Muscle spindle afferent neurons preferentially degenerate with aging

Muscle spindle afferent neurons preferentially degenerate with aging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Muscle spindle afferent neurons preferentially degenerate with aging Minako Kawai-Takaishi, Tohru Hosoyama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5487702/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 6 You are reading this latest preprint version Abstract Muscle spindles sense changes inmuscle length and transmit them to the central nervous system. Proprioception is essential for gait and postural maintenance, the abnormality of which has been linked to gait disorders and the risk of falling in older adults. However, the effects of aging on the muscle spindle structure remain nebulous. This study investigated age-related structural changes in themuscle spindles (from the equator to the polar) in the soleus and extensor digitorum longus (EDL) muscles of young, middle-aged, and aged mice. The findings indicated that the shape of the annulospiral endings of the sensory neurons began to deteriorate in middle-aged compared with young mice and was further exacerbated in aged mice. These changes were particularly pronounced in the nuclear bag fibers, whereas no significant age-related changes were observed in the intrafusal fibers or capsules. A decline in gait function due to changes in weight-bearing and weight-shifting in aged mice was also observed, suggesting that the deterioration of proprioceptive sensory neurons that innervate the nuclear bag fiber responsible for dynamic sensitivity prevents proper coordinated movement and contributes to movement disorders in aged animals including humans, together with the functional decline of extrafusal fibers. Health sciences/Anatomy/Musculoskeletal system Biological sciences/Neuroscience/Motor control/Neuromuscular junction aging muscle spindle intrafusal fibers proprioceptive sensory neuron annulospiral ending movement disorders Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Coordinated movement, including walking, standing, and sense of balance, is affected with aging 1 , increasing the incidence of falling and being bedridden and decreasing the quality of life of older adults 2 . Although activation of the α-motoneurons (α-MNs) and extrafusal fibers is essential for all voluntary movements, these functions are under the control of the nervous system. The proprioceptive neurons transmit information to α-MN and central nervous system about changes in the position and movement of body parts to adjust the contraction or relaxation of muscles, a part of the prerequisite for coordinating complex movements. 3 , 4 . Therefore, clarifying when and how the somatosensory system, including proprioception, is affected by aging will provide important information for future research plans aimed at preventing motor dysfunction in older adults. Muscle spindles, major proprioceptive receptors within skeletal muscles, sense changes in the length and velocity of stretched skeletal muscles, and intrafusal fibers within muscle spindles regulate their sensitivity 5 , 6 . Intrafusal fibers comprise nuclear bag (bag 1 and bag 2) and nuclear chain fibers, with the equatorial part innervated by two types of afferent proprioceptive sensory neurons (Ia and II afferents) and the polar part innervated by γ-motoneurons (γ-MN) 7 , 8 . Ia afferent innervates nuclear bag 1, bag2, and nuclear chain fibers, while II afferent innervates nuclear bag2 and nuclear chain fibers 9 . Equatorial Ia sensory endings have “annulospiral terminals,” a unique structure coiled around intrafusal fibers, while γ-MNs form neuromuscular junctions (NMJs) as well as α-MNs 6 , 10 – 13 . Nerve endings of proprioceptive sensory neurons can undergo detrimental structural changes due to disease or injury; however, our understanding of how aging affects proprioceptive sensory neurons and intrafusal fibers remains limited 14 – 17 . In this study, gait analysis was used to evaluate the motor function of mice. Although there are differences between bipeds and quadrupeds, the locomotor behavior of terrestrial mammals is achieved by rhythmic and coordinated movement of two or four legs with multiple joints 18 . Gait dysfunction and balance disorders are pervasive phenomena of human aging. Clinical studies demonstrate persistent reductions in gait speed and stride length 19 – 21 and increases in gait variability 22 in older adults when compared to young adults. The effects of aging on gait in mice are being elucidated, including evaluating the translational potential for humans 23 , 24 . In addition, the role of muscle spindle in walking ability has been investigated in mice lacking proprioception 25 – 28 , indicating that proprioceptive sensory feedback from muscle spindles contributes to the emergence of an alternation pattern of flexor and extensor muscle activity. The rhythmic stepping movement of the legs is divided into the stance phase when the foot is on the ground, and the swing phase when the foot is lifted off the ground and moving. The mutant mice, selectively eliminated muscle spindle activation, confronted with a walking task exhibited locomotor pattern degradation 25 , 26 . Although the mechanisms underlying age-related changes in gait function in older adults and aged mice include age-related mobility problems due to cardiopulmonary, musculoskeletal, and overt neurological conditions ( e . g . stroke and Parkinson’s disease), the analysis of age-related changes in muscle spindle and gait pattern would provide useful information to characterize age-related symptoms in aged mice and humans. In this study, we aimed to clarify age-related structural alteration in muscle spindles and its impact on gait functions. To achieve this objective, we analyzed age-related morphological changes in muscle spindles in detail by combining transverse and longitudinal sections of mouse skeletal muscles of three different age groups, including young (3 months old), middle-aged (12–15 months old), and aged (26 months old). This study will contribute to understanding the mechanisms of age-related increases in the risk of falls in older adults and establishing a novel strategy for achieving healthy longevity. 2. Results 2.1 Aging does not influence the number of muscle spindles and intrafusal fibers in muscle or the cross-sectional area of muscle spindles The length of the equatorial and juxta-equatorial regions of the muscle spindles of mouse leg muscles was approximately 1,000 µm, with an overall length of 2,000–3,000 µm 29 . Therefore, we evaluated muscle spindle morphology and numbers throughout the muscle by cross-sections made every 200 µm, a cutting interval suitable for evaluating the equatorial and polar morphologies with a minimum number of cross-sections (Fig. 1 A). Cross-sectional analysis of the whole muscle revealed that the soleus and EDL muscles possessed an average of 12 muscle spindles, two nuclear bag fibers per muscle spindle, and 1.7 nuclear chain fiber per muscle spindle (Table 1 ). These values were consistent across all the age groups. Table 1 Numbers of muscle spindle in muscle, nuclear bag fiber in muscle spindle, and nuclear chain fiber in muscle spindle. Young SOL Middle SOL Aged SOL Young EDL Middle EDL Aged EDL Muscle Spindle 13.3 ± 2.1 11.4 ± 1.0 11.2 ± 0.7 12.0 ± 1.1 12.4 ± 1.9 12.6 ± 1.9 NBF/Spindle 2.1 ± 0.1 2.1 ± 0.1 2.1 ± 0.1 2.1 ± 0.1 2.2 ± 0.1 2.0 ± 0.1 NCF/Spindle 1.6 ± 0.3 1.8 ± 0.3 1.7 ± 0.2 1.9 ± 0.1 1.5 ± 0.3 1.8 ± 0.2 NBF: Nuclear bag fiber. NCF: Nuclear chain fiber. SOL: Soleus. EDL: Extensor digitorum longus. The average length of muscle spindles determined from the analysis of muscle cross-sections was similar for all age groups, except for a decrease in the aged EDL (Fig. 1 B). Similarly, the capsule thickness and average cross-sectional area of the muscle spindle did not significantly differ between the age groups (Fig. 1 B-D). Next, we analyzed the age-related morphological changes in the intrafusal fibers. The average cross-sectional area of the intrafusal fibers, including nuclear bag and chain fibers, was calculated at the equator and polar of the muscle spindles. At the equator and polar muscle spindles, intrafusal fibers in the EDL of aged mice were larger than those in young and middle-aged mice, whereas no significant difference in the soleus was observed in either age group (Fig. 2 A, B). These results indicate that neither atrophy nor loss of intrafusal fibers occurs with age. 2.2 Annulospiral endings of the proprioceptive sensory neurons deteriorate with aging Analyzing muscle histology in longitudinal sections is important to clarify age-related structural changes in the annulospiral endings of proprioceptive sensory neurons. In this study, we attempted to construct the stacked images of muscle fibers from 150 µm of longitudinal muscle sections as our preliminary investigations showed that the maximum diameter of the muscle spindle was 50–100 µm. Annulospiral endings appeared to be progressively obscured in middle-aged and aged mice in representative immunofluorescence images (Fig. 3 A). The coil width and distance of the annulospiral endings were measured on the images and quantitatively compared by age (Fig. 3 B). Both the coil width and distance were similar for all age groups, except for the coil width of nuclear bag fiber of the soleus, which increased in middle-aged mice (Fig. 3 C, D). We then analyzed the number of coils in the annulospiral endings and compared them with age. The number of coiled annulospiral endings was reduced in both the nuclear bag and chain fibers of aged mice, indicating that sensory neuron coiling to intrafusal fibers decreased with increase in age (Fig. 4 A). Reductions in the number of coils included not only simple changes in number (decreased coils) but also fibers with indistinct coils (indistinct spirals) or complete loss of spirals (disappearance) (Fig. 4 B). Accompanying the disruption of the spiral-shaped structure, the number of blebbed sensory neurons in the annulospiral endings and unraveling fibers increased with age (Fig. 4 C, D). Unraveled fibers were particularly increased in nuclear bag fibers, even in middle-aged mice (Fig. 4 D). These results indicate that the spiral-shaped structure of the annulospiral endings disintegrates with age, especially in the nuclear bag fiber, where effects are observed in the early phase. Immunofluorescence analysis of vesicular glutamate transporter 1 (VGLUT1), which is abundant in annulospiral endings, and neurofilament protein, which occurs in neuronal axons, indicated the possible atrophy of afferent axons innervating intrafusal fibers with aging (Fig. 5 A; yellow arrowhead). In the soleus and EDL, we confirmed that the afferent annulospiral ending area and axonal diameter were decreased in aged mice (Fig. 5 B). 