Impulse Conduction Increases Mitochondrial Transport in Adult Mammalian Peripheral Nerves <i>In Vivo</i>

<div><p>Matching energy supply and demand is critical in the bioenergetic homeostasis of all cells. This is a special problem in neurons where high levels of energy expenditure may occur at sites remote from the cell body, given the remarkable length of axons and enormous variability of impulse activity over time. Positioning mitochondria at areas with high energy requirements is an essential solution to this problem, but it is not known how this is related to impulse conduction <i>in vivo</i>. Therefore, to study mitochondrial trafficking along resting and electrically active adult axons <i>in vivo</i>, confocal imaging of saphenous nerves in anaesthetised mice was combined with electrical and pharmacological stimulation of myelinated and unmyelinated axons, respectively. We show that low frequency activity induced by electrical stimulation significantly increases anterograde and retrograde mitochondrial traffic in comparison with silent axons. Higher frequency conduction within a physiological range (50 Hz) dramatically further increased anterograde, but not retrograde, mitochondrial traffic, by rapidly increasing the number of mobile mitochondria and gradually increasing their velocity. Similarly, topical application of capsaicin to skin innervated by the saphenous nerve increased mitochondrial traffic in both myelinated and unmyelinated axons. In addition, stationary mitochondria in axons conducting at higher frequency become shorter, thus supplying additional mitochondria to the trafficking population, presumably through enhanced fission. Mitochondria recruited to the mobile population do not accumulate near Nodes of Ranvier, but continue to travel anterogradely. This pattern of mitochondrial redistribution suggests that the peripheral terminals of sensory axons represent sites of particularly high metabolic demand during physiological high frequency conduction. As the majority of mitochondrial biogenesis occurs at the cell body, increased anterograde mitochondrial traffic may represent a mechanism that ensures a uniform increase in mitochondrial density along the length of axons during high impulse load, supporting the increased metabolic demand imposed by sustained conduction.</p></div

Outcome measures between BM-MSC treatment groups and control groups.

<p>SD – standard deviation;</p>*<p>Kruskal-Wallis test;</p>§<p>one way ANOVA, Tukey post-test;</p><p>HPF – high power field;</p

Lewis rat-derived MSCs do not improve clinical, electrophysiological or histological outcomes in experimental autoimmune neuritis.

<p>(A, B) Apidogenic and osteogenic potential of CD90+/CD45−/CD11b− Lewis BM-MSCs. Adipogenesis confirmed by Oil red O staining and osteogenesis confirmed by Alizarin Red staining. (C) Clinical course of EAN following various treatments. HBSS = Hank's buffered saline solution vehicle alone, BM-MSCs = 15×10⁶ bone marrow mesenchymal stem cells/kg, DF = 15×10⁶ dermal fibroblasts/kg, dead MSCs = 15×10⁶ dead bone marrow mesenchymal stem cells/kg, no treatment = no injection or anaesthesia. No significant differences in disease onset, severity or residual deficit were observed between groups. (D) Representative semithin sections from experimental groups. Inflammatory demyelination and axonal loss were observed in all groups. No histo-pathological differences were found between the treatment groups, as analysed in toluidine blue-stained micrographs of transverse sections (1 µm) through the sciatic nerves from animals in each group. (E–H) In cell tracing experiments, CFSE-labelled BM-MSCs were identified in bone marrow and lymphoid organs seven days after cell delivery (Hoechst – Blue; CFSE - Green; F & H show detail from E & G).</p

Human MSCs do not exert clinical effect in experimental autoimmune neuritis.

<p>(A) FACS profile of human MSCs confirms a CD90+/CD105+/CD34−/CD45− phenotype. (B) Human MSCs grow as confluent monolayers on tissue culture plastic and display mesenchymal differentiation (C) Osteogenesis: Alizarin red = orange, (D) Adipogenesis: Oil red O = red. (E) Human MSCs, delivered day 7 post immunisation at a dose of 25×10⁶ MSC/kg in 1 ml HBSS do not lead to a significantly different clinical outcome when compared with HBSS injection alone.</p

Mesenchymal stem cells derived from rat and human inhibit proliferation of myelin-reactive CD4+ T-cells <i>in vitro</i>.

<p>(A) CFSE proliferation assays, gated on CD4+ myelin reactive T-cells. Addition of a monoclonal myelin stimulus (MOG) leads to proliferation of T-cells, which is abolished by 1∶1 coculture with rat MSCs (rMSCs) and human MSCs (hMSCs). Representative FACS plots shown. (B) Coculture with rat mesenchymal stem cells and human mesenchymal stem cells leads to reduction in proliferation index of MOG-reactive CD4+ cells. (7.19+/−0.3 in absence of MSCs (n = 5), 1.4+/−0.1 with rat MSC coculture (n = 3), 1.4+/−0.1 with human MSC coculture (n = 3).</p

In saphenous nerve axons stimulated at high frequency (50 Hz) mitochondria accumulate at the peripheral sensory terminals.

