearance, with five or more acetylcholine re- scope software. Three to five sections were examined for ceptor (AChR) islands stained with α-Bgtx and/or unusually each muscle, in which NMJs from different randomly selected small and irregular clusters of AChR. Immunostaining for calci- visual fields were analysed. NMJ size was measured by deter- tonin gene-related peptide (CGRP), growth associated protein mining the area of manually outlined α-Bgtx-labelled 43 (GAP-43), agrin, fibroblast growth factor binding protein 1 Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1633 (FGFBP1), and transforming growth factor-β1 (TGF-β1) was es- measurement of randomly selected nerve fibres; the axon di- timated based on pixel intensity after background subtraction ameter was divided by the outer diameter of the myelin in NMJs, which postsynaptic site was visualized with α-Bgtx; sheath. At least 100 myelinated axons per mouse (4–5 mice an average of 50 NMJs per muscle was examined to assess per condition) were measured. the different morphometric parameters and the intensity of immunostaining. Any NMJ that was difficult to examine due to its location and/or orientation was excluded from the anal- Statistical analysis ysis. AChR extrasynaptic accumulations were quantified by es- timating the number of α-Bgtx labelled spots in muscle Data are reported as mean ± standard error of the mean. The sections after synaptic α-Bgtx exclusion; an average of 35 mus- statistical analysis was performed by either one-way or cle sections from 3–5 animals per condition were analysed. two-way analysis of variance followed by post hoc The analysis of central synapses, CGRP and matrix Bonferroni’s test or two-tailed Student’s t-test when only metalloproteinase-9 (MMP-9) in MNs, and glia in the ventral two different groups were compared. The level of significance horn was performed on confocal images taken from every was established at P ≤ 0.05. GraphPad Prism 6 software was 30th section of the entire lumbar spinal cord. The number used for statistical analysis and graph presentations of data. and area of vesicular acetylcholine transporter (VAChT), ve- sicular glutamate transporter 1 (VGluT1), VGluT 2, and vesic- ular GABA transporter (VGAT)-immunoreactive synaptic Results boutons on MN somata were counted and measured by de- lineating their periphery on the screen; only boutons that Reduction in the locomotor activity of mice during were in close contact with MNs displaying a large nucleus ageing and visible nucleolus were included in the counts, and num- bers were normalized to the perimeter of MN soma. Levels As expected, the body weight of animals was different with of immunoreactivity for ionized calcium-binding adaptor mol- age. A gradual increase in growth-related weight was ob- ecule (Iba1), Mac-2, CD206, glial fibrillary acidic protein served between 4 and 10 months of age (Supporting Informa- (GFAP), 14-3-3, and S100β in the spinal cord and those of tion, Figure S1A). However, the older animals (19–28 months CGRP and MMP-9 in MNs were quantified by analysing pixel of age) had significant lower average weight than the adults intensity following background subtraction. Spinal cord mi- (Supporting Information, Figure S1A). Moreover, as mice croglia morphology was quantified in confocal images from age, a gradual and significant decline in locomotor activity Iba1-immunostained sections by using an ImageJ software was detected with the open-field test, with this decline being for skeleton analysis [AnalyzeSkeleton (2D/3D) from http:// markedly prominent from 24 months onwards (Supporting imageJ.net/AnalyzeSkeleton]. Analysis was performed ac- Information, Figure S1B and C). In our hands, rotarod and grip cording to a previously described procedure.40 For this, digital strength tests were not suitable for monitoring the photomicrographs were transformed to 8-bit grayscale and age-associated progression of changes in motor skills; several then binarized to obtain a black and white image by means confounds and challenges in the interpretation of results of a formerly established threshold. Every image was manu- were found with these tests, particularly when performed ally edited to obtain an image with a continuous set of pixels in young and adult mice: a number of them fell soon after and gaps between processes belonging to neighbouring cells. they were placed on either the rod or the wire grid, or re- The image was then saved, and the plugin AnalyzeSkeleton fused the tests. This aspect is not unusual in longitudinal as- (2D/3D) was run. The accuracy of skeletonized images was sessments or repetitive tests, as those performed in the assessed, by creating an overlay of the obtained skeleton present study: after test repetitions, the animals end up with the corresponding original image. learning the innocuous consequences of falling.32 Although The number and soma area of CGRP, IB4, and parvalbumin these mice were excluded from the analysis, we think that (PV)-immunolabelled sensory neurons in the DRGs were mea- data from rotarod and grip strength tests were not reliable sured on digital images. enough to be conclusive. For this reason, we preferred to fo- The number and diameter of healthy and degenerating cus in results from open-field test. myelinated axons were determined on ×60 images taken from VR semithin cross sections stained with methylene blue. The images were joined together to obtain a whole picture of Age-related electrophysiological changes an entire nerve root transverse section; the diameter of the myelinated axons was measured by delineating the outer Data from the nerve conduction tests are detailed in Table 2. profile of the myelin sheath. Axon diameter and g-ratio mea- Compared with adult mice, old (24 months) animals exhibited surements were performed on electron micrographs of VRs. a reduction in conduction velocity, revealed by an increase in ImageJ g-ratio plugin was used to obtain semiautomated the latency time, in both motor and sensory nerves. Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1634 A. Blasco et al. Table 2 Results from motor and sensory nerve conduction tests number of apparently healthy MNs present in the whole lum- Parameter Adult (6 months, n = 10)Old (24 months, n = 8) bar spinal cord on serial transverse paraffin sections stained TA Lat (ms) 1.48 ± 0.06 1.54 ± 0.08 with H&E (Figure 1C–H). We found no significant changes in TA CMAP (mV) 54.6 ± 1.9 50.7 ± 2.3 the total number of lumbar MNs between young, adult, and SMUA (μV) 1,572 ± 39 1,240 ± 109* old mice (Figure 1J), indicating that no overt MN death oc- MUNE 42 ± 1.3 38 ± 1.7 PL Lat (ms) 1.89 ± 0.08 2.38 ± 0.17* curred during ageing. A detailed analysis of H&E-stained spi- PL CMAP (mV) 7.51 ± 0.50 5.24 ± 0.68* nal cord sections highlighted the presence of some MNs PL H wave Lat (ms)5.5 ± 0.3 6.7 ± 0.3* having an abnormal basophilic dark appearance (Figure 1F PL H/M ratio (%) 17 ± 3.4 32 ± 4.1* DgN Lat (ms) 1.27 ± 0.06 1.41 ± 0.05* and H). These ‘dark’ MNs could be clearly distinguished from DgN CNAP (μV) 31.6 ± 2.8 29.5 ± 2.5 commonly lighter stained (apparently healthy) MNs and per- Weight (g) 29.1 ± 0.6 29.5 ± 0.7 haps represent an injury-related change. Whereas dark MNs CMAP, compound muscle action potential; CNAP, compound nerve were very scarce and found occasionally in young animals, action potential; DgN, 4th digital nerve; Lat, latency at the peak of their presence increased with age, so in old animals, the pro- the wave; MUNE, motor unit number estimation; PL, plantar portion of dark MNs was significantly higher than that in interosseous muscle; SMUA, single motor unit amplitude; TA, tibialis anterior muscle. young mice (Figure 1K). In some cases, dark MNs were Statistical comparisons are made with Student’s t-test and Sidak– shrunken and displayed a deformed appearance, which Bonferroni’s * method for multiple comparisons. contrasted with other MNs that were hyperstained but main- P < 0.05 vs. adult group. tain a normal architecture. MNs located in the ventral horn of Moreover, a decline in the amplitude of the CMAP, which was lumbar spinal cord are organized in three major columns more evident in the distal PL muscle than in the more proximal (anteromedial, intermediate, and anterolateral), which are TA muscle, was observed. The CMAP amplitude decline was at- the reflection of muscular groups innervated by these MNs. tributable to a slight decrease in both the number and size of Thus, the anteromedial column MNs innervate axial muscles, motor units (MUNE and single motor unit amplitude, respec- whereas MNs located in the intermediate and anterolateral tively). Interestingly, the sensory CNAP recorded in the toes columns innervate proximal and distal hindlimb muscles, re- did not decrease significantly in old mice, suggesting that the spectively. By counting MNs in these three columns indepen- motor decline could be accounted, in part, to muscle loss dently, we observed that the number of apparently healthy and compressive forces on the foot sole occurring with age. MNs slightly, but not significantly, decreased in the anterolat- With regard to the H wave, the electrical counterpart of eral column of old mice with respect to adult animals (Figure the spinal stretch reflex, the latency of the H wave in the PL 1L). We also noticed that the number of dark MNs rose with muscle was found to be longer in old mice than in adult mice, age in the three MN columns, with this increase being more although the mean amplitude was similar in both groups. The pronounced in the intermediate column (Figure 1M). H/M ratio, indicative of the excitability of the Ia afferent syn- It has been shown that in MN diseases, spinal MNs exhibit apses on spinal MNs, was higher in old than in adult mice, differential vulnerability to degeneration.39,43,44 In this regard, likely due to compensatory mechanisms aimed to maintain MNs innervating slow-twitch muscles (slow MNs) exhibit a an adequate muscle reflex response. higher degree of resistance than those innervating fast-twitch muscles (fast MNs). MMP-9 has been reported to be selectively expressed in fast MNs and has been considered Age-related changes in spinal cord motoneurons as a determinant factor of specific vulnerable neuronal sub- populations.45 We wanted to examine whether ageing in- On the basis of contradictory data on age-associated MN loss duced changes in the degree of MN vulnerability to previously reported in humans and rodents,9,14–17,21,22,41,42 degeneration by analysing MMP-9 expression (a marker of we decided to re-examine this issue. We first analysed the vulnerability) in the spinal cord of young, adult, and old ani- morphology of lumbar spinal cord MNs in cryostat sections mals. Compared with young mice, the proportion of lumbar immunolabelled with an antibody against choline acetyltrans- spinal cord MNs showing MMP-9-positive immunolabelling ferase, as an MN marker, and counterstained with fluores- was moderately reduced in adult animals (Figure 2A). This re- cent Nissl for neuron visualization (Figure 1A1–B2). duction was accompanied by a modest, although significant, Compared with young MNs, adult MNs showed a slight (al- decrease in the intensity of MMP-9-immunostaining in adult though not significant) increase in soma size, consistent with MNs (Figure 2B). No further changes in either the number of their higher degree of maturity and the increase in the axonal MMP-9-positive MNs or MMP-9-staining intensity were no- length (Figure 1I). No significant reduction in the average ticed in old mice compared with adult mice (Figure 2A and soma size was found in old MNs with respect to adult ones B). In all the ages examined, MNs that were MMP-9 positive (adult: 536.30 ± 33.03 μm2; old: 508.42 ± 47.22 μm2; displayed a larger size than those which were MMP-9 negative P > 0.05; Figure 1I), indicating that ageing does not entail (Figure 