2.3 Neuromuscular junctions deteriorate with age In addition to the muscle spindles, we analyzed age-related morphological changes in NMJs (Fig. 6 A). In young mice, the presynaptic α-MN endings (NF + /Synapsin + ) innervate the postsynaptic α-bungarotoxin (α-BTX) + endplate. However, the percentage of nerve ending occupancy (innervation rate) was significantly decreased in middle-aged mice and further deteriorated in aged mice (Fig. 6 B). Endplate fragmentation and enlargement of the endplate area were observed, with deterioration of motor innervation (Fig. 6 C, D). These results support the widely accepted hypothesis that NMJs deteriorate with increasing age. 2.4 Aging alters gait function of mice We performed a footprint test to determine how age-related deleterious morphological changes in muscle spindles and neuromuscular junctions correlate with motor function (Fig. 7 A). The results showed that aging did not affect the stride, step length, base of support, or angle; however, the distance between the forelimb and hindlimb on the same side (anterior-posterior gap) was significantly increased in aged mice compared to young and middle-aged mice (Fig. 7 B; p = 0.051 and p = 0.0307, respectively). In addition, individual footprint parameters were measured to clarify the changes in the base of support of the forelimbs and hindlimbs (Fig. 7 C). The ground contact area (p = 0.0023 and p = 0.0184), perimeter area (p = 0.0065 and p = 0.0152), and plantar length (p = 0.0026 and p = 0.0079) of the hind limbs were significantly higher in aged than young and middle-aged mice (Fig. 7 D). Considering that young mice do not place their heels on the ground during leg grounding but shift their weight with their toes (Fig. 7 C), these results indicate a decline in gait functions, such as weight bearing and weight shifting, in aged mice. 3. Discussion Aging correlates with significant changes in α-MNs, extrafusal fibers, and NMJs in skeletal muscle 30 – 35 , yet the correlation between aging and changes in muscle spindle remains poorly understood. The lack of useful molecular markers for muscle spindle research has hindered the progression of research on muscle spindle maintenance and morphogenesis. Recently, several molecular markers that define muscle spindle structures have been identified using a combination of transcriptomic and proteomic analyses 36 . In this study, we used versican (VCAN), a recently identified molecular marker of muscle spindles, for morphological analysis, resulting in a more accurate and detailed structural analysis of the internal equatorial portion of muscle spindles using the intracapsular extracellular matrix as an indicator. In addition to VCAN, other molecular markers have been identified 36 , and a combination of these markers is expected to enable more detailed studies. This study demonstrated that the muscle spindle, an important structure for proprioceptive sensory feedback, was deleteriously affected by aging in addition to the well-known age-related degradation of NMJs 30 , 31 , 37 – 39 . Regarding NMJs, it has been demonstrated that age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission 40 . In other words, endplate fragmentation is an aging phenomenon but does not induce dysfunction. Although the annulospiral ending of proprioceptive nerve terminal is not a synapse, the same may be true for our result indicating age-related deterioration of afferents in muscle spindle. In fact, the percentage of the neurofilament + area to the VGLUT1 + area was significantly decreased with aging (Fig. 5 B). This result suggests that neurofilament, which plays essential roles in regulating neuronal diameter and axonal transport, decrease with age, making it difficult to maintain the structure of well-organized annulospiral endings in aged individuals. A previous study using aged rat has shown that the axonal diameter, and the dynamic response of Ia afferents both decreased with age 38 . Interestingly, our results indicated that age-related morphological changes occur in the nuclear bag fibers of the muscle spindles preceding any in nuclear chain fibers. Sensory neurons normally consist of only one Ia afferent fiber per spindle, and all intrafusal fibers within the spindle are innervated by that sensory neuron. Nuclear bag fibers consist of bag 1 and bag 2 fibers, and the sensory endings attached to each fiber are maximally responsive to dynamic and static sensitivity, respectively. Nuclear chain fibers respond to static sensitivity as do bag 2 fibers 41 , 42 . Taken together, although this study did not distinguish between bag 1 and bag 2 fibers and did not perform the physiological investigations, age-related deterioration of sensory terminals on nuclear bag fibers may hinder proprioceptive sensory feedback to the central nervous system in response to unanticipated body movements, contributing to increased motor impairment and fall risk in older adults. Atrophy of extrafusal fibers occurs with age 32 , 33 , and the number of muscle spindles and capsule thickness are altered with aging in rats and humans 37 , 38 , 43 , 44 . These reports clearly indicate that muscle spindles and extrafusal fibers are affected by aging. However, in this study, we observed no morphological changes in capsule thickness and intrafusal fibers. Morphological analysis of muscle spindles in other species and older individuals using similar research methods remains warranted. Whether these discrepancies were attributed to differences in experimental techniques, age, or animal species remains unclear. Nevertheless, our study, that combined transverse and longitudinal sections with immunofluorescence analysis, provides more accurate and precise results. Morphological analysis of muscle spindles in other species using similar research methods remains warranted. Previous studies using mouse skeletal muscle have shown that intrafusal fibers do not decrease in number or atrophy with age 39 , consistent with the findings of this study, validating our research methodologies. Furthermore, our data were obtained by experimenting with mice older (26 months old) than those previously utilized (15–17 months old) 39 , providing novel insights into age-related changes in muscle spindles and proprioceptive sensory neurons. In this study, we used numbers of animals of either sex. In the results of longitudinal-sectional muscle analysis using the same number of males and females, few parameters significantly differed between males and females (detailed data not shown). In addition, the study evaluating sexual dimorphism of the muscle spindle in rats has shown no significant differences in the number and morphological properties of intrafusal fibers or muscle spindles in male and female rats 45 . Therefore, at least in rodents, we assume that sex-related differences in the morphology of muscle spindle are negligibly small. The disease-related changes in gait patterns in rodents have been well documented using disease models, such as spinal cord injury, amyotrophic lateral sclerosis, and brain tumors 46 – 48 . Recently, data on age-related gait changes has also been accumulated 23 , 24 . In these studies, it has been demonstrated that aged mice exhibited significantly altered gait signatures compared to young animals and humans. It has also been reported that aged, freely moving mice exhibited significantly altered gait signatures compared to young animals, including changes in cadence, gait, variability, and footfall pattern distribution. In this study, we evaluated the gait performance of mice using the footprint test, a classical gait analysis. Unfortunately, since our gait analysis model is simple, it was limited to a few steps for technical reasons and may not have adequately captured subtle changes in locomotion dynamics. However, we found that the gait style changed with age. Interestingly, although the body weight, grip strength, muscle spindle afferents, and NMJs were already undergoing age-related changes in middle-aged mice (Table 2 ), the change in gait style only appeared as the mice aged. This suggests that deleterious changes in muscle spindles, such as poor afferent innervation, precede gait disturbance and are an early indicator of gait disturbance in older adults. Table 2 Summary of body weight, muscle wet weight, muscle wet weight/body weight, and grip strength/body weight in mice. Young Middle Aged Body weight (g) 25.5 ± 3.2 37.0 ± 9.8 36.9 ± 6.5 Muscle wet weight (mg) SOL 8.3 ± 1.1 9.9 ± 1.6 9.0 ± 0.7 EDL 9.8 ± 1.4 10.5 ± 0.9 9.8 ± 1.4 Muscle wet weight/Body weight (mg/g) SOL 0.32 ± 0.02 0.28 ± 0.03 0.25 ± 0.04 EDL 0.38 ± 0.01 0.30 ± 0.06 0.27 ± 0.02 Grip strength/Body weight Forelimb 6.4 ± 0.7 3.8 ± 0.5 3.2 ± 0.5 Four-limbs 11.6 ± 1.0 7.4 ± 1.3 5.7 ± 0.6 SOL: Soleus. EDL: Extensor digitorum longus. In this study, we did not evaluate movement speed, such as gait or swing speed; however, a decrease in gait speed has been demonstrated in aged mice 23 . The control