Macroscopic detection of demyelinated lesions in mouse PNS with neutral red dye

Lysophosphatidylcholine (LPC)-induced demyelination is a versatile animal model that is frequently used to identify and examine molecular pathways of demyelination and remyelination in the central (CNS) and peripheral nervous system (PNS). However, identification of focally demyelinated lesion had been difficult and usually required tissue fixation, sectioning and histological analysis. Recently, a method for labeling and identification of demyelinated lesions in the CNS by intraperitoneal injection of neutral red (NR) dye was developed. However, it remained unknown whether NR can be used to label demyelinated lesions in PNS. In this study, we generated LPC-induced demyelination in sciatic nerve of mice, and demonstrated that the demyelinated lesions at the site of LPC injection were readily detectable at 7 days postlesion (dpl) by macroscopic observation of NR labeling. Moreover, NR staining gradually decreased from 7 to 21 dpl over the course of remyelination. Electron microscopy analysis of NR-labeled sciatic nerves at 7 dpl confirmed demyelination and myelin debris in lesions. Furthermore, fluorescence microscopy showed NR co-labeling with activated macrophages and Schwann cells in the PNS lesions. Together, NR labeling is a straightforward method that allows the macroscopic detection of demyelinated lesions in sciatic nerves after LPC injection.

injury and diseases requires further studies, especially in the long terms typically required for regeneration of peripheral nerve neuropathy after PNS demyelination, including Guillain-Barre syndrome and CIDP. While mouse models of experimental demyelination are used to examine the molecular mechanisms and develop the drugs for demyelinating diseases, one of the challenges in the analysis of demyelination models has been the difficulty to identify and selectively acquire tissues of demyelinated lesions for quantitative analysis of transcripts, metabolites, and proteins. Moreover, selecting the demyelinated lesions for electron microscopy (EM), which allows examination of very limited volume of the collected tissues, has also been challenging in morphological investigation of myelin sheaths and evaluation of demyelination and remyelination.
Lysophosphatidycholine (LPC)-induced demyelination is a highly tractable demyelination/remyelination model in mice, in which LPC injection into the white matter of brain and peripheral nerves induces focally demyelinated lesions that spontaneously remyelinate over a reproducible timeframe [15][16][17] . LPC-induced demyelination is highly informative for evaluation of demyelinated condition and level of remyelination in lesions. Recently, it was shown that the administration of neutral red (NR) into mice allows the macroscopic visualization of demyelinated lesions in freshly dissected CNS tissue 18,19 . NR is a vital dye, which has preference for incorporating into live cells with low intracellular pH, and was previously shown to selectively label macrophages 20,21 . Single intraperitoneal (i.p) injection of NR before sacrifice allowed the detection of demyelinated lesions in the mouse CNS in vivo. However, it remained unknown if the NR labeling method would also be applicable to demyelination models of the PNS, and if the tissues with NR labeling could be used for subsequent ultrastructural analyses.
Here, we determined if the single i.p injection of NR can be used to detect demyelinated lesions in LPCinduced demyelination in the mouse PNS. We found that NR could be readily detected macroscopically on site of LPC-injection in sciatic nerves, and EM analysis of NR labeled tissue confirmed that LPC injection resulted in demyelination of sciatic nerve. Moreover, we observed NR co-labeling with activated macrophages and a subpopulation of Schwann cells in lesions. These results suggest that NR injection is a simple and powerful method to detect the lesions in demyelination animal models, and potentially applicable to the wide range of analyses in peripheral demyelinating disease models.

Results
Macroscopic detection of neutral red in sciatic nerve demyelination. To determine whether NR specifically labels PNS demyelinated lesions, focal demyelination was induced in mouse sciatic nerves by LPC injection. Intraperitoneal (i.p.) injection of NR was performed 2 h before sacrifice of LPC-treated mice. We found some of the mice exhibited a reduction in movement immediately after NR injection, possibly due to the volume of NR received (500 μl). However, these mice recovered within 30 min after injection, and none of the mice displayed any obvious signs of behavioral abnormality throughout the duration of the experiment. To detect NR labeling, LPC injected sciatic nerves and uninjured contralateral nerves were dissected at 7, 14 and 21 days post lesion (dpl) (Fig. 1a), corresponding to timepoints of remyelination 22,23 . We found that NR was readily detectable on the injured sciatic nerve, corresponding to the site of LPC injection at 7 dpl macroscopically or by light microscopy, and no NR labeling was observed on the contralateral nerve (Fig. 1b). Moreover, NR signal decreased at 14 dpl, and was barely detectable at 21 dpl (Fig. 1c). Moreover, longitudinal cryosections of LPC injected sciatic nerve revealed NR had incorporated into the nerve at the site of LPC demyelinated lesion (Fig. 1d,e). However, although NR was observed as a focal lesion on intact LPC-injected sciatic nerves, it displayed non-uniform staining in longitudinal sections. Note that the NR labeling is strongest at the lesion site from which it fades away. These results suggest that NR incorporated into the site of sciatic nerve injury, and the labeling gradually decreased over the course of PNS repair.