2C); this finding is in agreement with previous reports an atrophy of spinal cord MNs. We then counted the total and fits with the statement that MMP-9 is selectively Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1635 Figure 1 No significant motoneuron (MN) loss occurs in the lumbar spinal cord of mice with ageing. (A1–B2) Representative images of MNs in the spinal cord of adult mice immunolabelled with an antibody anti-choline acetyltransferase (ChAT) (green), as an MN marker, and counterstained with fluores- cent Nissl for neuron visualization (blue) as indicated. (C–H) Representative images of haematoxylin and eosin-stained MNs in the lumbar spinal cord of (C and D) young, (E and F) adult, and (G and H) old mice. The appearance of these MNs is shown at a higher magnification in (D, F, and H); black arrows in (F) and (H) indicate dark-stained and shrunken MNs; the red arrow in (H) points to a non-shrunken, hyperchromic, MN. (I) Average size (soma area in μm2) and (J) number of apparently healthy MNs and (K) proportion of dark MNs (expressed as the percentage of MNs), in the lumbar spinal cord of young, adult, and old mice. (L) Number of healthy and (M) dark MNs in anteromedial (AM), intermediate (IN), and anterolateral (AL) columns of ventral horn. The data in graphs are shown as mean ± SEM, *P < 0.05 and **P < 0.01 (one-way or two-way ANOVA and Bonferroni’s post hoc test); 4–5 mice per age. Scale bars: (A2) = 100 μm [valid for (A1)]; (B2) = 10 μm [valid for (B1)]; (G) = 100 μm [valid for (C) and (E)]; (H) = 40 μm [valid for (D) and (F)]. expressed in fast, more larger sized, and vulnerable α-MNs.45 in old mice compared with adult animals; old MNs, however, Moreover, our results suggest that once the degree of vulner- displayed a significant increase in CGRP content compared ability is achieved in adulthood, no overt changes in MN sus- with adult MNs (Figure 2E and F). Representative immuno- ceptibility to degeneration occur with age progression. staining for MMP-9 and CGRP in young, adult and old MNs In MNs, the neuropeptide CGRP is synthesized in the cell is shown in Figure 2G1–I4. body and subsequently transported to nerve terminals where it appears to play a role in the development and maintenance of muscle cells and NMJs.46–50 As previously reported by we Ageing is accompanied by significant depletion of and others,49,51 CGRP was found in subpopulations of spinal afferent inputs to motoneurons cord MNs and, in a particular MN pool, CGRP levels differed between cells, so that some cells exhibited higher contents We analysed the density of afferent synapses on lumbar spi- of this neuropeptide than others. As expected from previous nal cord MNs of old mice compared with adult animals. The studies (Matteoli et al.52 and our own observations), com- number of synaptic boutons on MN somata was quantified pared with young mice, adult animals showed decreased after immunostaining with anti-VAChT, VGluT1 and VGluT2, numbers of CGRP-immunostained MNs, with these exhibiting and VGAT antibodies for cholinergic (C-boutons), glutamater- lower levels of immunoreactivity (Figure 2D–F). No significant gic excitatory, and GABAergic inhibitory synapses, respec- changes in the number of MNs expressing CGRP were found tively (Figure 3C1–D2, G1–H2, K1–L2, and O1–P2). We Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1636 A. Blasco et al. Figure 2 Matrix metallopeptidase 9 (MMP-9) and calcitonin gene-related peptide (CGRP) immunoreactivity in lumbar spinal cord motoneurons (MNs) of young, adult, and old mice; MMP-9 immunostaining is used as a marker of MN vulnerability. (A) Proportion of MNs displaying positive MMP-9-im- munoreactivity (IR); the average intensity of the latter is shown in (B). (C) Size (in μm2) of MN somata exhibiting either positive or negative MMP-9 immunostaining. Age-related changes in the proportion of MNs showing (D) CGRP positivity and (E) in the immunoreaction (IR) intensity in these MNs. (F) Percentage of MNs displaying both CGRP and MMP-9 immunostaining. (G1–I4) Representative images taken from spinal cord sections double immunolabelled for MMP-9 (red) and CGRP (green) and counterstained with fluorescent Nissl staining (blue; for MN visualization). Data in the graphs are expressed as the mean ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001 vs. adult (one-way ANOVA and Bonferroni post hoc test); n = 4–6 animals per age; 50 MNs per animal were analysed. Scale bar: (I4) = 60 μm [valid for (G1–I3)]. found that compared with adult MNs, old MNs exhibited a F, I, and J). No differences in the number of GABAergic synap- significant decrease in the density of both C-boutons and glu- ses were observed between old and adult MNs, although a tamatergic afferent terminals on cell bodies [~40% (VAChT), significant reduction (~20%) in the size of this type of inhibi- 45% (VGluT1), and 43% (VGluT2) decrease] (Figure 3A, B, E, tory afferents was noticed on MNs of old animals (Figure Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1637 Figure 3 Age-related changes in synaptic inputs to lumbar spinal cord motoneurons (MNs). (A–P2) Sections were immunostained to identify cholin- ergic [vesicular acetylcholine transporter (VAChT)], glutamatergic [vesicular glutamate transporter 1 (VGluT1) and vesicular glutamate transporter 2 (VGluT2)], and GABAergic [vesicular GABA transporter (VGAT)] afferent synapses (green) and counterstained with fluorescent Nissl staining (blue) to visualize MN cell bodies. Graphs show the average density [(A, E, I, and M) number of puncta per 100 μm soma perimeter and (B, F, J, and N) size (in μm2)] of the different types of afferent terminals analysed. Representative confocal micrographs of (C1–D2) VAChT, (G1–H2) VGluT1, (K1–L2) VGluT2, and (O1–P2) VGAT terminals contacting (C1–C2, G1–G2, K1–K2, and O1–O2) adult and (D1–D2, H1–H2, L1–L2, and P1–P2) old MNs are shown. Data in the graphs are expressed as the mean ± SEM, *P < 0.05, **P < 0.01, and ****P < 0.0001 vs. adult (Student’s t-test); 50–60 MNs were analysed per animal (4–6 animals per age group). Scale bar in (P2) = 20 μm [valid for (C1–D2, G1–H2, K1–L2, and O1–P1)]. 3M and N). These results indicate that ageing results in a spinal cord, microglia and astroglia were analysed after marked loss of central synapses, mainly involving cholinergic immunolabelling for Iba1 and GFAP, respectively (Figure and glutamatergic inputs to MNs. 4A–F2). Compared with adult mice, old animals exhibited sig- nificantly higher levels of both Iba1 and GFAP immunoreactiv- ity [~93% and ~100% increase (P < 0.001 and P < 0.0001), Gliosis occurs in spinal cord during ageing respectively] in the ventral horn of spinal cords (Figure 4A and D), reflecting an increased density of microglial and Activation of glial cells in the ventral horn of spinal cord has astroglial profiles. This indicates that prominent microgliosis been described to occur in different motoneuronal patholo- and astrogliosis occur around aged MNs. The degree of gies including MN diseases and peripheral nerve injury.53–57 ramification and complexity of microglia was quantified by To explore whether glial activation also occurs in the aged using a skeleton analysis procedure,40 which measures Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1638 A. Blasco et al. Figure 4 Ageing is accompanied by prominent microgliosis and astrogliosis in the spinal cord. (A–F2) Serial sections of lumbar spinal cord were immu- nostained to visualize Iba1 and GFAP, for microglia and astroglia, respectively. Quantification of (A) ionized calcium-binding adaptor molecule 1 (Iba1)- positive microglia and (D) glial fibrillary acidic protein (GFAP)-positive astroglia expressed as the percentage of the ventral horn area occupied by Iba1-positive and GFAP-positive profiles. Representative confocal micrographs showing (B1–C2) Iba1 (red) and (E1–F2) GFAP (green) staining in the ventral horn spinal cord of (B1–B2 and E1–E2) adult and (C1–C2 and F1–F2) old mice; fluorescent Nissl staining (blue) was used for motoneuron visu- alization. (G–L3) Quantification of changes in microglial morphology by using a skeleton analysis procedure (see Materials and Methods section). The average (G) branch length, (H) number of endpoints, and (I) of triple and (J) quadruple points are shown. Representative images showing (K1 and L1) Iba1-stained profiles, (K2 and L2) the skeletonized images, and (K3 and L3) the overlay of the skeleton and the original image of microglia in (K1–K3) adult and (L1–L3) old mice. Data in the graphs are expressed as the mean ± SEM; the spinal cord of four animals per age group was used for analysis; a total of 40–45 sections (in A and D) and 7,000–12,000 cells (in G–J) per age were examined. *P < 0.05, ***P < 0.001, and ****P < 0.0001 vs. adult (Student’s t-test). Scale bar: In (F2) = 30 μm [valid for (B1–C2) and (E1–F1)]; in (L1) = 20 μm [valid for (K1)]. different parameters of microglia morphology in an entire re- processes and increased number of endpoints in association gion of ventral horn (Figure 4G–L3). Compared with adult mi- with significant greater amounts of branches, as indicated croglia, those of old mice displayed reduced lengths of by the increased numbers of triple and quadruple points Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1639 quantified (Figure 4G–J). These results indicated that with VRs. Interestingly, compared with adults, there was an in- ageing, gliosis involves structural changes in microglia, which crease in the proportion of small myelinated axons in become more ramified and complex. middle-aged VRs, but this did not reach statistical significance When activated, microglia and astroglia can attain one of (Figure 6F). In middle-aged VRs, these small axons were usu- two opposing maturation phenotypes: M1 and M2 for mi- ally seen clustered, contrasting with their homogeneous dis- croglia and A1 and A2 for astroglia.53,58 Whereas M1 and tribution in adult VRs (Figure 6G, H, and I). Clustered small A1 phenotypes have been related to inflammation and de- myelinated axons could represent newly formed, still imma- generation, the M2 and A2 phenotypes have been associated ture, nerve branches, which originate by sprouting from with anti-inflammatory and regenerative processes. To exam- healthy motor axons. These results suggest that early in the ine which type of microglial and astroglial phenotype is acti- ageing process, some motor axons undergo degeneration, vated in the spinal cord as a consequence of age-related and this is accompanied by the reactive generation of new reactive gliosis, Iba1 and GFAP immunocytochemistry was nerve fibres, in an attempt to replace those previously dam- combined with either Mac-2 or CD206 (for M1 or M2 microg- aged. G-ratio analysis allowed us to assess the relationship lia) and 14-3-3 or S100β (for A1 or A2 astroglia) between axon diameter and myelin thickness. Axon diameter, immunolabelling, respectively59–62 (Figure 5A–T). Compared by delineating the periphery of axon under the myelin with spinal cords of adult mice, those of old animals exhibited sheath, was found to be significantly longer in middle-aged a prominent increase in the density of harmful M1 (Mac-2- VRs than in those of adult VRs, suggesting that a certain de- positive) profiles and a modest reduction in M2 (CD206-pos- gree of motor axon swelling occurs during ageing (Figure itive) microglia (Figure 5A–D and I1–N). Similarly, the density 6J–N). In addition, the thickness of axon myelin was signifi- of A1 (14-3-3-immunostained) astroglia was found to be sig- cantly decreased in middle-aged VRs compared with adult nificantly higher in spinal cords of old mice compared with VRs, as indicated by the higher g-ratio average values found adult mice (Figure 5E, F, and O1–Q). Although S100β- in axons (g-ratios: adult age = 0.57 ± 0.12 and middle immunoreactivity (for A2 phenotype) only showed a moder- age = 0.61 ± 0.01; n = 105–285 and 100–180 axons per ani- ate, non-significant, decrease around old MNs, the propor- mal, four and five animals, respectively, P < 0.05). As shown tion of astroglia that stained positive for both GFAP and in the scatter plot of Figure 6N, motor axons of middle-aged S100β was significantly reduced in old mice compared with mice that were larger corresponded to those with higher g- adult animals (Figure 5G–H and R1–T). Overall, these findings ratios, indicating a thinner myelin sheath. The ultrastructural indicate that age-associated reactive gliosis implies an imbal- appearance of middle-aged motor axons at different stages ance in M1/M2 and A1/A2 phenotypes, leading to a polariza- of degeneration is shown in Figure 6O–Q. tion to harmful activated M1 and A1 states. Ageing is accompanied by alterations in dorsal root Early degenerative changes in motor nerves during ganglion sensory neurons ageing To ascertain whether ageing also affects to sensory neurons, Ventral nerve roots, which are only formed by the most prox- L4 DRGs of adult and old mice were examined. DRG sections imal part of motor axons, were examined to assess their po- were processed for immunocytochemistry with antibodies tential structural alterations over the age process. We found against CGRP and PV and also stained with IB4 lectin that compared with L4 VRs of adult animals, those of (Supporting Information, Figure S2A–O2). These are specific middle-aged mice exhibited significantly higher numbers of markers for different DRG neuronal subpopulations: CGRP is myelinated axons (Figure 6A). The average axonal diameter present in peptidergic, small and medium-sized sensory noci- (including the myelin sheath) of middle-aged VRs was signifi- ceptive neurons; IB4 stains nonpeptidergic, small-sized sen- cantly greater than that of adult VRs (Figure 6B and C). In ad- sory nociceptive neurons; and PV is present in large sensory dition, there was a significant increase in the proportion of proprioceptive neurons innervating muscle spindles.64–66 degenerating axons in middle-aged VRs compared with adult Compared with the DRGs of adults, old DRGs showed a higher VRs (Figure 6D, H, and I). Middle-aged VRs had a high number proportion of CGRP-positive, IB4-positive, and PV-positive of axons whose myelin sheath structure exhibited focal dis- neurons (Supporting Information, Figure S2A, F, and K). In ruption and, often, protruded into the axonal compartment DRGs of old mice, all these neuronal populations exhibited a to ultimately form myelin spheroidal inclusions (Figure 6E, significantly smaller soma size compared with those of adult H, and I); these inclusions, having the appearance of animals (Supporting Information, Figure S2B, C, G, H, L, and myelin-like multilaminar balls, were only occasionally seen M). These data suggest that ageing promotes significant in VRs from adult mice and had a similar morphology to that changes in the expression of CGRP, IB4, and PV in sensory observed during Wallerian degeneration.63 It was not unusual DRG neurons, which are accompanied by a marked atrophy to see myelin debris engulfed by macrophages in middle-aged of both proprioceptive and nociceptive neurons. Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1640 A. Blasco et al. Figure 5 Age-related changes in the phenotype of spinal cord microglia and astroglia. (A–J) Serial sections of lumbar spinal cord of adult and old mice were double immunostained for ionized calcium-binding adaptor molecule 1 (Iba1) and either (M1) Mac-2 or (M2) CD206 microglia, and glial fibrillary acidic protein (GFAP) and either (A1) 14-3-3 or (A2) S100β astroglia. Quantification of (A and C) Mac-2-positive and CD206-positive profiles surrounding motoneurons (MNs), based on the ventral horn area occupied by these immunostained profiles; the proportion of microglial profiles expressing both Iba1 and either Mac-2 or CD206 is shown in (B) and (D), respectively. Quantification of (E and G) 14-3-3-positive and S100β-positive profiles surround- ing MNs based on the ventral horn area occupied by these immunostained profiles; the proportion of astroglial profiles expressing both GFAP and ei- ther 14-3-3 or S100β is shown in (F) and (H), respectively. Representative confocal micrographs used for data analysis showing (I1–K) Mac-2, (L1–N) CD206, (O1–Q) 14-3-3, and (R1–T) S100β (all in green); microglial and astroglial markers were combined with Iba1 and GFAP, respectively (both in red), and fluorescent Nissl staining (blue) for MN visualization. Higher magnification images of Iba1-positive microglial profiles also expressing either (K) Mac-2 or (N) CD206 and GFAP-positive astroglial profiles also expressing either (Q) 14-3-3 or (T) S100β in the ventral horn of adult spinal cords are shown. Data in the graphs are expressed as the mean ± SEM; the spinal cord of four animals per age group was used for analysis; a total of 40–45 sections per age were examined. ***P < 0.001 and ****P < 0.0001 vs. adult (Student’s t-test). Scale bars: In (S) = 50 μm [valid for (I1–J4), (L1–M4), (O1–P4), and (R1–S4)] and in (T) = 10 μm [valid for (K, N, and Q)]. Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1641 Figure 6 Age-related changes in motor axons of mice. The analysis was performed in L4 ventral roots (VRs) of adult and middle-aged (middle) animals. (A) Number and (B) diameter of VR myelinated axons. (C) Relative frequency of myelinated axon diameter (in μm); note in adult VRs the bimodal pro- file indicative of axons originating from α-motoneurons (large axons) and γ-motoneurons (small axons). Proportion of axons showing a (D) degenerating appearance, (E) some myelin inclusions, and (F) a small (≤6 μm) diameter; the number of small axon clusters (≥3 axons) in adult and middle-aged VRs are shown in (G). Representative images of methylene blue-stained semithin cross sections of (H) adult and (I) middle-aged VRs used for axon analysis; in (I), arrows indicate degenerating axons (red), a myelin inclusion inside an axon (green), and clusters of small axons (orange). Elec- tron micrographs of (J) adult and (K) middle-aged VRs; note in (K) the distended appearance of some axons and the subtle changes in myelin structure seen in middle-aged motor axons; arrow in (K) points out a degenerating axon. (L) Diameter (in μm) of motor axons, showing a significant increase in axon size of middle-aged animals; the measurements were performed by delineating the axon periphery underneath the myelin sheath. Quantification of myelin thickness by (M) g-ratio analysis and (N) scatter plot depicting (N) g-ratios in relation to axon diameter in VRs; all the measurements in (L–N) were performed on electron micrographs taken from ultrathin cross sections of VRs at comparable levels. Representative electron micrographs of middle-aged VRs showing in (O) a focal disorganization of myelin sheath resulting in two multilamellar myelin balls located inside a motor axon and in (P) and (Q) motor axons in different stages of degeneration. Data in the graphs are expressed as the mean ± SEM, *P < 0.05 and **P < 0.01 vs. adult; n = 4 adult and 5 middle-aged VRs of different animals. Scale bars: In (I) = 25 μm [valid for (H)], (K) = 5 μm [valid for (J)], and (Q) = 5 μm [valid for (O and P)]. Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1642 A. Blasco et al. Structural and molecular changes in of GAP-43. In our study, adult muscles showed very low levels neuromuscular junctions of hindlimb muscles of GAP-43 compared with their young counterparts (Figure during ageing 9B1–B3 and C1–C3). The levels of this protein, however, were significantly increased in the NMJs of old muscles (Figure 9A The morphology and innervation of NMJs in TA, Sol, EDL, and and D1–D3). Gra muscles were examined following double immunostain- Agrin is a protein synthesized in the MN cell body and ing for NF and synaptic vesicle protein 2 (for visualization of axonally transported to the nerve terminal where once re- presynaptic nerve terminals) combined with α-Bgtx (for iden- leased concentrates at the synaptic basal lamina to play a cru- tification of postsynaptic sites). In adult muscles, both presyn- cial role in the process of NMJ formation and maintenance.73– 76 aptic and postsynaptic elements displayed a well-organized We analysed then whether agrin content in NMJs was al- appearance, with smooth and continuous nerve terminals en- tered in old muscles and found that NMJs of young and adult tering in endplates, the latter showing the ‘pretzel-like’ pat- muscles displayed low levels of agrin (Figure 9F1–F3 and G1– tern characteristic of mature, ‘healthy’ NMJs; postsynaptic G3). In comparison, NMJs of all muscles examined from old membranes exhibited numerous folds where α-Bgtx-labelled mice, with the exception of Gra, exhibited a significant in- AChRs were concentrated. In contrast, NMJs of old muscles crease in agrin expression (Figure 9E and H1–H3). displayed structural alterations, which are consistent with We next examined the expression levels of FGFBP1 and previous reports11,22,67–69: whereas some NMJs appeared TGF-β1 in muscles of adult and old mice. FGFBP1 is a secreted partially or fully denervated (Figure 7A and F1–H2), a high protein that binds fibroblast growth factor proteins present at proportion of innervated NMJs had overt signs of the extracellular matrix enhancing its biological activity.77 polyinnervation (Figure 7B, I1, and I2); moreover, many nerve FGFBP1 has been reported to be transcribed in muscle fibres, terminals displayed a more complex branching and sprouting where it appears to have a role in regenerating injured NMJs.78 (Figure 7E, J1, and J2). Motor endplates of old muscles were On the other hand, TGF-β1 has been shown to regulate also altered, showing increased size and fragmented appear- FGFBP1 expression in cultured myotubes.79 We found that ance (Figure 7C and D). Endplate denervation was frequently all the examined muscles from young mice expressed both associated with many small spots of clustered AChRs FGFBP1 and TGF-β1. Although a diffuse and discrete immuno- scattered along the myofibers (Figure 7K–N). These spots fluorescence was seen along the muscle fibres, both proteins concomitantly displayed a faint positive signal for presynaptic were mainly concentrated at the NMJs (not shown). However, markers, indicative of a reactive neoformation of neuromus- the expression levels of FGFBP1 and TGF-β1 changed with age. cular synaptic contacts (Figure 7O and P1–P4). We observed Muscles of adult mice exhibited lower content of both pro- some variations in these changes between muscle types stud- teins compared with that of young animals. Moreover, com- ied: in old mice, the proportion of denervated NMJs was pared with adult muscles, old muscles displayed an increased much lower in TA muscles than other muscle types (Figure expression of FGFBP1 and TGF-β1, showing a pattern of immu- 7A); moreover, TA muscles showed more polyinnervated noreactivity mostly restricted to the synaptic region of muscle NMJs than Sol and Gra muscles, with no obvious signs of (Figure 10A, B, and C1–F3). Although both proteins were polyinnervation in EDL muscles of old mice (Figure 7D). mainly detected within the postsynaptic α-Bgtx-stained area, some immunolabelling was also seen outside of this region, We and others have previously reported that during NMJ suggesting that FGFBP1 and TGF-β1 are in part located in the development, motor nerve terminals initially contain high extracellular matrix around the NMJ. levels of CGRP, which markedly decreases in mature NMJs; nevertheless, CGRP is up-regulated in NMJs during conditions of reinnervation and nerve sprouting.47,50,52,70 Consistent Age-related changes in hindlimb skeletal muscles with this, levels of CGRP in NMJs was very low in all adult muscles examined compared with their young counterparts Aged-related changes in skeletal muscles have been