of action potential activity by proprioceptive sensory feedback from muscle spindles is necessary for high-speed locomotion in mice 26 . Egr3 mutant mice, selectively eliminated muscle spindle activation, confronted with a walking task exhibited a pronounced extension in the duration of tibialis anterior (TA) muscle burst activity during the swing phase. This can cause co-contraction of TA and antagonist (gastrocnemius) muscle, possibly stiffening the ankle joint at the end of the swing phase 25 , 26 . This outcome is similar to the one previously found in humans, where the widening muscle activity emerged in response to aging 27 . Therefore, the age-related increase of plantar contact area in this study might be influenced by insufficient activity of the muscles controlling the ankle joint due to decline in muscle spindle function, the increase in body weight and the decrease in grip strength. Furthermore, the recent study using muscle spindle deficient mice has demonstrated that muscle spindle feedback is necessary for the smooth trajectory of the paw and the control of foot placement following the stumbling corrective reaction 26 . Since increased gait speed leads to increased dynamic stability 49 , it is possible that a series of these linkages result in gait disturbances and falls in older adults. Recently, a method was developed to activate proprioceptive functions in older adults using vibration stimulation in a unique frequency band specific to muscle spindles 50 , which may facilitate the establishment of a novel therapeutic strategy targeting poor afferent innervation of muscle spindles and the resulting gait disorders and falls. 4. Methods 4.1 Animals Young (3 months) and middle-aged (12–15 months) C57BL/6N mice were obtained from Clea Japan, Inc. (Tokyo, Japan), while aged (26 months) C57BL/6N mice were provided by the NCGG Aging Farm (Aichi, Japan) 51 . Both male and female mice were used. The mice were sacrificed at appropriate time points by cervical dislocation under anesthesia with Medetomidine hydrochloride (0.3 mg/kg), Midazolam (4 mg/kg), and Buprenorphine (5 mg/kg), and the hind limb muscles were collected and weighed. The dissected muscles, including the tibialis anterior (TA), soleus, and extensor digitorum longus (EDL) muscles, were frozen in liquid nitrogen-chilled isopentane and stored at -80 ˚C until use. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Center for Geriatrics and Gerontology (no. 6–6). The study was conducted in accordance with the relevant guidelines and carried out in compliance with the ARRIVE guidelines. The methods were carried out in accordance with the approved guidelines and the ARRIVE guidelines. 4.2 Morphological analysis of muscle spindle on muscle cross-sections Both the soleus and EDL muscles were used for cross-sectional muscle analysis. The breakdown of males and females for each muscle and age was as follows: young soleus (four males and one female), young EDL (two males and three females), middle-aged soleus and EDL (three males and two females), and aged soleus and EDL (five males). Transverse sections of 10-µm thickness were prepared every 200 µm from the proximal to distal ends of muscle and attached to a glass slide (25–30 sections for each muscle) (Fig. 1 A). After fixation in 4% paraformaldehyde (PFA), immunofluorescent analysis was performed on muscle cross-sections using the following antibodies: anti-Laminin α2 (1:1,000, Santa Cruz Biotechnologies, CA, USA), anti-S46 (1:2,000, the Developmental Studies Hybridoma Bank (DSHB), IA, USA), Alexa Fluor 594-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1,000, Thermo Fisher Scientific, MA, USA). Immunofluorescent images were obtained using a fluorescence microscope (Keyence Bz-X900 and Leica Mica). The number of muscle spindles per muscle, number of intrafusal fibers per muscle spindle, muscle spindle length, average cross-sectional area, and capsule thickness were calculated from the images. Regarding the cross-sectional area of the muscle spindle or each intrafusal fiber, the area of the equatorial region was calculated as the average of sections 1 to 3, which had large areas and wide intracapsular spaces ( i . e ., ECM rich), and the area of the polar region was calculated as the average of the other sections. 4.3 Morphological analysis of muscle spindle on muscle longitudinal sections Both the soleus and EDL muscles were used for longitudinal section muscle analysis. The sex-based breakdown in each muscle and age was three males and three females. Continuous longitudinal sections of 150-µm thickness were prepared, and sections were gently thawed in -10 ˚C-chilled 30% glycerin/Phosphate-buffered saline (PBS). Longitudinal sections were fixed in 4% PFA, suspended in solution, and immunofluorescent staining was performed using the following antibodies: anti-VGLUT1 (1:2,000; Synaptic Systems, Gottingen, Germany), anti-neurofilament (NF; 1:2,000; Abcam, Cambridge, UK), anti-versican (VCAN; 1:500; Abcam), anti-S46, Alexa Fluor 594-conjugated goat anti-rabbit IgG, and Cy5-conjugated goat anti-mouse IgG (1:1,000; Jackson ImmunoResearch Laboratories, PA, USA). S46 and VCAN were labeled with Alexa Fluor 488 using Zenon Alexa Fluor 488 labeling reagent (Thermo Fischer Scientific). NF and VGLUT1 were labeled with Cy5 and Alexa Fluor 594, respectively. Immunofluorescent images were obtained using a laser scanning confocal system (LSM780; Carl Zeiss, Oberkochen, Germany; STELLARIS, Leica, Wetzlar, Germany). Values were calculated as the average of 5–8 muscle spindles per muscle. 4.4 Morphological analysis of NMJs The TA muscles were used for the morphological analysis of NMJs, and the sex-based breakdown for each age group was five males. Continuous longitudinal sections of 150-µm thickness were prepared, and sections were gently thawed in -10˚C-chilled 30% PBS. Longitudinal sections were fixed in 4% PFA while suspended in the solution, and immunofluorescent staining was performed using the following antibodies: anti-synapsin (1:1,000; Cell Signaling Technology, MA, USA), anti-NF, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000). Alexa Fluor 594-conjugated α-BTX (1:500, Sigma-Aldrich, MO, USA) was used for NMJ staining. Immunofluorescent images were obtained using laser scanning confocal systems (LSM780 and STELLARIS), and the NMJ and endplate morphologies were evaluated. Values were calculated as an average of 50 plates per muscle. 4.5 Footprint test Before muscle sampling, the mice’s footprints were recorded using a homemade walking lane (40 cm × 8 cm). Mice were acclimated to the running lane in advance, had ink (black for forelimbs and green for hindlimbs) applied to their feet, and walked on graph paper. From the obtained footprints, we measured the parameters described below in the basis of the axis of the direction of motion; the stride length, step length, base of support, paw angle, distance between the ipsilateral front and rear limbs (anterior-posterior gap). In addition, the ground contact area of the palm, perimeter, finger openings, and plantar length were also calculated from each paw prints. The sex-based breakdown at each age was as follows: young mice (five males), middle-aged mice (two males and three females), and aged mice (five males). 4.6 Grip strength measurement According to the manufacturer's instructions, the grip strength of the fore limb and four limbs was measured using a GPM-101B (Melquest, Toyama, Japan). Each measurement was conducted three times with a 10-minute rest between each experimental set, and the maximum value of the three experiments was used as representative data for each mouse. 4.7 Statistical analyses Data are presented as mean ± standard deviation. Differences in means between groups were evaluated using one-way ANOVA and multiple comparison tests (significance level, p < 0.05). All statistical analyses were performed using the GraphPad Prism 9 software (GraphPad Software, CA, USA). Declarations Ethical approval All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Center for Geriatrics and Gerontology (no. 6–6). Funding JSPS-KAKENHI, 24K14396 to M.T-K. Japanese Ministry of Health, Labor, and Welfare; 21–44, 24 − 5, 24–28 and Japan Health Research Promotion Bureau; 2022-B-02 to T.H. Competing interests The authors declare no competing interests. Author Contribution M. K-T. contributed to conceptualization, investigation, data curation, formal analysis, funding acquisition, writing - original draft, and writing - review & editing. T. H. contributed to conceptualization, data curation, writing - original draft, writing - review & editing, and funding acquisition. Acknowledgement The authors thank Dr. Ken Watanabe for his helpful comments on this study. The anti-S46 antibody was obtained from the DSHB, developed under the NICHD, and maintained by the University of Iowa. 