Immune response in peripheral axons delays disease progression in SOD1G93A mice
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Increasing evidence suggests that the immune system has a beneficial role in the progression of amyotrophic lateral sclerosis (ALS) although the mechanism remains unclear. Recently, we demonstrated that motor neurons (MNs) of C57SOD1G93A mice with slow disease progression activate molecules classically involved in the cross-talk with the immune system. This happens a lot less in 129SvSOD1G93A mice which, while expressing the same amount of transgene, had faster disease progression and earlier axonal damage. The present study investigated whether and how the immune response is involved in the preservation of motor axons in the mouse model of familial ALS with a more benign disease course.
First, the extent of axonal damage, Schwann cell proliferation, and neuromuscular junction (NMJ) denervation were compared between the two ALS mouse models at the disease onset. Then, we compared the expression levels of different immune molecules, the morphology of myelin sheaths, and the presence of blood-derived immune cell infiltrates in the sciatic nerve of the two SOD1G93A mouse strains using immunohistochemical, immunoblot, quantitative reverse transcription PCR, and rotating-polarization Coherent Anti-Stokes Raman Scattering techniques.
Muscle denervation, axonal dysregulation, and myelin disruption together with reduced Schwann cell proliferation are prominent in 129SvSOD1G93A compared to C57SOD1G93A mice at the disease onset, and this correlates with a faster disease progression in the first strain. On the contrary, a striking increase of immune molecules such as CCL2, MHCI, and C3 was seen in sciatic nerves of slow progressor C57SOD1G93A mice and this was accompanied by heavy infiltration of CD8+ T lymphocytes and macrophages. These phenomena were not detectable in the peripheral nervous system of fast-progressing mice.
These data show for the first time that damaged MNs in SOD1-related ALS actively recruit immune cells in the peripheral nervous system to delay muscle denervation and prolong the lifespan. On the contrary, the lack of this response has a negative impact on the disease course.
KeywordsAmyotrophic lateral sclerosis SOD1G93A mice Immune system Peripheral nervous system
Amyotrophic lateral sclerosis
Chemokine (C-C motif) ligand 2
Low molecular mass 7
Major histocompatibility complex I
Peripheral nervous system
Cu/Zn superoxide dismutase
Transporter of antigen peptide 1
Amyotrophic lateral sclerosis (ALS) is the most common neuromuscular disorder, affecting individuals from all ethnic backgrounds, with an incidence of 2–3 cases per 100,000 population per year . Terminal axonal degeneration with neuromuscular junction (NMJ) denervation is an early component of motor neuron (MN) disease in ALS . In addition, it has now become clear that ALS is a non-cell autonomous disease involving activation of microglia and astrocytes in the CNS  and infiltrates of peripheral T cells and macrophages in the spinal cord and sciatic nerve [4, 5]. These non-neuronal cells play an active role in MN death. In fact, selective ablation of mutant SOD1 in astrocytes and microglial cells and neonatal bone marrow (BM) transplantation significantly increased MN survival and lifespan  of mutant SOD1 transgenic mice. In contrast, T cell deficiency accelerated the disease progression in these mouse models [4, 6]. These studies have been mainly focused on the CNS, while the contribution of immune cells in the peripheral nervous system (PNS) has not been fully analyzed.
ALS is a clinically heterogeneous disease with a high variability in the rate of symptom progression even in the familial cases [7, 8]. Growing evidence indicates that also in mutant SOD1 mice, the most widely used model of familial ALS, the severity of the disease is markedly influenced by their genetic background [9, 10, 11, 12]. In fact, we have recently obtained and characterized two mouse strains (C57BL6 and 129Sv) carrying the same copy number of the human SOD1G93A transgene that showed remarked difference in the speed of symptom progression [13, 14]. In particular, the fast-progressing mice (129SvSOD1G93A) compared to the slow progressors (C57SOD1G93A) showed an earlier impairment of motor function and shorten survival, which was unrelated to the extent of MN loss in the lumbar spinal cord, despite significant differences in their MN transcriptome [9, 13, 14]. Noteworthy is the fact that rapidly progressing mice, unlike the slow progressor, showed markedly decreased expression of transcripts linked to axonal transport and neuronal projection [9, 14] at disease onset, suggesting major involvement of the axonal compartment both at the central and peripheral levels to determine the different mice phenotypes. This was then demonstrated by the ex vivo diffusion tensor imaging and histopathological analysis showing an earlier axonal degeneration in the white matter of the spinal cord of fast- compared to the slow-progressor mice . In contrast, MNs of slow- but not fast-progressing mice specifically activated and translocated to the peripheral motor axons immune molecules (i.e., major histocompatibility complex I (MHCI)) directly involved in the induction of an adaptive immune response [9, 14].