reported (Figure 8C1–C3 and B1–B3). Conversely, a significant increase in some murine models at histological and electrophysiologi- in CGRP immunoreactivity was seen in NMJs of old muscles, cal levels68,80–84 (Hunter et al.85 and McKinnon et al.,86 as re- this augment being particularly prominent in the Gra muscle views). We examined the degree of histopathological (Figure 8A and F1–G3). alterations in different mouse muscles to assess the extent GAP-43 is a protein present in virtually all neurons during by which muscular changes could account for the impaired axonal growth, being particularly abundant in axonal growth motor performance observed in old animals. Because muscle cones (reviewed in Holahan71). It has been found that in denervation is typically associated with muscle atrophy, we the neuromuscular system, GAP-43 is down-regulated in determined the wet weight, as an indicator of muscle mass, MNs at the onset of synapse elimination in a process that is and the cross-sectional area of muscles. Wet weights of adult regulated by muscle activity.72 Consequently, mature MNs and old muscles did not exhibit significant differences, al- and their corresponding nerve terminals exhibit low levels though some reductions in the weight of Sol and EDL muscles Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1643 Figure 7 Age-related changes in neuromuscular junctions (NMJs). Proportion of (A) denervated and of (B) monoinnervated and polyinnervated NMJs in muscles of old mice. Changes in (C) NMJ size and (D) number of fragmented endplates in old muscles. All graphs show values as a percentage of change compared with adult muscles; 50–100 NMJs per muscle from four adult and five old mice were analysed. (E) Percentage of NMJs exhibiting terminal axonal sprouts in the muscles of adult and old mice; each point represents the percentage of sprouts found in 25–30 NMJs per muscle from four adult and five old mice. Data in all graphs are expressed as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. adult (one- way ANOVA and post hoc Bonferroni’s test). Maximal projections of confocal stacks of NMJs from (F1 and F2) adult and (H1–J2) old muscles, as indi- cated in panels. Muscle sections were stained with antibodies against neurofilament 68 KDa (NF) and synaptic vesicle protein 2 (SV2) (green) for pre- synaptic nerve terminals and α-bungarotoxin (α-Bgtx) (red) for postsynaptic acetylcholine receptor (AChR). Microphotographs in (G1–J2) correspond to representative old NMJs displaying different structural changes: (G1 and G2) partial innervation, (H1 and H2) denervation, (I1and I2, arrows) polyinnervation, and (J1 and J2, arrow) terminal sprouting, most of these showing signs of endplate fragmentation; compare with the typical appear- ance of an innervated adult NMJ in (F1 and F2). Representative images of α-Bgtx-stained (red) endplates in the (K and L) tibialis anterior (TA) and (M and N) soleus (Sol) muscles from adult and old mice; arrows in (L and N) point to patches of AChRs scattered along the myofibers. (O) Density (number per 1000 μm2) of extrasynaptic α-Bgtx-stained (AChR) spots in fibres of old muscles. (P1–P3) Representative images of the Sol muscle double immunolabelled for VAChT (green) and NF (blue) and stained with α-Bgtx (red); arrows point to (P3) α-Bgtx-stained AChR spots that exhibit both (P1) VAChT and (P2) NF positivity; the merged image for the three channels is shown in (P4). Scale bars: In (J2) = 20 μm [valid for (F1–J1)]; in (N) = 20 μm [valid for (K–M)]; and in (P4) = 30 μm [valid for (P1–P3)]. EDL, extensor digitorum longus; Gra, gracilis. Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1644 A. Blasco et al. Figure 8 Ageing is accompanied by an increased expression of calcitonin gene-related peptide (CGRP) in neuromuscular junctions (NMJs) of hindlimb muscles. (A) Changes in CGRP immunoreactivity (IR) in NMJs of old tibialis anterior (TA), soleus (Sol), extensor digitorum longus (EDL), and gracilis (Gra) muscles expressed as the percentage of increase compared with respective adult muscles. (B1–D3) Representative confocal micrographs of CGRP-immunostained (green) NMJs of Sol muscles from young, adult, and old mice; sections were also stained with α-bungarotoxin (α-Bgtx) (red) for endplate identification. Data in the graphs are expressed as the mean ± SEM, ***P < 0.001 and ****P < 0.0001 vs. adult (one-way ANOVA and post hoc Bonferroni’s test); one muscle for each muscle type taken from four adult and five old mice was used for analysis; 80–100 NMJs per muscle type were examined. Scale bar in (D3) = 10 μm [valid for (B1–D2)]. were seen in old animals compared with adults (muscle differential decline in the proportion of myofibers occurs in weight reduction: ~23% Sol and ~43% EDL) (Figure 11A). No distinct type of muscles with ageing. The effect of ageing on significant differences were obtained when muscle weights myofiber size was analysed in muscle cross sections were normalized to body weight (muscle mass/body weight) immunolabelled for laminin and showed no significant differ- (Figure 11B). Gra muscles from old mice exhibited even ences in the average myofiber size between adult and old an- higher (although not significant) normalized weights com- imals for all the muscles examined (Figure 11E). Nevertheless, pared with those from adults. Additionally, compared with compared with adult muscles, increased contents of connec- adult muscles, old muscles did not exhibit significant changes tive tissue were found in all muscles from old animals, this in- in cross-sectional areas (Figure 11C). We next analysed the crease being more marked in the EDL (Figure 11F). density of myofibers and found a reduction in the Sol and In agreement with previous reports,67 all the old hindlimb EDL muscles of old animals, with the most prominent de- muscles analysed exhibited high proportions of myofibers crease observed in the EDL muscle (myofiber density reduc- showing centrally located nuclei (Figure 11G–O). In young tion: ~24% Sol and ~37% EDL). This indicates that a muscles, only a small percentage (0.5–1.5%, depending on Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1645 Figure 9 Age-related changes in growth associated protein 43 (GAP-43) and agrin expression in in neuromuscular junctions (NMJs) of hindlimb mus- cles. Changes in (A) GAP-43-immunoreactivity (IR) and (E) agrin-IR (in arbitrary units [a.u.]) in NMJs of old tibialis anterior (TA), soleus (Sol), extensor digitorum longus (EDL), and gracilis (Gra) muscles expressed as the percentage of increase compared with adult respective muscles. Representative confocal images of NMJs of TA and Gra muscles from young, adult, and old mice immunostained for (B1–D3) GAP-43 and (F1–H3) agrin, both in green; muscle sections were also stained with α-bungarotoxin (α-Bgtx) (red) for endplate identification. Data in the graphs are expressed as the mean ± SEM, * P < 0.05; ***P < 0.001, and ****P < 0.0001 vs. adult muscles (one-way ANOVA and post hoc Bonferroni’s test); one muscle for each muscle type taken from four adult and five old mice was used for analysis; 80–100 NMJs per muscle type were examined. Scale bars: In (D3) = 10 μm [valid for (B1–D2)] and in (H3) = 10 μm [valid for (F1–H2)]. the muscle) of myofibers displayed central nuclei. These num- From studies mainly performed in rats, it has been docu- bers significantly increased in old muscles, this increase being mented that ageing is associated with changes in the fibre type more prominent in the TA and Gra muscles (~12% and 18%, profile of skeletal muscles. Thus, a transition from fast (type 2) respectively) (Figure 11G). Because the presence of central to slow (type 1) fibres, leading to a preponderance of the lat- nuclei in a myofiber can be considered as an indicator of its ter, appears to occur in aged muscles.91,92 We analysed the fi- regeneration following degeneration,87,88 these results sug- bre type (1, 2A, and 2B) composition of selected muscles by gest that over the ageing process, all limb muscles examined using different isoform-specific anti-myosin heavy chain (I, undergo, to some extent, fibre degeneration and attempt to IIA, and IIB ) antibodies. As previously reported,68,93 in adult regenerate these lost fibres. mice, the Sol muscle was seen to be mainly composed of Satellite cells (SCs) are muscle-resident stem cells required slower fibres (types 1 and 2A), whereas EDL and TA muscles for sustained muscle regeneration.89,90 SCs express paired showed a predominance of fast fibres (type 2B > 2A in EDL box protein 7 (Pax7), a transcription factor involved in the pro- and type 2A > 2B in TA); the Gra, which is the more proximal liferation of muscle precursor cells. To analyse age-related hindlimb muscle, also consisted predominantly of types 2A changes in SCs, immunocytochemistry for Pax7 was combined and 2B fibres (2A > 2B), with a lower proportion of type 1 fi- with DAPI staining in sections of adult and old mouse muscles. bres. These myofiber type profiles exhibited differential Compared with adult muscles, decreased numbers of changes with ageing. Compared with adult muscles, all old dis- Pax7-positive SCs were observed in all examined muscles from tal hindlimb muscles analysed (TA, EDL, and Sol) displayed a old animals, although SC depletion only attained statistical sig- significant reduction in the proportion of type 2B fibres, nificance in Sol and EDL muscles (Figure 11P–S3). whereas the amount of type 2A fibres was found significantly Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1646 A. Blasco et al. Figure 10 Age-related changes in fibroblast growth factor binding protein 1 (FGFBP1) and transforming growth factor-β1 (TGF-β1) expression in neu- romuscular junctions (NMJs) of hindlimb muscles. Quantification of changes in (A) FGFBP1-immunoreactivity (IR) and (B) TGF-β1-IR (in arbitrary units [a.u.]) in NMJs of tibialis anterior (TA), soleus (Sol), extensor digitorum longus (EDL), and gracilis (Gra) muscles of old mice; changes are expressed as the percentage of immunostaining increase compared with adult NMJs. Representative confocal micrographs of NMJs of Sol muscles from adult and old mice immunostained for (C1–D3) FGFBP1 and (E1–F3) TGF-β1, both in green, as indicated; muscle sections were also stained with α-bungarotoxin (α-Bgtx) (red) for endplate identification. Data in the graphs are expressed as the mean ± SEM, *P < 0.05, **P < 0.01, and ****P < 0.0001 vs. adult muscles (one-way ANOVA and post hoc Bonferroni’s test); one muscle for each muscle type taken from four adult and five old mice was used for anal- ysis; 80–100 NMJs per muscle type were examined. Scale bar in (F3) = 20 μm [valid for (C1–F2)]. decreased in the TA and EDL but dramatically increased in the structures.98 In our study, in comparison with muscles from Sol. Additionally, all these old muscles showed a reduced pro- adult animals in which no lipofuscin aggregates were seen, portion of type 1 myofibers with respect to adult muscles, but all the examined muscles from old mice exhibited prominent only in the case of the EDL, this decline attained significance lipofuscin accumulation (Figure 12N). This was especially (Figure 12A–M). Conversely, compared with adult mice, the prominent in the Gra muscle, compared with the Gra muscle of old animals exhibited a prominent increase in slow-twitch Sol muscle showing the smallest amount of the proportion of types 1 and 2B fibres and a reduction in lipofuscin aggregates. Within the muscle, the proportion of the content of type 2A fibres (Figure 12A–M). These findings lipofuscin granules varied considerably between myofibers, contrast with those obtained in previous studies performed and some of them appeared completely devoid of this pig- in rat skeletal muscles, in which it has been reported that age- ment (Figure 12O and P). Interestingly, in all muscles, the ing results in a switch from type 2A to