Electron microscopy analysis of NR-labeled lesion
To assess the identified NR labeled demyelinated lesions, electron microscopy (EM) analysis was performed on NR labeled sciatic nerves at 7 dpl. Transversely sectioned semithin and ultrathin sections were prepared from sciatic nerve tissues following postfixation and embedding into resins, and examined for myelin structure by light microscopic observation with toluidine blue staining and TEM observation (Fig. 2a). Compared with contralateral sciatic nerve semithin sections, the LPC-injected nerves displayed areas lacking compact myelin sheaths, as well as areas with intact myelin sheaths (Fig. 2b). The presence of patches of demyelinated and myelinated axons reflected the non-uniform access of LPC to the sciatic nerve, as was observed with NR labeling in Fig. 1d. TEM analysis of ultrathin sections in NR labeled tissue revealed demyelinated axons as well as myelin debris in lesions (Fig. 2c,d). We also observed the presence of myelin debris within the cytoplasm of some Schwann cells (Fig. 2e), confirming the ongoing process of myelin degradation by Schwann cells 24 . To determine that NR did not contribute directly to demyelination and the extent of myelin debris, we also performed EM analysis on LPC injected sciatic nerves from mice without NR injection at 7 dpl, and found that demyelinated lesion without NR displayed a similar pathology as tissues containing NR labeling (Additional file 1: Fig. S1). These results confirm that NR incorporated into sciatic nerve lesions, and that NR could be used in combination with EM studies for the detection and analysis of demyelinated lesions.
Fluorescence microscopy analysis localizes NR-labeling in Schwann cells and activated macrophages in demyelinated PNS lesions. NR displays inherent fluorescence with an excitation spectrum of 450-550 nm and a wide emission spectrum with the maximum range 550-650 nm 18 . To detect PNS myelin, lesioned sciatic nerves containing NR labeling were sectioned longitudinally, and immunofluorescence staining for myelin basic protein (MBP) was performed. We observed strong NR fluorescence in areas containing low MBP expression, corresponding to areas of demyelination, at 7 dpl (Fig. 3a,b). Furthermore, quantitative analysis demonstrated that the NR signal significantly decreased as immunoreactivity of MBP increased at 14 dpl, and www.nature.com/scientificreports/ became indistinguishable from uninjured tissue by 21 dpl when the MBP staining was restored (Fig. 3c,d). However, NR signals might be sometimes visible in what appears to be pericytes and tissue resident macrophages in non-lesioned areas. (see Fig. 5). These results demonstrate that NR incorporation into demyelinated lesions in sciatic nerve could be observed by fluorescence microscopy.
To examine Schwann cell distribution in NR labeled lesions, S100β 24 immunostaining was next performed. We observed decreased S100β labeled cells in areas of high NR labeling, compared to areas around the NRlabeled lesions and in non-lesion areas at 7 dpl (Fig. 4a). Some of NR signals were detected inside S100βpositive Schwann cells within the lesion, and no NR signals were detectable in S100β-positive Schwann cells in non-lesioned areas (Fig. 4b). Similar to MBP staining, NR signals decreased as immunoreactivity of S100β increased at 14 dpl, and the lesioned tissue was indistinguishable from uninjured tissue by 21 dpl (Fig. 4a,b). These results demonstrate that NR labeled demyelinated lesion exhibits decreased Schwann cells at 7 dpl that gradually repopulates back to the lesion by 21 dpl. Moreover, Schwann cells containing NR labeling in lesions at 7 dpl likely correspond to phagocytosing Schwann cells as was observed by EM.
Next, we examined macrophage distribution on NR labeled tissues by immunostaining for Iba1 expression, a pan-macrophage marker. We found that Iba1-positive macrophages accumulated in lesions at 7 dpl and strongly co-labeled with NR ( Fig. 5a,b). Moreover, NR intensity decreased with reduction of Iba1-positive macrophages between 7 and 21 dpl (Fig. 5a,b). In contrast, NR labeling was weakly detected or undetectable in Iba1-positive macrophages in non-lesioned peripheral nerve at 7 dpl (Fig. 5a,b), suggesting NR did not incorporate into resting macrophages. www.nature.com/scientificreports/ Macrophages under injury exhibit a spectrum of activation states, which includes pro-inflammatory M1-like and anti-inflammatory M2-like macrophages having distinctive gene expression patterns 25 . To determine whether NR labels M1-like or M2-like macrophages, we performed immunofluorescence staining using antibodies against iNOS, a pro-inflammatory macrophage marker (Fig. 6a,b), and CD163, an anti-inflammatory marker (Fig. 7a,b). We observed strong NR signals in both iNOS-positive and CD163-positive cells at 7 dpl, and the corresponding decrease of iNOS and CD163-positive cells with NR labeling in lesions by 21 dpl (Figs. 6b, 7b). These results suggest that NR dye labeled both M1 and M2 like macrophages in demyelinated lesion, and that NR labeling gradually decreased with the reduction of inflammation during remyelination. NR labeling colocalizes with lysosomes. Previously, it has been reported that NR incorporates into the lysosomes of live cells 20,21 . The detection of activated macrophages and phagocytic Schwann cells in lesions suggest these cells display high lysosomal content. To examine the intracellular localization of NR in the demyelinated lesion, we performed immunostaining using Lamp2 antibody as a lysosomal marker (Fig. 8a). Lamp2-positive signals were colocalized with NR signals in the lesion at 7 and 14 dpl (Fig. 8b). Both Lamp2 and NR signals were gradually decreased during remyelination. Furthermore, we also examined colocalization by 3D analysis. NR signals were colocalized with Lamp2-positive lysosomes in z-stack images (Fig. 8c-e: Movies S1-S3). These results suggest NR has a preference for cellular compartment with high lysosomal activity in the demyelinated lesion.