In the present study, we further extended these observations analyzing in more details the involvement of the PNS and the associated immune system in the modulation of disease progression. We clearly demonstrated that the dysregulation of PNS is the origin of the difference between the two transgenic mice strains and that immune cell infiltration in this compartment is essential to delay muscle denervation of SOD1G93A mice.
Female transgenic SOD1G93A mice on C57BL/6JOlaHsd or 129SvHsd genetic background, hereafter indicated as C57SOD1G93A and 129SvSOD1G93A, respectively, and corresponding non-transgenic (Ntg) littermates were used. Procedures involving animals and their care were conducted in conformity with institutional guidelines that comply with national (Legislative Degree 26, March, 2014) and international (EEC Council Directive 2010/63, August, 2013) laws and policies.
Animals studies were approved by the Mario Negri Institute Animal Care and Use Committee and by the Italian Ministerial decree no. 84-2013.
Laser-captured microdissection and microarray data analysis
We extrapolated data for β2m, Tap1, H2_K-D, and Lmp7 messenger RNA (mRNA) levels in C57-Ntg; 129Sv-Ntg, and C57-SOD1G93A laser-captured MNs from microarray analysis; the detailed procedure has been previously described in Nardo et al. . Additional information is supplied in the Additional file 1.
The spinal cord and sciatic nerve were processed as previously described . Briefly, the mice were perfused with Tyrode’s buffer, followed by Lana’s fixative (4 % formalin and 0.4 % picric acid in 0.16 M PBS, pH 7.2) at 20 °C. The lumbar spinal cord and sciatic nerves were quickly dissected out. The tissue was left in the same fixative for 90–180 min or overnight at 4 °C, rinsed, and stored 24 h in 10 % sucrose with 0.1 % sodium azide in 0.01 M PSB at 4 °C for cryoprotection, before mounting in optimal cutting temperature compound (OCT). The spinal cords and sciatic nerves were cut, respectively, in 30- and 14-μm sections. The following primary antibodies and staining were used: rat anti-MHC class I ER-HR 52 clone (1:100; Abcam), rat anti-CCL2 (1:50; Abcam), rabbit anti-β2m (1:500; Proteintech), rabbit anti-Lmp7 (1:500; AbD Serotec), mouse anti-SMI-31 (1:1000; Sternberger Inc), rabbit anti-CD8 (1:50; Abcam), rabbit anti S100β (1:400; Sigma-Aldrich), α-btx (5 μg/ml) conjugated with Alexa-594 (Invitrogen), and NeuroTrace conjugated with Alexa-488 or Alexa-594 (1:500; Invitrogen). Secondary antibodies were as follows: Alexa 488 or Alexa 594 goat anti-rat, Alexa 488 or Alexa 594 goat anti-rabbit, and Alexa 594 goat anti-mouse (Invitrogen). All immunohistochemistry followed an indirect immunostaining protocol whereas peroxidase-diaminobenzidine (DAB) reaction was used for detecting MHCI in the spinal cord and DAB (brown) plus DAB-NICHEL (blue) reactions in the sciatic nerve for the double labeling of MHCI (1:00) and CD8 (1:50).
Immunohistochemical evaluation of Lmp7 in human obturator nerve
All procedures in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 declaration of Helsinki and amendments, or comparable ethical standards. We studied a motor nerve sample from a sporadic ALS male patient, whose anterior branch of the obturator nerve had been previously biopsied for diagnostic purposes, as described . This patient developed progressive lower-limb weakness at the age of 52; motor nerve biopsy led to a neuropathological diagnosis of motor neuron disease. Subsequently, he developed upper motor neuron signs, allowing a clinical diagnosis of definite ALS . A normal nerve sample belonging to a 65-year-old patient with a final diagnosis of distal sensorimotor peripheral neuropathy was studied as a control. Although the histopathological analysis showed a normal nerve at time of biopsy, clinical worsening and neurophysiological follow-up subsequently allowed a final diagnosis of peripheral neuropathy. Neuropathological diagnosis was based on previous diagnostic criteria . Transverse 10-μm sections of the frozen nerve were cut with a cryostat and transferred to 0.1 % poly-d-lysine-coated glass slides. The cryosections were immunostained with affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat antibodies to human LMP7 (1:100; Southern Biotechnology Assoc.) and tetramethylrhodamine (TRITC) conjugated goat antibodies (Southern Biotechnology Assoc.) to human neurofilament 200 kDa (rabbit) (1:1000; Chemicon).