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Modular organization of murine locomotor pattern in the presence and absence of sensory feedback from muscle spindles. J Physiol 597 , 3147-3165, doi:10.1113/JP277515 (2019). Santuz, A., Laflamme, O. D. & Akay, T. The brain integrates proprioceptive information to ensure robust locomotion. J Physiol 600 , 5267-5294, doi:10.1113/JP283181 (2022). Lian, W. et al. Distribution Heterogeneity of Muscle Spindles Across Skeletal Muscles of Lower Extremities in C57BL/6 Mice. Front Neuroanat 16 , 838951, doi:10.3389/fnana.2022.838951 (2022). Larsson, L. et al. Sarcopenia: Aging-Related Loss of Muscle Mass and Function. Physiol Rev 99 , 427-511, doi:10.1152/physrev.00061.2017 (2019). Tomlinson, B. E. & Irving, D. The numbers of limb motor neurons in the human lumbosacral cord throughout life. J Neurol Sci 34 , 213-219, doi:10.1016/0022-510x(77)90069-7 (1977). Hepple, R. T. Sarcopenia--a critical perspective. Sci Aging Knowledge Environ 2003 , pe31, doi:10.1126/sageke.2003.46.pe31 (2003). Fiatarone, M. A. & Evans, W. J. The etiology and reversibility of muscle dysfunction in the aged. J Gerontol 48 Spec No , 77-83, doi:10.1093/geronj/48.special_issue.77 (1993). Fahim, M. A. & Robbins, N. Ultrastructural studies of young and old mouse neuromuscular junctions. J Neurocytol 11 , 641-656, doi:10.1007/BF01262429 (1982). Punga, A. R. & Ruegg, M. A. Signaling and aging at the neuromuscular synapse: lessons learnt from neuromuscular diseases. Curr Opin Pharmacol 12 , 340-346, doi:10.1016/j.coph.2012.02.002 (2012). Bornstein, B. et al. Molecular characterization of the intact mouse muscle spindle using a multi-omics approach. Elife 12 , doi:10.7554/eLife.81843 (2023). Swash, M. & Fox, K. P. The effect of age on human skeletal muscle. Studies of the morphology and innervation of muscle spindles. J Neurol Sci 16 , 417-432, doi:10.1016/0022-510x(72)90048-2 (1972). Kim, G. H., Suzuki, S. & Kanda, K. Age-related physiological and morphological changes of muscle spindles in rats. J Physiol 582 , 525-538, doi:10.1113/jphysiol.2007.130120 (2007). Vaughan, S. K., Stanley, O. L. & Valdez, G. Impact of Aging on Proprioceptive Sensory Neurons and Intrafusal Muscle Fibers in Mice. J Gerontol A Biol Sci Med Sci 72 , 771-779, doi:10.1093/gerona/glw175 (2017). Willadt, S., Nash, M. & Slater, C. R. Age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission in the mouse diaphragm. Sci Rep 6 , 24849, doi:10.1038/srep24849 (2016). Boyd, I. A., Gladden, M. H., McWilliam, P. N. & Ward, J. Control of dynamic and static nuclear bag fibres and nuclear chain fibres by gamma and beta axons in isolated cat muscle spindels. J Physiol 265 , 133-162, doi:10.1113/jphysiol.1977.sp011709 (1977). Bewick, G. S. & Banks, R. W. Mechanotransduction in the muscle spindle. Pflugers Arch 467 , 175-190, doi:10.1007/s00424-014-1536-9 (2015). Winarakwong, L., Muramoto, T., Soma, K. & Takano, Y. Age-related changes and the possible adaptability of rat jaw muscle spindles: immunohistochemical and fine structural studies. Arch Histol Cytol 67 , 227-240, doi:10.1679/aohc.67.227 (2004). Liu, J. X., Eriksson, P. O., Thornell, L. E. & Pedrosa-Domellof, F. Fiber content and myosin heavy chain composition of muscle spindles in aged human biceps brachii. J Histochem Cytochem 53 , 445-454, doi:10.1369/jhc.4A6257.2005 (2005). Gartych, M., Jackowiak, H., Bukowska, D. & Celichowski, J. Evaluating Sexual Dimorphism of the Muscle Spindles and Intrafusal Muscle Fibers in the Medial Gastrocnemius of Male and Female Rats. Front Neuroanat 15 , 734555, doi:10.3389/fnana.2021.734555 (2021). Hamers, F. P., Koopmans, G. C. & Joosten, E. A. CatWalk-assisted gait analysis in the assessment of spinal cord injury. J Neurotrauma 23 , 537-548, doi:10.1089/neu.2006.23.537 (2006). Samano, A. K. et al. Functional evaluation of therapeutic response for a mouse model of medulloblastoma. Transgenic Res 19 , 829-840, doi:10.1007/s11248-010-9361-1 (2010). Gerber, Y. N., Sabourin, J. C., Rabano, M., Vivanco, M. & Perrin, F. E. Early functional deficit and microglial disturbances in a mouse model of amyotrophic lateral sclerosis. PLoS One 7 , e36000, doi:10.1371/journal.pone.0036000 (2012). England, S. A. & Granata, K. P. The influence of gait speed on local dynamic stability of walking. Gait Posture 25 , 172-178, doi:10.1016/j.gaitpost.2006.03.003 (2007). Sakai, Y. et al. Targeted vibratory therapy as a treatment for proprioceptive dysfunction: Clinical trial in older patients with chronic low back pain. PLoS One 19 , e0306898, doi:10.1371/journal.pone.0306898 (2024). Kawaguchi, K., Asai, A., Mikawa, R., Ogiso, N. & Sugimoto, M. Age-related changes in lung function in National Center for Geriatrics and Gerontology Aging Farm C57BL/6N mice. Exp Anim 72 , 173-182, doi:10.1538/expanim.22-0109 (2023). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Accepted 19 Jun, 2025 Reviews received at journal 02 May, 2025 Reviewers agreed at journal 30 Apr, 2025 Reviewers invited by journal 30 Apr, 2025 Submission checks completed at journal 11 Apr, 2025 First submitted to journal 11 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5487702","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":450127289,"identity":"3a89c543-6378-451f-9ab5-2921471da5c1","order_by":0,"name":"Minako Kawai-Takaishi","email":"","orcid":"","institution":"National Center for Geriatrics and Gerontology","correspondingAuthor":false,"prefix":"","firstName":"Minako","middleName":"","lastName":"Kawai-Takaishi","suffix":""},{"id":450127290,"identity":"8b2c3017-5a95-4eac-9ab2-b7c283dff5e6","order_by":1,"name":"Tohru Hosoyama","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYJACZhDBj+AfwK+cB6ZFsoFkLQYEFCKAPXvvM+mCijo549vNj19+YaiVY2A8i183D89xM+kZZw4bm905ZmYtw3DcmIHhXAJ+LRJpbNK8bQcSt93IYTOWYDiW2MBwxoAYLXWJm2eQqIU5cYNEDvPDDww1RGg5c4zZmgfoF4kbaWbMwHAzZiPkF/b2NsbbPMAQ45+R/PjjDxBDgkCIIQM2aR6DwwxsEmeI1sHA/PEHQx0w6fQQr2UUjIJRMApGBAAAFDtAiCZLC4QAAAAASUVORK5CYII=","orcid":"","institution":"National Center for Geriatrics and Gerontology","correspondingAuthor":true,"prefix":"","firstName":"Tohru","middleName":"","lastName":"Hosoyama","suffix":""}],"badges":[],"createdAt":"2024-11-20 05:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5487702/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5487702/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-08270-1","type":"published","date":"2025-07-04T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81829085,"identity":"471b9240-0cad-470f-a429-5b0b8a4c6c7a","added_by":"auto","created_at":"2025-05-02 13:19:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1965806,"visible":true,"origin":"","legend":"\u003cp\u003eMuscle cross-sectional analysis showed no age-related morphological changes in muscle spindles. (\u003cstrong\u003eA\u003c/strong\u003e) Muscle cross-sections were made every 200 µm from top (start) to bottom (end), and muscle spindle morphology and numbers throughout the muscle were evaluated using immunofluorescent images. Arrowheads indicate muscle spindles at the equatorial region. Scale bar = 20 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Muscle spindle length and capsule thickness were evaluated in soleus and EDL and compared between young, middle-aged, and aged mice. Muscle spindle total area at the equatorial (\u003cstrong\u003eC\u003c/strong\u003e) and polar (\u003cstrong\u003eD\u003c/strong\u003e) regions were evaluated in soleus and EDL and compared between young, middle-aged, and aged mice. Five animals per age group and eight muscle spindles per muscle were analyzed.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/a97261e816ee4dc3f6a7604e.png"},{"id":81829081,"identity":"debb9ea0-f084-455b-9e0f-f4f003ff9508","added_by":"auto","created_at":"2025-05-02 13:19:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":707496,"visible":true,"origin":"","legend":"\u003cp\u003eNeither atrophy nor loss of intrafusal fibers occurred with aging. (\u003cstrong\u003eA\u003c/strong\u003e) The total area of the nuclear bag and chain fibers at the equatorial region of muscle spindles was evaluated in soleus (left 2 graphs) and EDL (right 2 graphs) and compared between young, middle-aged, and aged mice. (\u003cstrong\u003eB\u003c/strong\u003e) The total area of the nuclear bag and chain fibers at the polar region of muscle spindles was evaluated in soleus (left 2 graphs) and EDL (right 2 graphs) and compared between young, middle-aged, and aged mice. Five animals per age group and eight muscle spindles per muscle were analyzed. Individual muscle spindles had a mean of 2.1 nuclear bag fibers and 1.7 nuclear chain fibers (Table 1), so at least sixty fibers were included in each bar.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/041106555842f8ff8f64e0ec.png"},{"id":81829091,"identity":"47f4d0c3-fd7a-4f60-868b-3793342a70e1","added_by":"auto","created_at":"2025-05-02 13:19:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3544469,"visible":true,"origin":"","legend":"\u003cp\u003eBoth coil width and distance of the annulospiral endings appeared normal, even in aged mice. (\u003cstrong\u003eA\u003c/strong\u003e) Representative immunofluorescent images of the proprioceptive sensory neurons and muscle spindles in soleus and EDL. NF: neurofilament, VGLUT1: vascular glutamate transporter 1, VCAN: versican. Scale bar = 20 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Coil width (length of the red bar) and distance (length of the blue bar) of the annulospiral endings. Scale bar = 10 µm. Coil width (\u003cstrong\u003eC\u003c/strong\u003e) and distance (\u003cstrong\u003eD\u003c/strong\u003e) were evaluated in soleus (upper 2 graphs) and EDL (lower 2 graphs) and compared between young, middle-aged, and aged mice. Six animals per age group and at least five muscle spindles per muscle were analyzed. Ten coils (all coils of unraveling fiber) were measured per nerve endings.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/c53e59b5d2ebb1f2e1c7880e.png"},{"id":81829904,"identity":"03c2f30d-3f1f-4929-ae89-f763eafa9fc8","added_by":"auto","created_at":"2025-05-02 13:27:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2200955,"visible":true,"origin":"","legend":"\u003cp\u003eAnnulospiral endings of the proprioceptive sensory neurons deteriorated \u0026nbsp;with aging. (\u003cstrong\u003eA\u003c/strong\u003e) The coil number of the nuclear bag and chain fibers was measured in soleus (left 2 graphs) and EDL (right 2 graphs) and compared between young, middle-aged, and aged mice. (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of the annulospiral endings in young, middle-aged, and aged mice. Scale bar = 20 µm. (\u003cstrong\u003eC\u003c/strong\u003e) The number of muscle spindles with blebbed sensory neurons was measured in soleus and EDL and compared between young, middle-aged, and aged mice. Red arrowheads indicate the blebbing of neurons. Scale bar = 10 µm. (\u003cstrong\u003eD\u003c/strong\u003e) Unraveling fibers in the nuclear bag and chain fibers were calculated and compared between young, middle-aged, and aged mice. Six animals per age group and at least five muscle spindles per muscle were analyzed.