Discussion
In this study, we determined if i.p injection of NR can be used to detect demyelinated lesions in the PNS after LPC injection. We found that NR readily labels demyelinated sciatic nerve, and that NR labeling diminishes following remyelination. We detected NR incorporation in activated macrophages and phagocytic Schwann cells in demyelinated lesions, and the decrease of NR labeling coincided with decrease of the activated macrophages. Furthermore, we found that NR labeled tissue could be successfully applied for EM analysis of demyelinated axons and myelin debris in lesions. Since demyelinated lesions could be readily identified macroscopically, NR labeling facilitates the straightforward acquisition of small tissue pieces containing a focal demyelinated lesion for subsequent sample preparation and EM analysis.
Our data indicated that activated macrophages along with significant myelin loss were observed in NRlabeled lesions, and NR-labeling was detected in both iNOS-positive (M1-like) and CD163-positive (M2-like) macrophages as well as S100β-positive Schwann cells. In PNS demyelinated lesions, Schwann cells, monocytes or hematogenous, endogenous or mesenchymal cells have been reported to display phagocytic activity 24,26,27 . www.nature.com/scientificreports/ Our data showing S100β-positive cells with NR-labeling at 7 dpl suggest that NR incorporates into phagocytotic Schwann cells in lesions. Moreover, the decreased NR labeling at 21 dpl coincided with the reduction of macrophage activity and myelin repair. These results suggest that the NR labeling method is also applicable to visualize damaged regions in PNS demyelination or injury.
While it remained to be elucidated if NR labeling can be applied to demyelination models other than LPC injection, the macroscopic identification of demyelinated PNS lesions by NR labeling would be useful to reduce the error in lesion detection and facilitate subsequent experimental analyses to elucidate molecular mechanisms of pathology in demyelination as well as recovery through remyelination. Indeed, previous study has reported . Neutral red (NR) signals were gradually decreased during remyelination. (a) Representative confocal images of NR (magenta) and immunofluorescence staining for myelin with anti-MBP (green) in areas with lysophosphatidylcholine injection at 7, 14, 21 dpl and a nonlesion area at 7 dpl (n = 3 mice per timepoint examined). (b1-4) Enlarged images of (a1-4) (white dotted square). The demyelinated lesion with weak MBP immunoreactivity is shown in 7 dpl image (a, b; arrowhead). Quantification of fluorescence intensity based on MBP staining (c) and NR (d) labeling in lesion at 7, 14, and 21 dpl and a nonlesion area at 7 dpl (n = 3). Nuclei were counterstained with Hoechst (blue). Scale bar, 50 µm (a) and 20 µm (b). Mean ± s.e.m. One-way ANOVA, Tukey-Kramer test. *P < 0.05, **P < 0.01, ***P < 0.001. Graphs were created using Prism 7 (GraphPad Software), https:// www. graph pad. com. www.nature.com/scientificreports/ that NR labeling could be used for RT-PCR, Western blot and flow cytometry analysis of unfixed spinal cord demyelinated lesions 18 . Although fluorescence microscopy can be applied to examine NR incorporated cells in lesions and the strong NR staining was observed in areas of demyelination, we have also observed fluorescent NR labeling outside of the lesion site. It is possible that NR is also incorporated into activated macrophages or other phagocytic cells outside of lesions. Moreover, a caveat with fluorescence imaging is that weak NR-positive signals, due to the wide emission fluorescence spectrum of NR, could also be observed extracellularly, and autofluorescence in the perineurium or epineurium containing connective and adipose tissue surrounding the sciatic nerve 28,29 may also be detected under fluorescence microscopy even after destaining with 50% ethanol/1% glacial acetic acid. On the other hand, since demyelinated fibers are non-uniformly distributed in LPC injected sciatic nerves, inhomogeneous NR labeling might be detected at the lesioned tissue. Moreover, lactic acidosis is characteristic for inflammatory processes 30 . Therefore, the extracellular NR signals could represent general low pH within the microenvironment of the nerve lesion. While combinations of these reasons might be responsible for the diffuse NR labeling, optimization of the tissue processing procedure and the staining procedure may improve background staining under fluorescence microscopy for future studies.