type 1 fibres in the slow largest fibres were those which exhibited the highest levels Sol muscle and from type 2B to type 2X (a type of fibre having a of lipofuscin accumulation (Figure 12Q). The correlation be- resistance to fatigue intermediate between type 2A and type tween fibre type and accumulation of lipofuscin was explored 2B fibres) in fast muscles.94,95 in the old TA muscle. We observed that type 1 fibres exhib- Lipofuscin, often called ‘age pigment’, consists of polymor- ited a significantly lower content of the pigment compared phous and non-degradable autophagocytosed material that, with types 2A and 2B fibres (Figure 12R). coming from different intracellular structures, accumulates in lysosomes. The presence of lipofuscin in a cell is considered the result from a oxidative stress process, in which oxidized Discussion compounds, mainly of lipid and protein origin, are resistant to hydrolysis by lysosomal enzymes.96,97 Lipofuscin has a Despite the notable progresses made in the knowledge of the broad autofluorescence and, when excited by lights of di- etiological factors leading to ageing sarcopenia, the precise verse wavelengths in combination with proper barrier filters, causative mechanisms are far from fully understood. The in- is easy to distinguish from immunolabelled fluorescent tricate interactions between the distinct types of cells and Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1647 Figure 11 Effect of ageing on the histology of tibialis anterior (TA), soleus (Sol), extensor digitorum longus (EDL), and gracilis (Gra) muscles. (A) Wet weight of muscles (in mg) and (B) weight of muscles relative to body weight (expressed in mg/g). (C) Changes in the total cross-sectional area of old muscles (expressed as the percentage with respect to adult muscles). (D) Density of fibres in muscles (expressed as the number of myofibers in 1000 μm2 muscle cross-sectional area). (E) Average myofiber size (cross-sectional area in μm2) of the muscles. (F) Changes in the content of connective tissue in old muscles (expressed as the percentage with respect to adult muscles). (G) Proportion of myofibers exhibiting central nuclei. (H–O) Repre- sentative images of transversal muscle sections, double labelled with an antibody against laminin (lam) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for DNA. (P) Percentage of paired box protein 7 (Pax7)-immunostained cells (satellite cells) with respect to DAPI-positive nuclei in adult and old muscles. Representative confocal images of Pax7 immunostaining (red) combined with immunolabelling for laminin (green) and DAPI staining (blue) in (Q1–Q3) adult and (R1–R3 and S1–S3) old EDL muscle sections. A higher magnification view of Pax7 immunostaining in old EDL muscle is shown in (S1–S3). Data in the graphs are expressed as the mean ± SEM, *P < 0.05, **P < 0.01, and ***P < 0.001 vs. adult; n = 5–6 muscles for muscle type from different animals and age; number of fibres examined per muscle and mouse: (D and E) = 200, (G) = 3,000, and (P) = 100. Scale bar in (O) = 50 μm [valid for (H–N)]; (R3) = 40 μm [valid for (Q1–R2)]; and (S3) = 10 μm [valid for (R1–R2)]. tissues shaping the neuromuscular system are complex and performed in murine models has been restricted to specific hard to interpret in isolation. Indeed, results reported in the constituents of the neuromuscular system, providing a partial literature are sometimes ambiguous, with strong divergences view of cellular and molecular alterations leading to in results obtained from different ageing models.99 Rodents, sarcopenia. In an attempt to unify and clarify discrepant pub- mainly various rat and mouse strains, have been widely used lished data, we conducted a detailed analysis of in studies of sarcopenia. Although it is unlikely that the ageing ageing-related structural and functional alterations in the neu- processes in these animal models are entirely comparable with romuscular system of the C57BL/6JRj mouse, a those in humans, rodents provide an experimental paradigm well-established model for sarcopenia. Here, all the main cellu- that allow us to overcome the obvious problems inherent to lar components were simultaneously examined in each individ- human long lifespan, environmental and nutrition conditions, ual animal at specific time points. Additionally, changes in some and tissue sampling.99 However, most of the ageing research key molecules with well-established roles in the development Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1648 A. Blasco et al. Figure 12 Age-related changes in fibre type composition and lipofuscin accumulation. (A) Changes in myofiber type composition in the old tibialis an- terior (TA), soleus (Sol), extensor digitorum longus (EDL), and gracilis (Gra) muscles expressed as the proportion of types 1, 2A, and 2B fibres compared with respective adult muscles (*P < 0.05 and **P < 0.01 vs. respective adult muscles; Student’s t-test). Representative images of (B–G) TA and (H–M) Gra muscles from (B–D and H–J) adult and (E–G and K–M) old mice, showing myosin heavy chain I, IIA, and IIB immunostaining to demonstrate types 1, 2A, and 2B myofibers, as indicated. (N) Percentage of fibres containing lipofuscin granules in muscles from adult and old mice. (O) Percentage of old TA muscle fibres with central or peripherally located nuclei containing lipofuscin aggregates. (P) Average size (cross-sectional area in μm2) of myofibers with or without lipofuscin granules. (Q) Percentage of types 1, 2A, and 2B myofibers containing lipofuscin in the TA muscle of old mice. (R and S) Rep- resentative fluorescent images of EDL and Gra muscle sections processed for laminin immunostaining (green); lipofuscin autofluorescence was excited using 510–560 nm excitation and 590 nm emission filters; note that fibres with a small size are devoid of lipofuscin aggregates (arrows). Data in graphs are shown as the mean ± SEM of fibres analysed from four adult and five old muscles of different animals (number of examined myofibers per muscle: (N and O) = 400–700; (P and Q) = 300–500 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; one-way or two-way ANOVA and post hoc Bonferroni’s test). Scale bars: (M) = 50 μm [valid for (B–L)] and (S) = 100 μm [valid for (R)]. and maintenance of neuromuscular innervation were also ex- degeneration at advanced ages.21,22,100 The disparity in all amined. A summary of the main results of this study is depicted these results could be ascribed to differences in the animal in Figure 13. model species and age of animals included in the studies, seg- We reported that ageing is not accompanied by significant ments of the spinal cord analysed, and the procedures uti- MN death in the lumbar spinal cord of mice. Although the lized for MN counting. Thus, most of the mentioned studies presence of a certain degree of MN degeneration cannot be have been performed in different rat strains and, in some excluded, our results suggest that MN loss should be consid- cases, without gender segregation. In the present analysis, ered as a minor event in the context of the neuromuscular we only used male mice classified into three age groups, with changes occurring during ageing. This finding is in contrast a strict age delimitation. Moreover, we counted the total with results from previous works in rodents9,15–17 and number of MNs in serial sections of the entire lumbar spinal humans,14,41 in which significant MN death has been re- cord by using a standard and reliable procedure widely ap- ported with ageing. Our results, however, are consistent with plied in the literature (see Clark and Oppenheim37 and also data from studies that have shown the absence of MN Calderó et al.38,101), which allows an accurate estimation of Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1649 Figure 13 Overview of age-associated changes in the mouse neuromuscular system. (A1–D1) The structural organization of the different components of the system, including spinal cord motoneurons (MNs), neuromuscular junctions (NMJs), skeletal muscles, and dorsal root ganglion (DRG) sensory neurons, is schematically represented. MNs, located in the ventral horn, project their axons throughout the ventral roots; motor nerve terminals es- tablish synaptic contacts with skeletal muscle fibres (NMJs). (B1) In normal conditions, adult spinal MNs receive abundant synaptic inputs (synaptic boutons), which can be identified by means of vesicular acetylcholine transporter (VAChT) (excitatory cholinergic), vesicular glutamate transporter 1 (VGluT1) and vesicular glutamate transporter 2 (VGluT2) (excitatory glutamatergic), and vesicular GABA transporter VGAT (inhibitory GABAergic) markers. (A1) Glutamatergic (VGluT1-positive) afferents to MNs come from proprioceptive (parvalbumin-positive) sensory neurons located in the DRG; cholinergic afferents (C-boutons) to MNs come from a small cluster of cholinergic interneurons (V0C), which are located near the central canal of spinal cord. (B1) Spinal MNs are surrounded by abundant microglial and astroglial cells, which are in a resting state. The vast majority of MNs show a ‘healthy’, lightly haematoxylin and eosin-stained appearance; intermixed with them, some abnormally dark MNs can be occasionally seen in adult spi- nal cords; these dark MNs could account for the presence of some degenerating motor axons that can be already observed in ventral roots of adult mice. In adult muscles, both presynaptic and postsynaptic elements of NMJs exhibit a well-organized and ‘healthy’ appearance: smooth nerve termi- nals enter in endplates displaying a ‘pretzel-like’ pattern; (C1) adult NMJs exhibited low levels of agrin, growth associated protein 43 (GAP-43), calci- tonin gene-related peptide (CGRP), fibroblast growth factor binding protein 1 (FGFBP1), and transforming growth factor-β1 (TGFβ1). (D1) Diagram of adult myofibers surrounded by connective tissue as can be seen in a transverse muscle section. (A2–D2) In the course of ageing, there is (i) an increase in the number of dark MNs; (ii) a reactive gliosis in ventral horn with a raise in the proportion of harmful M1 microglia and A1 astroglia; (iii) a marked loss of excitatory (cholinergic and glutamatergic) synaptic afferents and reduction in the size of GABAergic synaptic boutons on MNs; (iv) an increase in the proportion of degenerated motor axons and the existence of signs of nerve sprouting and regeneration; and (A2 and B2) (v) an atrophy of sensory proprioceptive and nociceptive DRG neurons and increased expression of their markers. (C2) Muscles of old mice exhibit signs of either polyinnervation or denervation and fragmentation of endplates; these changes are accompanied by the increased expression of agrin, GAP-43, CGRP, FGFBP1, and TGF- β1, suggesting and active process of NMJ remodelling and muscle reinnervation. Moreover, old muscles show numerous fibres with lipofuscin accu- mulation and centrally positioned nuclei, indicative of muscle regeneration, and fibrotic tissue deposition between fibres; (D2) the proportion of sat- ellite cells were also markedly reduced in old muscles. Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1650 A. Blasco et al. neurons that die. This systematic approach is not often axons located in VRs of adult animals displayed signs of de- employed in other studies. Although we did not find a signif- generation and myelin inclusions. These alterations markedly icant reduction in the total number of MNs in old mice com- increased in VRs of middle-aged mice, in which numerous pared with that in adult animals, we noticed a modest myelin spheroidal inclusions and Wallerian-like degenerative decrease in the number of MNs located in the anterolateral changes are often seen. Interestingly, middle-aged column, without changes in the anteromedial and intermedi- (14 months) VRs exhibited increased numbers of clusters of ate columns. Because MNs from the anterolateral column in- small myelinated axons, which likely are new nerve sprouts nervate distal hindlimb muscles, this finding is consistent with originated from healthy axons. Overall these findings suggest the more prominent age-related involutive changes occurring that sustained cycles of degeneration/regeneration and in these muscles. sprouting from healthy axons occur, to greater or lesser de- Additionally, it is important to note the presence of MNs grees, throughout the lifespan. We assume that these with an altered histological appearance (dark MNs) scattered degeneration/regeneration cycles are exacerbated in older across the lumbar spinal cord, particularly in the intermediate mice, in an attempt to compensate for the loss of motor ter- column. Dark neurons have been described in healthy and minal branches and synaptic sites in muscle. pathological brains of different animal models,102 but it re- Although MN death does not appear to be a relevant path- mains unclear whether they are the result from a pathological ological process over the course of ageing, we cannot rule out change or merely represent an artefact. In this regard, an in- the possibility that MNs with a normal histological appear- appropriate tissue specimen fixation, particularly the time in- ance may have impaired function. In fact, we provide here tervals between collecting samples and fixation, appears to strong evidence that MNs of old animals suffer a prominent provoke an increase in the proportion of dark cells in the ner- loss of excitatory cholinergic and glutamatergic inputs, which vous tissue.103 Although we cannot completely exclude that is in agreement with recent reports.100,106 Although we no- dark MNs are a consequence of tissue fixation defect, this ticed no age-associated changes in the density of inhibitory possibility appears unlikely because all our animals were in- GABAergic synapses, their size on old MNs was found signifi- tracardially perfused and the spinal cords were rapidly re- cantly reduced. MN deafferentation has also been widely re- moved and subsequently postfixed. The fact that dark MNs ported by us and others in murine models of MN diseases, might reflect damaged cells is supported by the presence of such as spinal muscular atrophy (SMA)107–111 and amyotro- this type of neurons in the brain cortex and hippocampus fol- phic lateral sclerosis (ALS),109,112–115 and also following nerve lowing traumatic brain injury or focal cerebral ischemia.104,105 injury.116,117 Data from studies performed in these paradigms Nevertheless, the fate of dark neurons is not still clear. Al- support the idea that the loss of synaptic inputs is a process though it has been generally assumed that dark neurons are that precedes MN degeneration and that a significant degree ‘sick’ cells prone to die and would be subsequently elimi- of deafferentation is not necessarily associated to MN nated, it is presently proposed that a number of them un- death.107 This assumption is in agreement with the absence dergo reactive mechanisms for cell death prevention. of MN loss we found in the present work. It is reasonable Whether or not surviving neurons can conduct their appropri- to assume that alterations in central synapses may negatively ate functions remains to be elucidated.102,104 In our analysis, impact on MN integrity and function. It is well known that the proportion of dark MNs, although increased with age, did MN excitability is finely regulated by afferent synaptic inputs, not significantly change in old animals compared with adult which mediate the generation of co-ordinated firing patterns mice. Even considering that a certain number of dark MNs leading to muscle contractions.118 Because the accurate con- undergo degeneration, only a minor proportion of them trol of MN excitability is critical for a proper motor behaviour, would finally die. Indeed, we found that the number of it is obvious that the latter can be markedly impaired by MN ‘healthy’, lighter stained, MNs did not exhibit a significant de- deafferentation. Thus, overall, our results indicate that MN cline in the course of ageing. On the other hand, we noticed dysfunction, rather than cell death, could explain the changes no significant reduction in the proportion of MMP-9 MNs in in motor skills and in electromyography recordings seen in spinal cord of old animals, suggesting that vulnerable fast our study. MNs do not necessarily die during the ageing process. An interesting finding in the present study is the prominent The presence of dark, presumably sick, MNs in the spinal microgliosis and astrogliosis found around aged MNs. This re- cord of adult mice fits well with the pattern of changes we sult indicates that ageing is accompanied by a observed in VRs. Previous work performed in young (2– neuroinflammatory response in spinal cord ventral horn, as 4 months) and old (22–25 months) mice has shown that in it has been documented in different brain regions in healthy the latter, VR motor axons displayed alterations in diameter elderly (reviewed in previous studies119–121). Ageing-related ratios and myelin, indicative of age-associated axonal degen- chronic reactive gliosis has been associated to an increased eration82; in this study, however, no significant decline in the vulnerability of aged brain to neurodegenerative diseases. In- number of motor axons has been found with ageing. Here, deed, neuroinflammation has been reported to occur in a we noticed that although in a low proportion, some motor number of neurodegenerative processes, including the MN Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1651 disorders ALS and SMA.56,107,108,110,122–124 The role of neuro- spinal cord is a primary event leading to the loss of normal inflammation in chronic neurodegeneration, and particularly functional synaptic inputs to MNs with a subsequent neuro- in MN diseases, has still to be clarified. It is now accepted that nal damage or, conversely, is a secondary response to an al- glial reaction in these pathologies may have either detrimen- tered synaptic function,129 as appears to occur in damaged tal (cytotoxic) or beneficial (neuroprotective) effects on MNs after peripheral nerve transection.57 MNs.124 In this regard, depending on the type of stressor trig- In this context, we found that ageing-related spinal cord gering the gliosis process, both microglia and astroglia can central synapse loss is accompanied by changes in DRG sen- achieve M1 and A1 (pro-inflammatory) or M2 and A2 (anti-in- sory neurons. We noticed that with ageing, nociceptive flammatory) reactive functional phenotypes, respec- (CGRP-positive and IB4-positive) and proprioceptive (PV-pos- tively.53,58 Moreover, M1 and A1 reactive cells can turn into itive) DRG neurons exhibited significant changes in their num- M2 and A2 phenotypes, respectively, and vice versa, accord- bers and expression levels of their markers and atrophy of ing to the environment in which they are activated. In fact, cell soma. Sensory neurons showed increased expression of M1–A1 and M2–A2 phenotypes represent the extremes of a CGRP and PV and higher staining with IB4. The increased pro- continuum range of distinct reactive sates. The dynamic and portion of DRG PV-positive neurons differs from previous bidirectional transformation of M1/M2 and A1/A2 pheno- data by Vaughan et al.130 showing no significant changes in types seems to play a critical role in neurodegeneration, in the numbers of DRG PV-positive neurons between which neuronal damage has been related to a sustained acti- middle-aged (11- to 13-month-old) and old (15- to 21- vation of M1 and A1 cells resulting in an imbalance between month-old) mice. Differences between both studies could the two microglial (M1/M2) and astroglial (A1/A2) subpopu- be related to mouse strains used because genetically modi- lations. Additionally, activated M1 microglia can induce neu- fied mice were utilized in Vaughan’s work. In our study, how- rotoxic A1 astrocytes, which have been found to be ever, neuron atrophy in old DRGs was particularly evident in abundant in diverse human neurodegenerative diseases,61 in- proprioceptive large, PV-positive neurons, which are the dicating a cross-talk between microglia and astrocytes in neu- source of the major synaptic inputs (Ia VGluT1 afferents) to roinflammation. We show here that gliosis in the aged spinal MNs. Ia afferents establish selective excitatory monosynaptic cord is accompanied by a prominent increase in harmful M1 connections with spinal MNs that, in turn, project their axons microglia and A1 astroglia, with a reduction in the proportion to the same or related (homonymous or synergistic, respec- of neuroprotective M2 and A2 phenotypes. A recent study tively) muscles from which they receive sensory feedback.131 has reported that the depletion of microglia is able to prevent This circuitry, which is essential for motor coordination and or reverse the loss of muscle innervation in aged mice125; this posture, appeared to be impaired in old animals, as inferred finding suggests an important pathogenic role of neurotoxic from progressive age-related alterations in mobility observed microglia in the age-related neuromuscular decline, although in open field analysis. On the other hand, changes in the pro- the mechanism by which microglia exert their detrimental ac- portion of DRG neurons expressing CGRP and in the levels of tions in ageing is not known. It has been shown that both mi- this neuropeptide have been reported to occur in various in- croglia and astrocytes are involved in synapse elimination flammatory pain models.132–135 Therefore, the increased during neurodegeneration.126 A similar function has been as- numbers of DRG neurons expressing CGRP found in our study cribed to astrocytes in actively eliminating defective synap- are in concordance with the state of general, low-grade, ses.127 The correlation between gliosis and MN chronic inflammation that appears to be linked to elderly, deafferentation, found here in the old spinal cord, suggests which has been considered an etiological factor of ageing a possible function of glial cells in the synapse elimination sarcopenia. In this regard, a systemic increase in during ageing, as it has also been reported for ALS and pe- pro-inflammatory mediators coming from different sources ripheral nerve injury.57,126 However, to our knowledge, no has been reported to occur in advanced ages.2 published data exist on whether neuroinflammatory glial cells Nerve conduction studies allow the functional quantitative actually eliminate synapses on diseased MNs. In a recent assessment of large myelinated fibres. Thus, the amplitude of study using a model of sciatic nerve axotomy, we found no the evoked action potentials is an index of the number of evidence of synaptic bouton enwrapping and phagocytosis conducting nerve fibres and the peripheral innervation, by microglia but noticed that microglial processes, recruited whereas the nerve conduction velocity is related to axonal by lesioned MNs, are in close contact with degenerating af- calibre and myelination.136 The nerve conduction tests per- ferent synaptic terminals, suggesting an active role of microg- formed indicate that the conduction of impulses is reduced lia in the disintegration of adjacent presynaptic terminals on in advanced age, which is in agreement with previous reports MNs.57 It has been reported that activated microglia can pro- in laboratory animals137–139 and in humans.140–142 In this re- duce molecules (i.e. nitric oxide, oxygen radicals, proteases, gard, a previous longitudinal evaluation in mice has shown and cytokines), which can induce neuronal damage and/or that motor and sensory nerve conduction velocity increased the extracellular digestion of synaptic debris.128 A question during young adult life, and then remained unchanged until that needs to be answered is whether glial