Coherent Anti-Stokes Raman Scattering (CARS) analysis of the sciatic nerves
The animals were sacrificed with cervical dislocation, and their sciatic nerves were rapidly surgically explanted and frozen in dry ice in a WillCo dish (GWSt-3522; WillCo Wells, Amsterdam, The Netherlands). The samples were then thawed by immersion into warm (~38 °C) Dulbecco’s phosphate buffer saline (D8537; Sigma-Aldrich, Saint Luis, Missouri, U.S.A.) and immobilized with a custom-made electrophysiology-type anchor. The explanted sciatic nerves from C57SOD1G93A, 129SvSOD1G93A, and relative NTG mice were observed with an RP-CARS microscope  tuned to CH2 molecular vibrations yielding the average in-plane orientation anisotropy (α) of the chosen molecular vibrations and their average orientation (φ) within the sub-micrometric excitation volume . The acquired z-stacks were analyzed with a custom-made Python software (Python Software Foundation, Beaverton, OR, USA) to threshold the myelin walls and to extract the stack distribution of the α values (we employed the same algorithm described in , but computed the threshold on the α value images). The α value distributions are shown in Additional file 1: Figure S1. Finally, the same z-stacks were analyzed with a second custom-made Python software to extract the stack-based average dispersion (β value) of the myelin-associated φ values over a chosen spatial scale (see Additional file 1). The β value is an indicator of the average micrometric longitudinal straightness of the myelin sheaths.
Tibialis anterior muscles were dissected out and snap-frozen in isopentane cooled in liquid nitrogen. Twenty-micrometer serial longitudinal cryosections were collected on poly-lysine objective slides (VWR International). The sections were stained with anti-synaptophysin (SYP) (1:100; Synaptic System) as pre-synaptic marker. The secondary antibody was FITC-labeled goat anti-rabbit (1:500, Invitrogen). Subsequently, the sections were stained with α-bungarotoxin (α-BTX) coupled to Alexa Fluor 594 (1:1200; Invitrogen) as post synaptic marker. Fluorescence images along the z-axis were taken by Olympus confocal microscopy using a ×20 objective, and z-stacking was done using Fiji software (with Z-Stack Projection/SUM command) (ImageJ, US National Institutes of Health, Bethesda, MD, USA). End plates were scored as innervated if there was complete overlap between SYP and a-BTX staining and scored as denervated if they showed only α-bungarotoxin staining.
The mice were decapitated, and the spinal cord and sciatic nerve were rapidly dissected. The spinal cord was processed as previously described . The sciatic nerves and muscles were powdered in liquid nitrogen then homogenized by sonication in ice-cold homogenization buffer centrifuged at 13,000 rpm for 15 min at 4 °C, and the supernatants were collected and stored at −80 °C. Equal amounts of total protein homogenates were loaded on polyacrylamide gels and electroblotted onto PVDF membrane (Millipore) as previously described . Densitometric analysis was performed with Progenesis PG240 v2006 software (Nonlinear Dynamics). Immunoreactivity (IR) was normalized to β-tubulin, β-actin, or the total amount of protein detected by red Ponceau (Sigma-Aldrich) or SYPRO Ruby Blot (Thermo Fisher scientific). Additional information is supplied in the Additional file 1.
Quantitative reverse transcription PCR
The spinal cords, sciatic nerves, and muscles were perfused in 0.1 M PBS, freshly collected and then frozen on dry ice. The total RNA from spinal cord was extracted using the Trizol method (Invitrogen) and purified with PureLink RNA columns (Life Technologies). For fibrous tissues (sciatic nerve and muscles), the RNeasy® Mini Kit (Qiagen) was used. The RNA samples were treated with DNase I, and reverse transcription was done with a High Capacity cDNA Reverse Transcription Kit (Life Technologies). For quantitative reverse transcription PCR (RT-qPCR), we used the TaqMan Gene expression assay (Applied Biosystems) following the manufacturer’s instructions, on complementary DNA (cDNA) specimens in triplicate, using 1X Universal PCR master mix (Life technologies) and 1X mix containing specific receptor probes. The levels of transcripts were normalized to β-actin. The mean values of the triplicate results for each animal were used as individual data for 2−ΔΔCt statistical analysis. Additional information is supplied in the Additional file 1.