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/db0d2ec8bd2d3e009406e42f.png"},{"id":81829906,"identity":"616e8f19-e41f-4c40-806f-fc9d7383c8bf","added_by":"auto","created_at":"2025-05-02 13:27:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2190374,"visible":true,"origin":"","legend":"\u003cp\u003eAfferent neurons were atrophied in aged mice. (\u003cstrong\u003eA\u003c/strong\u003e) Representative immunofluorescent images of afferent neurons (yellow arrowhead). Scale bar = 10 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Afferent axon area, axon diameter, and NF\u003csup\u003e+\u003c/sup\u003e occupancy were measured in soleus (upper 3 graphs) and EDL (lower 3 graphs) and compared between young, middle-aged, and aged mice. NF\u003csup\u003e+\u003c/sup\u003e occupancy was calculated as follow; (NF\u003csup\u003e+\u003c/sup\u003e area)/(VGLUT1\u003csup\u003e+\u003c/sup\u003e area) x100. Six animals per age group and at least five muscle spindles per muscle were analyzed.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/3430d40c54dabe74faca5217.png"},{"id":81829092,"identity":"fd358dd7-5699-41bf-b114-e79d906d1f78","added_by":"auto","created_at":"2025-05-02 13:19:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1401874,"visible":true,"origin":"","legend":"\u003cp\u003eNeuromuscular junctions deteriorated with age. (\u003cstrong\u003eA\u003c/strong\u003e) Representative fluorescent images of NMJs in young, middle-aged, and aged mice. Scale bar = 20 µm. Innervation rate (\u003cstrong\u003eB\u003c/strong\u003e), the number of fragments (\u003cstrong\u003eC\u003c/strong\u003e), and endplate area (\u003cstrong\u003eD\u003c/strong\u003e) were calculated from fluorescent images of NMJs and compared each parameter between 3 age categories. Innervation rate was defined as the percentage of nerve ending occupancy, which was calculated as follows: (NF + Synapsin\u003csup\u003e+\u003c/sup\u003e area/α-BTX\u003csup\u003e+\u003c/sup\u003e area) x100. Five animals per age group and fifty endplates per muscle were analyzed.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/bf36ae83fff79061d89ced58.png"},{"id":81830381,"identity":"1d16043f-7526-41b3-8251-9f21ac69cf0d","added_by":"auto","created_at":"2025-05-02 13:35:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1792883,"visible":true,"origin":"","legend":"\u003cp\u003eAging alters the gait function of mice. (\u003cstrong\u003eA\u003c/strong\u003e) Representative footprint trails of young, middle-aged, and aged mice on a homemade walking lane (40 × 8 cm). Strength, sway, stance, angle, and anterior-posterior gap were measured from footprint trails. (\u003cstrong\u003eB\u003c/strong\u003e) Five parameters of footprints were compared between young, middle-aged, and aged mice. (\u003cstrong\u003eC\u003c/strong\u003e) Representative individual footprints of young, middle-aged, and aged mice. Ground contact area, perimeter area, finger spread, and plantar length were measured from individual footprints. (\u003cstrong\u003eD\u003c/strong\u003e) Four parameters of footprints were compared between young, middle-aged, and aged mice. Five animals per age group were analyzed.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/ad01d32b6a6ad68d2da6a83c.png"},{"id":86179113,"identity":"8a4165c3-3112-4ca3-8b4e-8ae7dfcc3a8e","added_by":"auto","created_at":"2025-07-07 16:15:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14569017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5487702/v1/d1bb9ce2-20e9-4003-bd74-8b6a3283958d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Muscle spindle afferent neurons preferentially degenerate with aging","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCoordinated movement, including walking, standing, and sense of balance, is affected with aging\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, increasing the incidence of falling and being bedridden and decreasing the quality of life of older adults\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although activation of the α-motoneurons (α-MNs) and extrafusal fibers is essential for all voluntary movements, these functions are under the control of the nervous system. The proprioceptive neurons transmit information to α-MN and central nervous system about changes in the position and movement of body parts to adjust the contraction or relaxation of muscles, a part of the prerequisite for coordinating complex movements.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Therefore, clarifying when and how the somatosensory system, including proprioception, is affected by aging will provide important information for future research plans aimed at preventing motor dysfunction in older adults. Muscle spindles, major proprioceptive receptors within skeletal muscles, sense changes in the length and velocity of stretched skeletal muscles, and intrafusal fibers within muscle spindles regulate their sensitivity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Intrafusal fibers comprise nuclear bag (bag 1 and bag 2) and nuclear chain fibers, with the equatorial part innervated by two types of afferent proprioceptive sensory neurons (Ia and II afferents) and the polar part innervated by γ-motoneurons (γ-MN)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Ia afferent innervates nuclear bag 1, bag2, and nuclear chain fibers, while II afferent innervates nuclear bag2 and nuclear chain fibers\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Equatorial Ia sensory endings have \u0026ldquo;annulospiral terminals,\u0026rdquo; a unique structure coiled around intrafusal fibers, while γ-MNs form neuromuscular junctions (NMJs) as well as α-MNs\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Nerve endings of proprioceptive sensory neurons can undergo detrimental structural changes due to disease or injury; however, our understanding of how aging affects proprioceptive sensory neurons and intrafusal fibers remains limited\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, gait analysis was used to evaluate the motor function of mice. Although there are differences between bipeds and quadrupeds, the locomotor behavior of terrestrial mammals is achieved by rhythmic and coordinated movement of two or four legs with multiple joints\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Gait dysfunction and balance disorders are pervasive phenomena of human aging. Clinical studies demonstrate persistent reductions in gait speed and stride length\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003eand increases in gait variability\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003ein older adults when compared to young adults. The effects of aging on gait in mice are being elucidated, including evaluating the translational potential for humans\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In addition, the role of muscle spindle in walking ability has been investigated in mice lacking proprioception\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, indicating that proprioceptive sensory feedback from muscle spindles contributes to the emergence of an alternation pattern of flexor and extensor muscle activity. The rhythmic stepping movement of the legs is divided into the stance phase when the foot is on the ground, and the swing phase when the foot is lifted off the ground and moving. The mutant mice, selectively eliminated muscle spindle activation, confronted with a walking task exhibited locomotor pattern degradation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Although the mechanisms underlying age-related changes in gait function in older adults and aged mice include age-related mobility problems due to cardiopulmonary, musculoskeletal, and overt neurological conditions (\u003cem\u003ee\u003c/em\u003e.\u003cem\u003eg\u003c/em\u003e. stroke and Parkinson\u0026rsquo;s disease), the analysis of age-related changes in muscle spindle and gait pattern would provide useful information to characterize age-related symptoms in aged mice and humans.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to clarify age-related structural alteration in muscle spindles and its impact on gait functions. To achieve this objective, we analyzed age-related morphological changes in muscle spindles in detail by combining transverse and longitudinal sections of mouse skeletal muscles of three different age groups, including young (3 months old), middle-aged (12\u0026ndash;15 months old), and aged (26 months old). This study will contribute to understanding the mechanisms of age-related increases in the risk of falls in older adults and establishing a novel strategy for achieving healthy longevity.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e \u003cb\u003e2.1 Aging does not influence the number of muscle spindles and intrafusal fibers in muscle or the cross-sectional area of muscle spindles\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe length of the equatorial and juxta-equatorial regions of the muscle spindles of mouse leg muscles was approximately 1,000 \u0026micro;m, with an overall length of 2,000\u0026ndash;3,000 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, we evaluated muscle spindle morphology and numbers throughout the muscle by cross-sections made every 200 \u0026micro;m, a cutting interval suitable for evaluating the equatorial and polar morphologies with a minimum number of cross-sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Cross-sectional analysis of the whole muscle revealed that the soleus and EDL muscles possessed an average of 12 muscle spindles, two nuclear bag fibers per muscle spindle, and 1.7 nuclear chain fiber per muscle spindle (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These values were consistent across all the age groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumbers of muscle spindle in muscle, nuclear bag fiber in muscle spindle, and nuclear chain fiber in muscle spindle.