activation in aged the last third of the lifespan, when it tended to decline.139 Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1652 A. Blasco et al. Additionally, age-associated reduction in the amplitude of nerve terminals, where they are released and exert their motor and sensory nerve action potentials has also been re- function. Whereas GAP-43 is highly expressed in the growth ported in animals138,139 and humans.140,141,143 Our present cones of motor axons and are involved in developmental ax- results also indicate that the amplitude of CMAPs and CNAPs onal outgrowth,71 agrin plays a crucial role in the initiation of tend to decrease with ageing, particularly in distal targets, NMJ formation by clustering AChRs and, also, in the preserva- such as foot muscles and toe nerves. In fact, a slow loss of tion and regeneration of the adult NMJ.74,75 On the other myelinated fibres in peripheral nerves with ageing has been hand, in the neuromuscular system, CGRP has been proposed demonstrated in several animal species.140,144–146 Indeed, in to act as a communication molecule between MNs and skel- a detailed morphometrical study in mice, we have found that etal muscles and is involved in the development and mainte- after 20 months of age, the sciatic nerve showed a noticeable nance of NMJs and muscle cells155,156; motoneuronal CGRP is loss of both unmyelinated and myelinated axons, accompa- up-regulated following nerve injury, but its raised levels de- nied by a decrease in axonal size and myelin thickness.146 cline with the morphological and functional recovery of mo- The effect of sustained pressure on the paws over life has tor innervation, suggesting a function of the neuropeptide been attributed as one of the determining factors for the par- in axonal growth and NMJ plasticity.49,50,157,158 On the whole, ticular functional loss of plantar muscles compared with more these three molecules were found in low levels in muscles of proximal and tail muscles,145 as we have observed here in old adult mice compared with young animals but were clearly mice. up-regulated in motor nerve terminals of old muscles. This MUNE analysis reflects well the potential loss of MNs with finding suggests an active role for these proteins in the regu- ageing.147 The reduction in MUNE found here in old mouse lation of the compensatory nerve terminal sprouting and re- muscles was only mild and is in agreement with the absence innervation of denervated muscles in ageing. of significant MN degeneration. Following loss of an MN, mo- The FGFBP1 has been found to be up-regulated in skeletal tor axons are able to sprout collateral branches within the muscles of a mouse model for SMA.159 More recently, it has own muscle and thus increase the size of the corresponding been described that NMJs of normal adult muscles express motor unit,148 allowing an efficient compensatory mechanism and concentrate FGFBP1, but its levels decrease when NMJs for maintaining muscle force. However, with advanced age- undergo pathophysiological changes as occurred in the ing, the capability for collateral sprouting149 and maintenance SOD1G93A mouse model for ALS.79 Compared with the rela- of size after expansion of the motor unit150 is defective. In ad- tive high intensity of immunostaining for FGFBP1 observed dition, alterations of NMJ function as a consequence of alter- in NMJs of young muscles (not shown), we found that ations in fast-twitch, fast-fatigable motor units have been FGFBP1-immunolabelling markedly declined in adult NMJs described in aged rodents,151 which cause a decrease in the but increased again in old NMJs, particularly in those of the mean size of remaining motor units, as we found here. Gra muscle. The increased content of FGFBP1 in old muscles Whether NMJs are stable over the lifespan and if they is in concordance with its proposed function in promoting change their morphology with ageing are debatable. Some NMJ repair78 and suggests a role of this protein in the process early studies in rabbits and cats have shown that a certain de- of age-related NMJ remodelling and muscle reinnervation. gree of nerve sprouting and a continuous turnover of NMJs The elevated levels of FGFBP1 noticed by immunocytochem- occur in normal muscles.152 However, more recent data in istry in old muscles contrast with the findings by Valdez’s mice and humans support the idea that NMJs are generally group,79 which have reported a significant reduction in stable for most of the lifespan.28,153 In agreement with previ- FGFBP1 transcripts in the TA muscle of 24-month-old mice. ous published reports,67–69,82,154 we found, in old muscles, a This divergence could be explained by the differences in significant amount of NMJs exhibiting conspicuous plastic methodology and animals used in this latter study,79 in which changes, including partial or complete denervation, the FGFBP1 expression levels have been examined by quanti- polyinnervation, increased nerve terminal branching com- tative PCR in aged wild-type heterozygous (FCFBP1+/ ) ani- plexity, and sprouting. Moreover, overt signs of endplate mals of homozygous (FCFBP1 / ) FGFBP1 knockouts. fragmentation and ectopically innervated AChR clusters Nevertheless, in agreement with data from this same study, throughout the myofiber were seen in old muscles. Overall, we found higher contents of TGF-β1 in muscles of young these observations fit with the hypothesis that ageing entails and old animals than in those of adult mice. Our findings a continuous process of muscle denervation/reinnervation showing increased levels of both TGF-β1 and FGFBP1 in old (reviewed in Larsson et al.6). In this regard, we noticed that muscles in vivo are difficult to reconcile with the suggested aged muscles displayed a significant increase in some mole- role of TGF-β1 in inhibiting FGFBP1 expression found in cul- cules having specific roles in development, maturation, plas- tured myotubes.79 However, the increased levels of TGF-β1 ticity, and stability of NMJs. These molecules include CGRP, could explain some changes found in muscles of old mice. Al- GAP-43, agrin, FGFBP1, and TGF-β1. CGRP, GAP-43, and agrin terations in the expression of TGF-β1 and its signalling path- are developmentally regulated proteins, which are ways have been associated with inherited and acquired synthetized in MN cell bodies and axonally transported to myopathies (reviewed in Burks and Cohn160). In this regard, Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1653 an increase in circulating TGF-β1 levels has been found to be comparable age-associated alterations in both muscles. These associated with sarcopenia. TGF-β1 is considered a potent observations point to the fact that the specific function and regulator of tissue wound healing and fibrosis in the skeletal the degree of activity of muscles could be main determinants muscle161 and appears to play an important role in promoting of muscle wasting severity in elderly. the transformation of myoblasts in myofibroblastic cells. All the old muscles examined in our study displayed fibres Myofibroblasts have been involved in tissue fibrosis and having central nuclei, with much higher proportions of them could be responsible for the increased proportions of connec- in TA and Gra muscles. The existence of increased amounts tive tissue we found in old muscles. It has been proposed that of fibres with centrally located nuclei has been associated to elevated systemic or local levels of TGF-β1 and skeletal mus- different muscle disorders and repair.88 In the case of muscle cle fibrosis could interfere with the activation and prolifera- repair, the fusion of activated SCs with the damaged myofiber tion of SCs with the subsequent impairment of myofiber causes the incorporation of new nuclei, which move first to the regeneration and muscle remodelling in sarcopenia.160,162 In centre of the myofiber before moving out to its periphery. this regard, it should be expected that the reduction in the Therefore, the presence of centrally positioned nuclei has proportion of SCs we found in old muscles negatively affects been considered as an indicator of myofiber regeneration. In the regenerative capacity of muscles and exacerbates muscle this regard, the finding of elevated numbers of fibres with cen- fibrosis.163,164 Connective tissue deposition was found in all tral nuclei in old muscles might reflect a process of continual muscles from old mice, although it was particularly conspicu- myofiber regeneration in response to a persistent muscular ous in the EDL and Sol. Although the fibrotic tissue deposition impairment occurring with ageing. Changes in the NMJs lead- would contribute to worsen the impaired muscle function in ing to denervation/reinnervation could be one of the main ageing, fibrosis could explain the absence of significant drivers of fibre degeneration/regeneration cycles in aged mus- age-related changes in the muscle weight we observed in cles. However, an intrinsic alteration in myofibers as a cause of EDL and Sol muscles despite myofiber loss. muscle dysfunction in elderly cannot be excluded. Thus, nu- Interestingly, compared with adult mice, no statistically clear misposition should be considered not only a sign of mus- significant differences in muscle mass were found in old mus- cle pathology but also a cause of it.88 In any case, the finding of cles, although in the latter, some decrease in the average myofibers with centrally positioned nuclei in all the old mus- weight was observed in Sol and EDL muscles. Overall, these cles, but at different proportions, suggests that myofiber re- findings suggest that ageing has only a minor impact on mus- generation in ageing is a general feature shared by distinct cle mass of mice, a result which contrasts with reported data types of hindlimb muscles, although they exhibited a highly from human studies but is in agreement with previous analy- variable degree of repair. Interestingly, muscles with the low- sis conducted in ageing rats and mice: in these models, only est numbers of myofibers with central nuclei (EDL and Sol) also moderate or very small decreases in muscle mass have been appeared to display the lowest capacity of myofiber regenera- observed compared with humans (reviewed by Ballak tion: both these muscles from old animals, predominantly the et al.99). Moreover, the fact that Sol and EDL muscles, two EDL, exhibited a reduction in the density of myofibers, indicat- distal hindlimb muscles with different fibre type composition, ing that regeneration cannot fully counteract myofiber loss. In have a greater tendency for age-related mass reduction than fact, we failed to detect any decline in the fibre density in the the distal TA or the proximal Gra muscles corroborates the old TA and Gra muscles, which have the highest number of fi- hypothesis that different muscles are impacted by varying de- bres displaying central nuclei. grees over the ageing process. This may suggest that muscle Studies performed in rat models have reported that wasting in advanced ages could be more linked to both mus- age-related changes in muscle innervation results in a de- cle location and specific function than to fibre type composi- creased proportion of type 2 myofibers, and particularly of tion. In fact, others have reported differences in age-related those having a glycolytic metabolism (type 2B)94,95 (see also muscle mass loss between mouse hindlimb (Sol and EDL) McKinnon et al. and Schiaffino and Reggiani86,92 as reviews). and the cervical (sternomastoid and cleidomastoid) muscles, Type 2 myofibers are innervated by fast, MMP-9-positive, vul- with no changes in the latter.165 In addition, it has been nerable MNs.43,45 The age-related decrease in the proportion shown in rats166 and also in humans167 that hindlimb (leg) of type 2 myofibers appears to be compensated by the in- muscles are more severely affected than forelimb (arm) mus- creased