The Mann-Whitney test was used to compare differences between two groups and one-way or two-way ANOVA was used to compare differences between more than two groups. For the CARS analysis, z-stack-based indicators (i.e., the α values and the β values) were statistically analyzed with a general linear mixed model implemented in R (R Foundation, Vienna, Austria), using the strain (C57 vs. 129Sv) and the mutation (Ntg vs. G93A) as fixed factors and the nerve as a random factor. Post hoc comparisons between the experimental groups were instead performed with Mann-Whitney test on the specimen-based medians.
The severity of disease in SOD1G93A mice correlates with earlier muscle denervation
Axonal dysregulation and reduced Schwann cell proliferation are prominent in fast-progressing mice at disease onset
Sustained activation of the MHCI pathway, centrally and peripherally, occurs in slow- but not fast-progressing mice
We have previously demonstrated that MHCI is activated more in MNs of slow-progressing than fast-progressing mice at disease onset . The present study extended the analysis to other components for MHCI presentation such as Lmp7, H2_D-K, Tap1, and β2m. Basal mRNA levels of H2_D-K and β2m were significantly higher in the C57Ntg than 129SvNtg MNs, with fold changes, respectively, of 3.6 and 4.7 times (Additional file 1: Figure S3a). When we measured the mRNA expression of Lmp7, H2_D-K, Tap1, and β2m obtained by microarray analysis of laser-captured MNs from the C57SOD1G93A mice (n = 4) compared to the Ntg littermates (n = 4) at the presymptomatic (56 days), symptom onset (135 days), and symptomatic (147 days) stages of the disease, all four genes showed similar patterns of expression during disease progression, with a peak at disease onset and a progressive decrease at the symptomatic stage (Additional file 1: Figure S3b). No differences were found for the 129Sv mice . For this reason, we examined the expression and distribution of the components of this pathway in more detail in the CNS and PNS of the C57SOD1G93A mice.
Peripheral activation of CCL2 and complement subunit C3 is responsible for greater macrophage recruitment in the sciatic nerve of slow-progressing mice
This study demonstrates that the integrity of PNS plays an essential role in governing the onset of the disease and the speed of its progression in two mouse models of familial ALS carrying the same amount of human SOD1G93A transgene on a different genetic background and suggests that this phenomenon is influenced by the immune system. In fact, while both mouse ALS strains present similar loss of MNs during the disease course , the rapid-progressor 129SvSOD1G93A mice present earlier axonal impairment and denervation of hind limb muscles as indicated by the marked upregulation of the AChRγ-subunit mRNA levels in the TA muscles compared to the C57SOD1G93A mice. Normally, the AChRγ-subunit it is almost undetectable or at very low levels in innervated, normally active muscle, while it becomes abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked . This is in line with the hypothesis that alterations at the NMJ might precede the massive MN loss . Muscle denervation is associated with a with marked disruption of motor axons in 129SvSOD1G93A mice demonstrated by the reduced levels of acetylated tubulin and neurofilaments, two indices of axonal transport and structure [24, 25] and by the increase of β-importin, a marker of axonal stress  in their sciatic nerve.
The extent of the de-differentiation and proliferation of SCs during regeneration of the damaged motor axons in SOD1G93A mice also seems to play a relevant role modulation the disease course in these ALS mouse models. In fact, the basal expression in sciatic nerves of P-ERK, which is fundamental to stimulate SCs de-differentiation during reinnervation , is much lower in the 129Sv NTg than in the C57 NTg mice. This difference was reflected in a reduced level of activation in 129SvSOD1G93A mice in comparison to C57SOD1G93A mice. The same difference is evident for the expression of GFAP in the sciatic nerves, which is essential to promote the proliferation of SCs after damage [27, 30]. In fact, depletion of this protein in the sciatic nerves has been reported to delay nerve regeneration after axotomy in mice . RP-CARS structural analysis of the explanted sciatic nerves further supports this concept at morphological level showing significant differences in myelin anisotropy between the C57 and 129sv background. Statistically significant differences were also found in the β value between the two strains (and, in particular, between SOD1G93A mice) highlighting the contribution of mouse genetic background in promoting the integrity of the myelin structural architecture during disease progression.
