Striking Denervation of Neuromuscular Junctions without Lumbar Motoneuron Loss in Geriatric Mouse Muscle

Reasons for the progressive age-related loss of skeletal muscle mass and function, namely sarcopenia, are complex. Few studies describe sarcopenia in mice, although this species is the mammalian model of choice for genetic intervention and development of pharmaceutical interventions for muscle degeneration. One factor, important to sarcopenia-associated neuromuscular change, is myofibre denervation. Here we describe the morphology of the neuromuscular compartment in young (3 month) compared to geriatric (29 month) old female C57Bl/6J mice. There was no significant difference in the size or number of motoneuron cell bodies at the lumbar level (L1–L5) of the spinal cord at 3 and 29 months. However, in geriatric mice, there was a striking increase (by ∼2.5 fold) in the percentage of fully denervated neuromuscular junctions (NMJs) and associated deterioration of Schwann cells in fast extensor digitorum longus (EDL), but not in slow soleus muscles. There were also distinct changes in myofibre composition of lower limb muscles (tibialis anterior (TA) and soleus) with a shift at 29 months to a faster phenotype in fast TA muscle and to a slower phenotype in slow soleus muscle. Overall, we demonstrate complex changes at the NMJ and muscle levels in geriatric mice that occur despite the maintenance of motoneuron cell bodies in the spinal cord. The challenge is to identify which components of the neuromuscular system are primarily responsible for the marked changes within the NMJ and muscle, in order to selectively target future interventions to reduce sarcopenia

Age-related changes in the Schwann cells (SCs) at the NMJs. Low (A,B) and high (C,D,E,F,G,H) power views.

<p>Confocal images of NMJs and SCs in the EDLs of 3 (A,C,E,G) and 29 (B,D,F,H) month old mice. High power views show that SCs of young mice are structured and completely overlay with the muscle endplates (C,E,G). SCs of old mice are disorganised, partially cover muscle endplates and have swollen endings (white arrows) (D,F,H). Scale bars are 150 µm.</p

Lumbar spinal cord α-motoneurons.

<p>α-motoneurons stained with toluidine blue in the ventro-lateral quarter of the spinal cord between the bold lines were counted (A). The maximum diameter of α-motoneurons was obtained by measuring the longest axis through the nucleolus (B). Motoneurons with no visible nucleolus (*) or with diameters <25 µm were not included. Total number of α-motoneuron profiles (C) and average diameter (D) of α-motoneurons were analyzed in 20 sections of a 1 in 20 series of the lumbar region (L1–L5) in spinal cords for each mouse. There was no significant change in the average number (C) and diameter (D) of α-motoneurons between mice aged 3 and 29 months. N = 4 mice per age group. Values are mean ± s.e.m.</p

Whole mount immunohistochemical preparations of EDL (A–F) and soleus (G–L) muscles from 3 and 29 month old mice.

<p>Muscles were stained with synaptophysin (A,D,G,J; red) to detect pre-synaptic neuronal compartments and with α-bungarotoxin (B,E,H,K; green) to detect acetylcholine receptors at the muscle endplates. Overlays are shown in (C,F,I,L; yellow). Muscle endplates that are positive for only α-bungarotoxin (green) are not innervated. One such endplate is indicated (white circle) in the 29 month old EDL (D,E,F). NMJs in the 3 month old EDL appear compact and well defined (A–C), while many NMJs have a diffused, irregular and fragmented appearance in the 29 month old EDL (D–F). In contrast, the NMJs in soleus of geriatric mice (J–L) did not show morphological changes when compared to 3 month old NMJs (G–I). Scale bars are 75 µm.</p

Phenotypic characterisation of 3 and 29 month old mice: tibial bone length, body weight, limb muscle weights and abdominal fat pad weight.

<p>For body and muscle weights absolute values as well as values standardised to tibial bone length are shown.</p><p>**P<0.05. All values are mean ± s.e.m.</p

Fast 2B, Fast 2A and slow myofibres in the inner TA, EDL and soleus muscles.

<p>Antibodies for MHCIIB, MHCIIA and MHCI were used to detect three different types of myosin respectively: fast 2B (A,E,I,M,Q,U), fast 2A (B,F,J,N,R,V) and slow (C,G,K,O,S,W). The overlay of these is shown in D,H,L,P,T and X. Myofibres not detected with either of these antibodies were presumed to be fast 2× (MHCIIX) (a few are indicated by asterisks * in D, H, X). Along with the slow type myofibres, antibody for MHCI also stains muscle spindles (arrow in O). Grouping of slow type 1 myofibres was seen in 29 month soleus muscles (outlined in W). Scale bars are 50 µm.</p

Percentage (A,C,E) and average cross-sectional area (B,D,F) of different myofibre types in the TA, EDL and soleus muscles of 3 and 29 month old mice.

<p>There are no error bars on some of the graphs (D,F) as the myofibre types were present in less than 3 animals (i.e. variation in myofibre type distribution occurred in different animals within the same age group). N = 4 animals per age group. *P<0.05, **P<0.005. Values are mean ± s.e.m.</p