numbers of type 1 myofibers,94,95 which, innervated cles. Our results indicate that even muscles in proximity of by slow MNs, have an oxidative metabolism and are more re- each other, such as TA and EDL, also display differential sistant to degeneration. In fact, several analyses conducted in age-related histological alterations, with a significant de- models of MN diseases have reported that MNs and synapses crease in fibre density in the EDL muscle and no overt innervating type 2B myofibers degenerate earlier than those changes in the TA muscle of old animals. These observations innervating types 2A and 1 myofibers.168,169 These results have are somehow surprising because EDL and TA muscles have a been correlated with differences in the degree of plasticity of similar fibre type composition and belong to the same func- synapses: synapses exhibiting a low sprouting competence tional group, and, consequently, we should expect are the most vulnerable, whereas those which react with a Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 1654 A. Blasco et al. robust sprouting are selectively resistant.168 Thus, it has been and rhesus monkeys as a marker of neuronal ageing.100 In proposed that fibre type 2 to 1 transition in ageing is the con- this work, authors have shown a variable degree of lipofuscin sequence of a preferential loss of fast, vulnerable, MNs with accumulation among MNs, suggesting that not all MNs aged the resulting denervation of 2B fibres and their subsequent in- either at a comparable rate or at the same time. In a similar nervation by slow, resistant, MNs with high sprouting capacity. way, we noticed that in sharp contrast with the absence of In fact, myofiber type 2 to type 1 switching has been reported lipofuscin in adult muscles, all old muscles from C57BL/6JRj to occur in models of MN diseases, such as SMA mice.39,170 Be- mice accumulate this pigment in the myofiber cytosol. We cause skeletal muscles with a higher content of type 1 fibres observed, however, remarkable variations in the amounts of are more resistant to wasting than those with a predominance lipofuscin between the different muscles examined, with of type 2 fibres,92,171 it has been proposed that fibre type 2 to the slow-twitch Sol muscle exhibiting the lowest proportion type 1 transition in these diseases could result from the activa- of the lipid pigment and the fast-twitch Gra muscle the tion of metabolic oxidative myogenic compensatory pro- highest. These differences correlate with the finding that gramme aimed at limiting muscle dysfunction. Our results the fast-twitch types 2A and 2B myofibers are those which indicate that a similar mechanism does not likely operate in exhibited the highest content of lipofuscin in old muscles, muscles of C57BL/6J mice with ageing. In divergence with data with type 1 fibres almost devoid of the pigment granules. This from ageing rat studies, with the exception of Gra muscle, we indicates that vulnerable (types 2A and 2B) fibres are more did not observe any significant augment in the proportion of fi- prone to ageing than type 1 fibres. Large numbers of bre type 1 in old muscles. Conversely, a trend to an age-related lipofuscin granules have been reported to be present in dif- decrease in the amount of type 1 fibres was seen in all the dis- ferent muscular dystrophies in humans and young tal hindlimb muscles, this reduction being particularly promi- dystrophin-deficient mdx mice.180–182 It has been proposed nent in the EDL muscle. Additionally, in the old slow-twitch that cellular lipofuscin accumulation is the consequence of Sol muscle, the proportion of type 2A fibres was found promi- an oxidative stress process making cellular components nently increased at the expense of a reduction in the number undegradable by lysosomal enzymes.96 Indeed, the treatment of types 1 and 2B fibres. These results corroborate previous of young mdx mice with antioxidants reduces lipofuscin accu- findings indicating that fibre type 2 to 1 switching in ageing is mulation and improves muscle function. Oxidation, as a cause not a common feature shared by all muscles, with profound of lipofuscin formation, seems to be also linked to a de- differences existing between fast-twitch and slow-twitch mus- creased degradative capacity of lysosomes, which would oc- cles.165 Additionally, our data suggest that differences in cur with age or under pathological conditions. It is not aged-related myofiber type transition also occur between clear, however, whether lipofuscin really interferes with nor- muscles having similar fibre type composition but different mal cellular function or, conversely, the accumulation of this limb topography (i.e. the distal TA or EDL and the proximal pigment is a mere result from a previous cellular disturbance. Gra muscle; all of them are fast-twitch muscles). It is important Several pieces of evidence suggest that a detrimental feed- to note that there is a substantial inconsistency between the back loop leading to lysosomal and ubiquitin/proteasome age-related changes in fibre type composition reported in the pathway inhibition may occur as a result of lipofuscin aggre- literature. Some studies in human muscles have found a type gation. In any case, it seems plausible that the accumulated 2 to type 1 fibre switching with a reduction in the former, lipofuscin makes cells more vulnerable to pathologic stimuli whereas other analyses have conversely reported and decreases the cellular adaptability and response capacity aged-related increases in type 2 fibres; other works, however, to injury.96 In the case of aged MNs, lipofuscin accumulation have found no changes in muscle-fibre type profiles in humans does not associate with cell atrophy or death, although it ap- with ageing.172–175 Similar disparities in age-associated fibre pears to correlate with the unequivocal alterations occurring type transitions have been found in rodent studies.165,176–178 with ageing in motor nerve terminals.100 Moreover, although The absence of a consistent pattern of fibre type alterations, the lipofuscin aggregation is significantly higher in muscle fi- we found in old muscles, correlates with the absence of sub- bres with peripheral nuclei, the presence of a certain propor- stantial MN death and, more specifically, with the lack of a de- tion of fibres with central nuclei indicates the existence a cline in the proportion of vulnerable (MMP-9-positive) MNs. newly regenerated myofibers already containing the age pig- Overall, our data suggest that fibre type changes are more of ment. Whether or not lipofuscin granules in these fibres cor- a consequence of MN dysfunction than cell death. relates with an impaired cellular function already present in The accumulation of lipofuscin in postmitotic cells is widely the initial steps of myogenic programme should be analysed. considered a hallmark of ageing (reviewed by Brunk and In summary, ageing in mice is accompanied by defects in Terman96 and Gray and Woulfe97). In this respect, the pres- motor skills and changes in the electromyographic patterns. ence of lipofuscin granules in brain cells, particularly in cere- These alterations occur in the absence of significant MN bral cortex and motor areas, is one of the most consistent death but are associated to prominent MN deafferentation features of ageing.179 In a recent study, the content of and reactive gliosis, which possibly contributes to MN impair- lipofuscin aggregates has been analysed in MNs of old mice ment. Therefore, age-related MN dysfunction rather than Journal of Cachexia, Sarcopenia and Muscle 2020; 11: 1628–1660 DOI: 10.1002/jcsm.12599 Regressive neuromuscular changes in aged mice 1655 death could be responsible of structural and molecular alter- authorship and publishing in the Journal of Cachexia, ations in motor axons, MNJs, and skeletal muscles found in Sarcopenia and Muscle.183 senescence. The ageing progress appears to entail continuous cycles of motor axon degeneration/regeneration and sprouting, as deduced by the signs of morphologically altered Online supplementary material motor axons in adult mice and the increased numbers of clus- tered small myelinated axons found in VRs of middle-aged Additional supporting information may be found online in the mice. Although the distinct types of old muscles examined Supporting Information section at the end of the article. share some common features indicative of the ageing pro- cess, the profile of some alterations is markedly variable be- tween muscles. This variability exists even between muscles located in close vicinity and having similar fibre type compo- Figure S1. Aging is accompanied by a decline in body weight sition, suggesting that the degree of activity and specific func- and motor performance measured with the open-field test. tion of muscles, rather than their topography and fibre A) Body weight in adult (4–10 months) and old (19– typology, has greater impact on age-related muscular 28 months) mice; ***p < 0.001 vs. adult 46 weeks. B and changes. Further research is needed to draw more reliable C) Motor performance assessed by open-field test in mice conclusions concerning the pathogenic mechanisms underly- from 24 months of age on; the distance (in cm, B) and aver- ing neuromuscular alterations during ageing and the extent age speed (cm/s, C) in the course of the 16 week-period in of their contribution to development of sarcopenia. which measurements were performed. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. 0 weeks. Data in the graphs Funding are shown as the mean ± SEM, n = 15 (84 to 112 weeks); n = 8 (~116 weeks onwards). This work was supported by Abbott and a grant from the Ministerio de Ciencia, Innovación y Universidades cofinanced Figure S2. Age-related changes in DRG sensory neurons. A-C, by Fondo Europeo de Desarrollo Regional ( RTI2018-099278- F-H and K-M) Proportion (A, F, K) and soma area (B, G, L) of B-I00 to J.C. and J.E.). sensory neurons expressing CGRP (A, B), IB4 (F, G) and PV (K, L) in DRGs. C, H, M) Relative size-frequency (expressed as soma area in μm2) histogram of DRG neurons showing CGRP- Author contributions (C), IB4- (H) and PV- (M) positive staining. D1-O2) Representa- tive micrographs taken from DRG cryostat sections processed J.C., J.E.E., S.L.P., A.Ba., T.D., and R.R. contributed to concep- for CGRP, IB4 and PV staining (green), as indicated. Sections tion of the research, and J.C. and J.E.E. designed the study. A. were also counterstained with fluorescent Nissl (red) for neu- B., S.G., G.M.-C., O.T., A.C., and L.P. performed the research. ron visualization. Data in the graphs are expressed as the A.B., S.G., O.T., X.N., and J.C. analysed the data. J.C., J.E.E., X. mean ± SEM, **p < 0.01 and ***p < 0.001 vs. adult; n = 4 N., and S.L.P. contributed to writing the paper. ventral roots of different animals per age group (Student’s t-test). Scale bar: in O2 = 100 μm2 (valid for D1-E2, I1-J2 and N1-O1). Acknowledgements We thank Sara Salvany, Sara Hernández, Alaó Gatius, Mònica Esteve, Maria Beltrán, and Clara Paniagua for collaborating in Conflict of interest some experiments, Anaïs Panosa from the SCT of Microscopy A.Ba., S.L.P., T.D., and R.R. are employees of Abbott. The of the Universitat de Lleida for technical support with confo- other authors declare that they have no conflict of interest. cal microscopy, and the staff from the SCT Animal Facility of the Universitat de Lleida for mouse care and housing. The au- thors certify that they comply with the ethical guidelines for References 1. Fried LP, Tangen CM, Walston J, Newman 2. Cardoso AL, Fernandes A, Aguilar- age-related diseases. Ageing Res Rev AB, Hirsch C, Gottdiener J, et al. Frailty in Pimentel JA, de Angelis MH, Guedes JR, 2018;47:214–277. older adults: evidence for a phenotype. J Brito MA, et al. Towards frailty bio- 3. 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