This result is in accordance with the studies by Bieber et al.  who, by using different inbred mouse strains model of chronic progressive multiple sclerosis, clearly demonstrated that the axonal integrity and neurologic function is mainly affected by genetic background-related loci inherited as dominant traits. In parallel, the study of different experimental mouse models of axon remyelination has allowed identification of potential genetic factors able to enhance axonal remyelination . For instance, fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF) promote the proliferation of oligodendrocyte precursor cells (OPCs) [33, 34], while insulin growth factor 1 (IGF1) stimulates both OPCs differentiation and axon myelination . In addition, P-ERK, which we observed to be more expressed in the C57 than the 129Sv mice, is essential to coordinate the stress response of myelinating oligodendrocytes following insult .
The mouse genetic background also profoundly influences the immune system response. In fact, inflammatory cell recruitment is markedly lower in the 129Sv than in the C57 mice following thioglycolate intraperitoneal injection . This is consistent with our observation of a reduced macrophage and T cell infiltration in the peripheral nerves of the 129SvSOD1G93A compared to C57SOD1G93A mice. The striking activation of the immunoproteasome and MHCI in motor axons and SCs of C57SOD1G93A mice correlates with the specific infiltration of CD8+ T cells in sciatic nerves. The recruitment of macrophages is related to the coordinated and specific activation of both CCL2 and the complement C3 subunit. Apparently, this reactive immune response is not accompanied by inflammation as no significant upregulation of pro-inflammatory factors (TNFα; IL-6; Ly6c) were observed in the sciatic nerves of the C57SOD1G93A mice. By contrast, an increase in the M2 marker, Ym1, suggested a shift towards a protective macrophage phenotype. Thus, these findings indicate a beneficial role of immune cell infiltrates during the disease progression of SOD1-related ALS mice.
Several observations suggest the importance of immune infiltration in CNS repair [38, 39, 40, 41, 42]. For instance, recent studies demonstrated a beneficial role of macrophage infiltration in mouse models of experimental autoimmune encephalitis (EAE). In this regard, the transfer of M2-polarized monocytes resulted in a suppression of EAE [43, 44]. Additional studies in EAE mouse models and other disorders indicate that T cells responses can facilitate CNS repair at a later stage by restricting a prominent secondary wave of damage [45, 46, 47, 48].
This scenario resembles that previously reported in experimental mouse models of axon remyelination in which the proliferation and differentiation of oligodendrocytes precursor cells were accompanied by immune cells infiltration [52, 53, 54]. Notably, the depletion or pharmacological inhibition of macrophages following toxin- or virus-induced demyelination leads to an impairment of remyelination [55, 56, 57], as does an absence of T cells [58, 59]. This evidence suggests that protective autoimmunity is a physiologically evoked mechanism through which the body harnesses the immune system in order to protect itself against neuronal degeneration.
This mechanism is more prominent in the PNS where immune cell infiltration is not restricted, in contrast to the CNS . Barrette et al. recently demonstrated that peripheral macrophages were essential to promote the production of neurotrophins (NT-3; BDNF) in regenerating motor axons and macrophage depletion significantly slowed axon regeneration and functional recovery after sciatic nerve injury . Moreover, the CCL2-mediated neuron-macrophage interaction is vital for amplification and maintenance of enhanced regenerative capacity by preconditioning peripheral nerve injury . In vivo imaging in a SOD1G93A mice revealed distinct inflammatory activity of CNS microglia compared to PNS macrophages, the latter showing no substantial morphological reaction during degeneration of peripheral motor axons, but a passive role in clearing debris, particularly lipid-rich myelin . In keeping with this, myelin ingestion by myeloid cells in vitro induces a foamy appearance and confers an anti-inflammatory function . This evidence indicates about an essential role of hematogenous macrophages in promoting the remyelination of defective motor fibers. In addition, CD8+ T cells infiltrating the peripheral nerves may be essential not only to promote the phagocytosis of myelin debris but also for limiting the activity of effector T cells and macrophages once the remyelination is completed (recovery phase) . In fact, it is well known that the CD8+ T cells include a subset of regulatory T cells (CD8+ Tregs) expressing specific regulatory markers such as CD103, FoxP3, or CD122 . These were the first described subset of T cells capable of inducing immune suppression in vitro  and showing a greater immunosuppressive effect than CD4+ Tregs in a number of models including EAE [66, 67].