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYoung SOL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMiddle SOL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAged SOL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eYoung EDL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMiddle EDL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAged EDL\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle Spindle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNBF/Spindle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNCF/Spindle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eNBF: Nuclear bag fiber. NCF: Nuclear chain fiber. SOL: Soleus. EDL: Extensor digitorum longus.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe average length of muscle spindles determined from the analysis of muscle cross-sections was similar for all age groups, except for a decrease in the aged EDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similarly, the capsule thickness and average cross-sectional area of the muscle spindle did not significantly differ between the age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eNext, we analyzed the age-related morphological changes in the intrafusal fibers. The average cross-sectional area of the intrafusal fibers, including nuclear bag and chain fibers, was calculated at the equator and polar of the muscle spindles. At the equator and polar muscle spindles, intrafusal fibers in the EDL of aged mice were larger than those in young and middle-aged mice, whereas no significant difference in the soleus was observed in either age group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). These results indicate that neither atrophy nor loss of intrafusal fibers occurs with age.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Annulospiral endings of the proprioceptive sensory neurons deteriorate with aging\u003c/h2\u003e \u003cp\u003eAnalyzing muscle histology in longitudinal sections is important to clarify age-related structural changes in the annulospiral endings of proprioceptive sensory neurons. In this study, we attempted to construct the stacked images of muscle fibers from 150 \u0026micro;m of longitudinal muscle sections as our preliminary investigations showed that the maximum diameter of the muscle spindle was 50\u0026ndash;100 \u0026micro;m.\u003c/p\u003e \u003cp\u003eAnnulospiral endings appeared to be progressively obscured in middle-aged and aged mice in representative immunofluorescence images (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The coil width and distance of the annulospiral endings were measured on the images and quantitatively compared by age (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Both the coil width and distance were similar for all age groups, except for the coil width of nuclear bag fiber of the soleus, which increased in middle-aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then analyzed the number of coils in the annulospiral endings and compared them with age. The number of coiled annulospiral endings was reduced in both the nuclear bag and chain fibers of aged mice, indicating that sensory neuron coiling to intrafusal fibers decreased with increase in age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Reductions in the number of coils included not only simple changes in number (decreased coils) but also fibers with indistinct coils (indistinct spirals) or complete loss of spirals (disappearance) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Accompanying the disruption of the spiral-shaped structure, the number of blebbed sensory neurons in the annulospiral endings and unraveling fibers increased with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Unraveled fibers were particularly increased in nuclear bag fibers, even in middle-aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results indicate that the spiral-shaped structure of the annulospiral endings disintegrates with age, especially in the nuclear bag fiber, where effects are observed in the early phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence analysis of vesicular glutamate transporter 1 (VGLUT1), which is abundant in annulospiral endings, and neurofilament protein, which occurs in neuronal axons, indicated the possible atrophy of afferent axons innervating intrafusal fibers with aging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; yellow arrowhead).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the soleus and EDL, we confirmed that the afferent annulospiral ending area and axonal diameter were decreased in aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Neuromuscular junctions deteriorate with age\u003c/h2\u003e \u003cp\u003eIn addition to the muscle spindles, we analyzed age-related morphological changes in NMJs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In young mice, the presynaptic α-MN endings (NF\u003csup\u003e+\u003c/sup\u003e/Synapsin\u003csup\u003e+\u003c/sup\u003e) innervate the postsynaptic α-bungarotoxin (α-BTX)\u003csup\u003e+\u003c/sup\u003e endplate. However, the percentage of nerve ending occupancy (innervation rate) was significantly decreased in middle-aged mice and further deteriorated in aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Endplate fragmentation and enlargement of the endplate area were observed, with deterioration of motor innervation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). These results support the widely accepted hypothesis that NMJs deteriorate with increasing age.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Aging alters gait function of mice\u003c/h2\u003e \u003cp\u003eWe performed a footprint test to determine how age-related deleterious morphological changes in muscle spindles and neuromuscular junctions correlate with motor function (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The results showed that aging did not affect the stride, step length, base of support, or angle; however, the distance between the forelimb and hindlimb on the same side (anterior-posterior gap) was significantly increased in aged mice compared to young and middle-aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB; p\u0026thinsp;=\u0026thinsp;0.051 and p\u0026thinsp;=\u0026thinsp;0.0307, respectively). In addition, individual footprint parameters were measured to clarify the changes in the base of support of the forelimbs and hindlimbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The ground contact area (p\u0026thinsp;=\u0026thinsp;0.0023 and p\u0026thinsp;=\u0026thinsp;0.0184), perimeter area (p\u0026thinsp;=\u0026thinsp;0.0065 and p\u0026thinsp;=\u0026thinsp;0.0152), and plantar length (p\u0026thinsp;=\u0026thinsp;0.0026 and p\u0026thinsp;=\u0026thinsp;0.0079) of the hind limbs were significantly higher in aged than young and middle-aged mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Considering that young mice do not place their heels on the ground during leg grounding but shift their weight with their toes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), these results indicate a decline in gait functions, such as weight bearing and weight shifting, in aged mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eAging correlates with significant changes in α-MNs, extrafusal fibers, and NMJs in skeletal muscle\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, yet the correlation between aging and changes in muscle spindle remains poorly understood. The lack of useful molecular markers for muscle spindle research has hindered the progression of research on muscle spindle maintenance and morphogenesis. Recently, several molecular markers that define muscle spindle structures have been identified using a combination of transcriptomic and proteomic analyses\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this study, we used versican (VCAN), a recently identified molecular marker of muscle spindles, for morphological analysis, resulting in a more accurate and detailed structural analysis of the internal equatorial portion of muscle spindles using the intracapsular extracellular matrix as an indicator. In addition to VCAN, other molecular markers have been identified\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and a combination of these markers is expected to enable more detailed studies.\u003c/p\u003e \u003cp\u003eThis study demonstrated that the muscle spindle, an important structure for proprioceptive sensory feedback, was deleteriously affected by aging in addition to the well-known age-related degradation of NMJs\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Regarding NMJs, it has been demonstrated that age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In other words, endplate fragmentation is an aging phenomenon but does not induce dysfunction. Although the annulospiral ending of proprioceptive nerve terminal is not a synapse, the same may be true for our result indicating age-related deterioration of afferents in muscle spindle. In fact, the percentage of the neurofilament\u003csup\u003e+\u003c/sup\u003e area to the VGLUT1\u003csup\u003e+\u003c/sup\u003e area was significantly decreased with aging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This result suggests that neurofilament, which plays essential roles in regulating neuronal diameter and axonal transport, decrease with age, making it difficult to maintain the structure of well-organized annulospiral endings in aged individuals. A previous study using aged rat has shown that the axonal diameter, and the dynamic response of Ia afferents both decreased with age\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Interestingly, our results indicated that age-related morphological changes occur in the nuclear bag fibers of the muscle spindles preceding any in nuclear chain fibers. Sensory neurons normally consist of only one Ia afferent fiber per spindle, and all intrafusal fibers within the spindle are innervated by that sensory neuron. Nuclear bag fibers consist of bag 1 and bag 2 fibers, and the sensory endings attached to each fiber are maximally responsive to dynamic and static sensitivity, respectively. Nuclear chain fibers respond to static sensitivity as do bag 2 fibers\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Taken together, although this study did not distinguish between bag 1 and bag 2 fibers and did not perform the physiological investigations, age-related deterioration of sensory terminals on nuclear bag fibers may hinder proprioceptive sensory feedback to the central nervous system in response to unanticipated body movements, contributing to increased motor impairment and fall risk in older adults.