<p>Skin innervated by saphenous nerve was immunohistochemically labelled with VDAC1 (red) and neuron-specific β-III-tubulin (green). In comparison with sham-stimulated animals (A), axons were strongly labelled for VDAC1 in nerves stimulated at 1 Hz (B) and 50 Hz (C). (C′–C′″) Saphenous nerve from a YFP⁺ mouse (shown in low power in C), which expresses YFP (green) in a proportion of fibres, was stimulated with 50 Hz and labelled with VDAC1 (red). (D′–D′″) Saphenous nerve from a YFP⁻ mouse was stimulated with 50 Hz and double labelled with VDAC1 (red) and β-III-tubulin. The two markers were often found to co-localise. (E) Intensity of VDAC1 labelling was significantly higher within cutaneous fibres of saphenous nerve stimulated at 50 Hz (<i>n</i> = 5, <i>p</i><0.05) than in sham-stimulated (<i>n</i> = 3) or fibres stimulated at 1 Hz (<i>n</i> = 3). (F) There was no difference in VDAC1 labelling intensity in DRGs of saphenous nerves between the groups. Scale bars in (A–C) = 100 µm, in (C′–C′″) = 20 µm, in (D′–D′″) = 10 µm.</p

Higher frequency (50 Hz) conduction is associated with shortening of stationary, and an increase in distance between stationary mitochondria.

<p>(A) The total number of stationary mitochondrial profiles did not change significantly in response to impulse conduction (<i>p</i>>0.05, Kruskal-Wallis test with Dunn's multiple comparison test). (B) Average length of stationary mitochondria was significantly lower in axons conducting impulses at 50 Hz (<i>n</i> = 366, <i>p</i><0.01), or at 1 Hz following 50 Hz (<i>n</i> = 231, <i>p</i><0.001) than in naive (<i>n</i> = 231) or sham-stimulated (<i>n</i> = 366) axons. (C). Frequency distribution of length of stationary mitochondria between groups showed a significantly lower number of long mitochondria (i.e., 4 µm) and higher number of short (i.e., 2 µm) in axons conducting at high frequency, than in sham-stimulated axons. (D) The number of mitochondria separated by 1.5 µm (measured between their mid-points) decreased in axons conducting at 50 Hz (<i>n</i> = 15 axons, <i>n</i> = 288 mitochondria), and was significantly lower for mitochondria separated by 3 µm (<i>p</i><0.001), than in sham-stimulated group (<i>n</i> = 13 axons, <i>n</i> = 309 mitochondria; three animals per group), i.e., mitochondria were less clustered in stimulated axons. In all groups axons were pulled from three independent experiments (animals).</p

Mitochondria do not accumulate in the vicinity of Nodes of Ranvier.

<p>(A and B) Nodes of Ranvier in sham-stimulated (<i>n</i> = 35) and axons stimulated at high frequency (50 Hz, <i>n</i> = 28) appeared similar upon ultrastructural examination (bar = 1 µm), and (C) possessed similar numbers of mitochondria (<i>p</i> = 0.22, Mann-Whitney test).</p

Mitochondrial traffic during impulse conduction.

<p>(A) Confocal image of saphenous nerve exposed in an anaesthetised mouse, labelled with IB4-isolectin (green) and TMRM (red). (B) Typical image of saphenous fibres labelled with TMRM. Asterisks denote Schwann cell nuclei. (C) Kymograph analysis of mitochondrial movement in representative axons. (D) CAPs averaged every 30 s during 50 Hz stimulation for 75 min during time-lapse confocal imaging remained similar in form. (E) Impulse conduction significantly increased the number of trafficking mitochondria. (F) Anterograde and retrograde mitochondrial trafficking increased in axons conducting at 1 Hz (<i>n</i> = 30 axons) and 50 Hz (<i>n</i> = 31 axons) versus naive (<i>n</i> = 60 axons) (<i>p</i><0.001) and sham-stimulated animals (<i>n</i> = 32 axons) (<i>p</i><0.01): anterograde trafficking is selectively and markedly raised with 50 Hz stimulation (<i>p</i><0.001). (G) Trafficking velocity is significantly higher at 50 Hz than in naive, sham, and 1 Hz-stimulated axons (<i>p</i><0.001), and it decreases upon reducing stimulation to 1 Hz (<i>n</i> = 23 axons) (<i>p</i><0.05): anterograde velocity is particularly increased (H). In all groups axons were pulled from three independent experiments (animals). Data that followed normal (Gaussian) distribution are represented as mean ± SEM, whereas data that did not follow normal distribution are represented as median ± IQR. Kruskal-Wallis test with Dunn's multiple comparison test; *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001.</p