It is generally accepted that terminal axonal degeneration and NMJ denervation occur before the loss of MNs in ALS . However, this evidence was largely neglected for years with the result that no preclinical trials designed to inhibit degeneration of MN cell bodies in SOD1G93A mice yielded the expected outcomes in terms of therapeutic benefit. There is growing evidence now that even complete rescue of MN somata may not be enough to counteract the disease progression in SOD1G93Amice . This agrees with our findings which highlight that the extent of axonal and neuromuscular dysfunction, rather than MNs loss, seems predictive of a more aggressive phenotype. The level of degeneration (or preservation) of this compartment may therefore underlie the heterogeneity of ALS, which is a powerful confounding factor in evaluating the real efficacy of treatments .
Our results demonstrate that genetic background influences the basal levels of proteins that are essential to better preserve the myelination of peripheral motor axons during disease progression. In addition, we found that the maintenance of PNS function through the interaction with a specific immune response is essential for delaying disease progression in transgenic mouse models suggesting that genetic differences in immune responsiveness could affect the outcome of ALS. Accordingly, it will be extremely relevant to conduct genetic linkage analysis on F1 backcross generation between fast and slow mouse strains to search for genetic loci linked to enhanced remyelination and immune activation.
With respect to the translation of these findings to human ALS, so far there are no studies comparing the PNS of fast- and slow-progressing ALS patients due to the limited availability of tissues. However, here we demonstrate for the first time that one of the main components of the MHCI pathway, the LMP7, is upregulated in the motor axon of a sporadic ALS patient indicating that this phenomenon is not strictly related to the overexpression of mutant SOD1. We have not been able to investigate whether this variation correlates with a protective or detrimental effect on motor axons. Nevertheless, the failure of anti-inflammatory and anti-immune therapies in clinical trials  highlights the incomplete knowledge of the inappropriate knowledge of the dynamic immunological changes that occur during the disease progression and indirectly supports a reconsideration of the immune system in ALS.
The SOD1G93A mouse used in this study is representative of a low percentage of ALS patients . However, so far it is the only model that well recapitulates the clinical and pathological characteristics of the human disease . For this reason, we expect that a better understanding of the molecular mechanisms of cross-talk between damaged axon signaling and the immune response in SOD1G93A mice should help in finding new approaches for promoting axonal regeneration and muscle reinnervation in ALS. In parallel, the discovery of genetic factors that may be important for slowing disease progression could allow identification of molecular signatures as potential druggable targets and disease biomarkers for homogeneous subsets of ALS patients.
We thank Dr. Beatrice Bosani, Dr. Martina Rossi, and Dr. Mattia Verderio for the technical assistance.
This work was supported by the Thierry Latran Foundation, the Motor Neurone Disease Association (MNDA), “Amici del Mario Negri” Association and Regione Lombardia under Institutional Agreement no. 14501, and the European Community (FP7/2007-2013) under grant EuroMOTOR no. 259867. PJS is supported as an NIHR Senior Investigator.
Availability of data and materials
Data, materials, and software information supporting the conclusions of this article are included within the article and its Additional file 1.
GN and PH analyzed the gene expression of the MN extracted by laser dissection microscopy. MCT and GN conducted the recruitment of the mouse tissue and the immunohistochemical and biochemical analysis of the spinal cord sciatic nerve and muscles. VP supervised the CARS experiments. GDV developed the statistical analysis algorithms and analyzed the CARS data. RC acquired the CARS images. NR and GD analyzed the human biosamples by immunohistochemistry. CB, GN, AQ, NR, and PJS supervised and designed the experiments. GN wrote the manuscript. All authors have read and edited the manuscript and approved the final version.
The authors declare that they have no competing interests.
Consent for publication
The patients’ consent for publication is in the archives of the San Raffaele Scientific Institute and can be made available on request.
Ethics approval and consent to participate
All human nerve biopsies had been previously obtained for diagnostic purposes, after informed consent, and stored in our tissue bank. All experimental protocols were approved by San Raffaele Scientific Institute Ethical Committee (Milan, Italy), and the methods were carried out in accordance with the approved guidelines. All procedures performed in studies involving animals were in accordance with the ethical standards of the Mario Negri Institute at which the studies were conducted.
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