\u003c/p\u003e \u003cp\u003eAtrophy of extrafusal fibers occurs with age\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and the number of muscle spindles and capsule thickness are altered with aging in rats and humans\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These reports clearly indicate that muscle spindles and extrafusal fibers are affected by aging. However, in this study, we observed no morphological changes in capsule thickness and intrafusal fibers. Morphological analysis of muscle spindles in other species and older individuals using similar research methods remains warranted. Whether these discrepancies were attributed to differences in experimental techniques, age, or animal species remains unclear. Nevertheless, our study, that combined transverse and longitudinal sections with immunofluorescence analysis, provides more accurate and precise results. Morphological analysis of muscle spindles in other species using similar research methods remains warranted. Previous studies using mouse skeletal muscle have shown that intrafusal fibers do not decrease in number or atrophy with age\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, consistent with the findings of this study, validating our research methodologies. Furthermore, our data were obtained by experimenting with mice older (26 months old) than those previously utilized (15\u0026ndash;17 months old)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, providing novel insights into age-related changes in muscle spindles and proprioceptive sensory neurons.\u003c/p\u003e \u003cp\u003eIn this study, we used numbers of animals of either sex. In the results of longitudinal-sectional muscle analysis using the same number of males and females, few parameters significantly differed between males and females (detailed data not shown). In addition, the study evaluating sexual dimorphism of the muscle spindle in rats has shown no significant differences in the number and morphological properties of intrafusal fibers or muscle spindles in male and female rats\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Therefore, at least in rodents, we assume that sex-related differences in the morphology of muscle spindle are negligibly small.\u003c/p\u003e \u003cp\u003eThe disease-related changes in gait patterns in rodents have been well documented using disease models, such as spinal cord injury, amyotrophic lateral sclerosis, and brain tumors\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Recently, data on age-related gait changes has also been accumulated \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In these studies, it has been demonstrated that aged mice exhibited significantly altered gait signatures compared to young animals and humans. It has also been reported that aged, freely moving mice exhibited significantly altered gait signatures compared to young animals, including changes in cadence, gait, variability, and footfall pattern distribution. In this study, we evaluated the gait performance of mice using the footprint test, a classical gait analysis. Unfortunately, since our gait analysis model is simple, it was limited to a few steps for technical reasons and may not have adequately captured subtle changes in locomotion dynamics. However, we found that the gait style changed with age. Interestingly, although the body weight, grip strength, muscle spindle afferents, and NMJs were already undergoing age-related changes in middle-aged mice (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the change in gait style only appeared as the mice aged. This suggests that deleterious changes in muscle spindles, such as poor afferent innervation, precede gait disturbance and are an early indicator of gait disturbance in older adults.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of body weight, muscle wet weight, muscle wet weight/body weight, and grip strength/body weight in mice.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYoung\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMiddle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAged\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e36.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle wet weight (mg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSOL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEDL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle wet weight/Body weight (mg/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSOL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEDL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrip strength/Body weight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForelimb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFour-limbs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cem\u003eSOL: Soleus. EDL: Extensor digitorum longus.\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn this study, we did not evaluate movement speed, such as gait or swing speed; however, a decrease in gait speed has been demonstrated in aged mice\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The control of action potential activity by proprioceptive sensory feedback from muscle spindles is necessary for high-speed locomotion in mice\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Egr3 mutant mice, selectively eliminated muscle spindle activation, confronted with a walking task exhibited a pronounced extension in the duration of tibialis anterior (TA) muscle burst activity during the swing phase. This can cause co-contraction of TA and antagonist (gastrocnemius) muscle, possibly stiffening the ankle joint at the end of the swing phase\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. This outcome is similar to the one previously found in humans, where the widening muscle activity emerged in response to aging\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Therefore, the age-related increase of plantar contact area in this study might be influenced by insufficient activity of the muscles controlling the ankle joint due to decline in muscle spindle function, the increase in body weight and the decrease in grip strength. Furthermore, the recent study using muscle spindle deficient mice has demonstrated that muscle spindle feedback is necessary for the smooth trajectory of the paw and the control of foot placement following the stumbling corrective reaction\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Since increased gait speed leads to increased dynamic stability\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, it is possible that a series of these linkages result in gait disturbances and falls in older adults. Recently, a method was developed to activate proprioceptive functions in older adults using vibration stimulation in a unique frequency band specific to muscle spindles\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, which may facilitate the establishment of a novel therapeutic strategy targeting poor afferent innervation of muscle spindles and the resulting gait disorders and falls.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Animals\u003c/h2\u003e \u003cp\u003eYoung (3 months) and middle-aged (12\u0026ndash;15 months) C57BL/6N mice were obtained from Clea Japan, Inc. (Tokyo, Japan), while aged (26 months) C57BL/6N mice were provided by the NCGG Aging Farm (Aichi, Japan)\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Both male and female mice were used. The mice were sacrificed at appropriate time points by cervical dislocation under anesthesia with Medetomidine hydrochloride (0.3 mg/kg), Midazolam (4 mg/kg), and Buprenorphine (5 mg/kg), and the hind limb muscles were collected and weighed. The dissected muscles, including the tibialis anterior (TA), soleus, and extensor digitorum longus (EDL) muscles, were frozen in liquid nitrogen-chilled isopentane and stored at -80 ˚C until use.\u003c/p\u003e \u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Center for Geriatrics and Gerontology (no. 6\u0026ndash;6). The study was conducted in accordance with the relevant guidelines and carried out in compliance with the ARRIVE guidelines. The methods were carried out in accordance with the approved guidelines and the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Morphological analysis of muscle spindle on muscle cross-sections\u003c/h2\u003e \u003cp\u003eBoth the soleus and EDL muscles were used for cross-sectional muscle analysis. The breakdown of males and females for each muscle and age was as follows: young soleus (four males and one female), young EDL (two males and three females), middle-aged soleus and EDL (three males and two females), and aged soleus and EDL (five males). Transverse sections of 10-\u0026micro;m thickness were prepared every 200 \u0026micro;m from the proximal to distal ends of muscle and attached to a glass slide (25\u0026ndash;30 sections for each muscle) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). After fixation in 4% paraformaldehyde (PFA), immunofluorescent analysis was performed on muscle cross-sections using the following antibodies: anti-Laminin α2 (1:1,000, Santa Cruz Biotechnologies, CA, USA), anti-S46 (1:2,000, the Developmental Studies Hybridoma Bank (DSHB), IA, USA), Alexa Fluor 594-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1,000, Thermo Fisher Scientific, MA, USA). Immunofluorescent images were obtained using a fluorescence microscope (Keyence Bz-X900 and Leica Mica).