Scientific Reports
In summary, our results suggest that NR labeling is a tractable method for evaluation of demyelinated state and recovery from demyelination in PNS tissues. The method may be used to identify the molecular mechanisms of Schwann cell remyelination, and applied to investigate drug targets or future drug screening for PNS repair.
These mice were kept in standard cages with fewer than 5 mice per cage at 20-25 °C on a 12 h light/dark cycle. All animal experiments were performed in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. All efforts were made to use only the number of animals necessary. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Jichi Medical University (approval number 19034-03) and performed in accordance with the guidelines on the care and use of animals of this committee. www.nature.com/scientificreports/

Focal demyelination of the sciatic nerve and NR injection. Demyelination was induced by injection
of 1% LPC (Sigma-Aldrich) in phosphate buffered saline (PBS) into sciatic nerve 31 . Mice were anesthetized by the mixture of 3 drugs (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol) and the right sciatic nerve was exposed. Hamilton syringe (31 gauge) connected to the micro injector (IMS-3; Narishige) and manipulator (YOU-1; Narishige) was inserted into the nerve and kept for 3 min before injection of LPC. Sciatic nerve was injected with 1 µl of 1% LPC using the micro injector, and the needle was kept for 5 min after injection to reduce leak along the needle track. The mice were sacrificed at 7, 14 or 21 days post lesion (dpl). Prior to the sacrifice, 500 µl of 1% NR (10 mg/ml; Sigma-Aldrich) in PBS was injected by intraperitoneal (i.p) injection for each mouse as described previously 18,19 .
Tissue processing and staining procedures. Two hours after the NR injection, mice were transcardially perfused with 1 ml/g body weight of PBS. Sciatic nerves were rapidly dissected and immersed overnight in 4% (w/v) paraformaldehyde (PFA) in PBS at 4 °C for fixation. Fixed sciatic nerves were sequentially immersed overnight in 15% and 30% (w/v) sucrose (Fujifilm Wako Pure Chemical Co.) in PBS at 4 °C for cryoprotection, and frozen in OCT compound (Sakura Finetec). Cryosections (10 µm thick) were prepared using a cryostat (CM3050; Leica Microsystems), collected on coated glass slides (Matsunami), and stored at -30 °C. Immunofluorescence staining of cryosections were performed according to a previously described protocol with slight modifications 32 . Before immunofluorescence staining, cryosections were incubated with a destain solution of 50% ethanol/1% glacial acetic acid to reduce the background for 30 min 33 . For immunofluorescence staining, cryosections were permeabilized and blocked in blocking buffer (0.3% TX-Tris-Buffered Saline (TBS) and 10% normal goat serum) for 1 h. Then the sections were incubated overnight at 4˚C with primary antibodies in the blocking buffer. Thereafter, cryosections were incubated at RT for 1 more hour, washed in 0.3% TX-TBS, incubated for 3 h at RT with secondary antibodies in 0.3% TX-TBS, washed in 0.3% TX-TBS, and embedded in Fluoromount-G (Southern Biotech) 32 . Some other cryosections were stained with 1% toluidine blue. The stained sections were observed by light microscopy (BX63; Olympus) or confocal microscopy (FV1000; Olympus).

Statistical analysis
Quantitative analysis is presented as mean ± standard error of the mean (s.e.m) from each image. For quantification, the images of areas with LPC injection and fluorescent intensities of the images captured by confocal microscopy were determined by Fiji-imageJ. Note that the images of different conditions (7, 14, 21 dpl and non lesioned) have been obtained using the same image acquisition settings for quantitative analysis. The intensities of the positive signals were measured per unit areas and compared in arbitrary units (AU). Statistical analyses were www.nature.com/scientificreports/ performed using Prism 7 (GraphPad Software). Statistical comparisons were performed using one-way ANOVA, followed by a Tukey Kramer test. The significance was indicated as a P value: *P < 0.05, **P < 0.01, ***P < 0.001.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.