\u003c/p\u003e \u003cp\u003eThe number of muscle spindles per muscle, number of intrafusal fibers per muscle spindle, muscle spindle length, average cross-sectional area, and capsule thickness were calculated from the images. Regarding the cross-sectional area of the muscle spindle or each intrafusal fiber, the area of the equatorial region was calculated as the average of sections 1 to 3, which had large areas and wide intracapsular spaces (\u003cem\u003ei\u003c/em\u003e.\u003cem\u003ee\u003c/em\u003e., ECM rich), and the area of the polar region was calculated as the average of the other sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Morphological analysis of muscle spindle on muscle longitudinal sections\u003c/h2\u003e \u003cp\u003eBoth the soleus and EDL muscles were used for longitudinal section muscle analysis. The sex-based breakdown in each muscle and age was three males and three females. Continuous longitudinal sections of 150-\u0026micro;m thickness were prepared, and sections were gently thawed in -10 ˚C-chilled 30% glycerin/Phosphate-buffered saline (PBS). Longitudinal sections were fixed in 4% PFA, suspended in solution, and immunofluorescent staining was performed using the following antibodies: anti-VGLUT1 (1:2,000; Synaptic Systems, Gottingen, Germany), anti-neurofilament (NF; 1:2,000; Abcam, Cambridge, UK), anti-versican (VCAN; 1:500; Abcam), anti-S46, Alexa Fluor 594-conjugated goat anti-rabbit IgG, and Cy5-conjugated goat anti-mouse IgG (1:1,000; Jackson ImmunoResearch Laboratories, PA, USA). S46 and VCAN were labeled with Alexa Fluor 488 using Zenon Alexa Fluor 488 labeling reagent (Thermo Fischer Scientific). NF and VGLUT1 were labeled with Cy5 and Alexa Fluor 594, respectively. Immunofluorescent images were obtained using a laser scanning confocal system (LSM780; Carl Zeiss, Oberkochen, Germany; STELLARIS, Leica, Wetzlar, Germany). Values were calculated as the average of 5\u0026ndash;8 muscle spindles per muscle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Morphological analysis of NMJs\u003c/h2\u003e \u003cp\u003eThe TA muscles were used for the morphological analysis of NMJs, and the sex-based breakdown for each age group was five males. Continuous longitudinal sections of 150-\u0026micro;m thickness were prepared, and sections were gently thawed in -10˚C-chilled 30% PBS. Longitudinal sections were fixed in 4% PFA while suspended in the solution, and immunofluorescent staining was performed using the following antibodies: anti-synapsin (1:1,000; Cell Signaling Technology, MA, USA), anti-NF, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1,000). Alexa Fluor 594-conjugated α-BTX (1:500, Sigma-Aldrich, MO, USA) was used for NMJ staining. Immunofluorescent images were obtained using laser scanning confocal systems (LSM780 and STELLARIS), and the NMJ and endplate morphologies were evaluated. Values were calculated as an average of 50 plates per muscle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Footprint test\u003c/h2\u003e \u003cp\u003eBefore muscle sampling, the mice\u0026rsquo;s footprints were recorded using a homemade walking lane (40 cm \u0026times; 8 cm). Mice were acclimated to the running lane in advance, had ink (black for forelimbs and green for hindlimbs) applied to their feet, and walked on graph paper. From the obtained footprints, we measured the parameters described below in the basis of the axis of the direction of motion; the stride length, step length, base of support, paw angle, distance between the ipsilateral front and rear limbs (anterior-posterior gap). In addition, the ground contact area of the palm, perimeter, finger openings, and plantar length were also calculated from each paw prints. The sex-based breakdown at each age was as follows: young mice (five males), middle-aged mice (two males and three females), and aged mice (five males).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Grip strength measurement\u003c/h2\u003e \u003cp\u003eAccording to the manufacturer's instructions, the grip strength of the fore limb and four limbs was measured using a GPM-101B (Melquest, Toyama, Japan). Each measurement was conducted three times with a 10-minute rest between each experimental set, and the maximum value of the three experiments was used as representative data for each mouse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.7 Statistical analyses\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Differences in means between groups were evaluated using one-way ANOVA and multiple comparison tests (significance level, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All statistical analyses were performed using the GraphPad Prism 9 software (GraphPad Software, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of the National Center for Geriatrics and Gerontology (no. 6\u0026ndash;6).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eJSPS-KAKENHI, 24K14396 to M.T-K.\u003c/p\u003e \u003cp\u003eJapanese Ministry of Health, Labor, and Welfare; 21\u0026ndash;44, 24\u0026thinsp;\u0026minus;\u0026thinsp;5, 24\u0026ndash;28 and Japan Health Research Promotion Bureau; 2022-B-02 to T.H.\u003c/p\u003e \u003cp\u003eCompeting interests\u003c/p\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM. K-T. contributed to conceptualization, investigation, data curation, formal analysis, funding acquisition, writing - original draft, and writing - review \u0026amp; editing. T. H. contributed to conceptualization, data curation, writing - original draft, writing - review \u0026amp; editing, and funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Dr. Ken Watanabe for his helpful comments on this study. The anti-S46 antibody was obtained from the DSHB, developed under the NICHD, and maintained by the University of Iowa.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll the data analyzed and presented in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFried, L. P., Ferrucci, L., Darer, J., Williamson, J. D. \u0026amp; Anderson, G. 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Age-related changes in lung function in National Center for Geriatrics and Gerontology Aging Farm C57BL/6N mice. \u003cem\u003eExp Anim\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 173-182, doi:10.1538/expanim.22-0109 (2023).\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"aging, muscle spindle, intrafusal fibers, proprioceptive sensory neuron, annulospiral ending, movement disorders","lastPublishedDoi":"10.21203/rs.3.rs-5487702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5487702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMuscle spindles sense changes inmuscle length and transmit them to the central nervous system. Proprioception is essential for gait and postural maintenance, the abnormality of which has been linked to gait disorders and the risk of falling in older adults. However, the effects of aging on the muscle spindle structure remain nebulous. This study investigated age-related structural changes in themuscle spindles (from the equator to the polar) in the soleus and extensor digitorum longus (EDL) muscles of young, middle-aged, and aged mice. The findings indicated that the shape of the annulospiral endings of the sensory neurons began to deteriorate in middle-aged compared with young mice and was further exacerbated in aged mice. These changes were particularly pronounced in the nuclear bag fibers, whereas no significant age-related changes were observed in the intrafusal fibers or capsules. A decline in gait function due to changes in weight-bearing and weight-shifting in aged mice was also observed, suggesting that the deterioration of proprioceptive sensory neurons that innervate the nuclear bag fiber responsible for dynamic sensitivity prevents proper coordinated movement and contributes to movement disorders in aged animals including humans, together with the functional decline of extrafusal fibers.\u003c/p\u003e","manuscriptTitle":"Muscle spindle afferent neurons preferentially degenerate with aging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-02 13:19:50","doi":"10.21203/rs.3.rs-5487702/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-06-19T22:35:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-02T12:01:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83523151393781237007630171845343254862","date":"2025-04-30T09:48:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-30T08:11:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-12T02:10:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-11T09:44:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f480357a-b9f3-4fdb-81b3-ccde3ae55de5","owner":[],"postedDate":"May 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47899599,"name":"Health sciences/Anatomy/Musculoskeletal system"},{"id":47899600,"name":"Biological sciences/Neuroscience/Motor control/Neuromuscular junction"}],"tags":[],"updatedAt":"2025-07-07T16:04:58+00:00","versionOfRecord":{"articleIdentity":"rs-5487702","link":"https://doi.org/10.1038/s41598-025-08270-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-04 15:57:14","publishedOnDateReadable":"July 4th, 2025"},"versionCreatedAt":"2025-05-02 13:19:50","video":"","vorDoi":"10.1038/s41598-025-08270-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-08270-1","workflowStages":[]},"version":"v1","identity":"rs-5487702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5487702","identity":"rs-5487702","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}