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Neutrophils promote VLA-4–dependent B cell antigen presentation and accumulation within the meninges during neuroinflammation

Significance A distinct murine model of multiple sclerosis used to examine factors involved in ectopic lymphoid tissue formation during central nervous system autoimmunity reveals that infiltration and aggregation of B cells within the leptomeninges is dependent upon B cell expression of VLA-4 and is preceded by neutrophil migration. This finding establishes the early mechanisms involved in the establishment of chronic inflammatory changes within the meninges during autoimmune inflammation that promote the formation of ectopic lymphoid tissue associated with disease progression and disability in multiple sclerosis.

The success of B cell depletion therapies and identification of leptomeningeal ectopic lymphoid tissue (ELT) in patients with multiple sclerosis (MS) has renewed interest in the antibodyindependent pathogenic functions of B cells during neuroinflammation. The timing and location of B cell antigen presentation during MS and its animal model experimental autoimmune encephalomyelitis (EAE) remain undefined. Using a new EAE system that incorporates temporal regulation of MHCII expression by myelin-specific B cells, we observed the rapid formation of large B cell clusters in the spinal cord subarachnoid space. Neutrophils preceded the accumulation of meningeal B cell clusters, and inhibition of CXCR2-mediated granulocyte trafficking to the central nervous system reduced pathogenic B cell clusters and disease severity. Further, B cell-restricted very late antigen-4 (VLA-4) deficiency abrogated EAE dependent on B cell antigen presentation. Together, our findings demonstrate that neutrophils coordinate VLA-4-dependent B cell accumulation within the meninges during neuroinflammation, a key early step in the formation of ELT observed in MS.
B cell | EAE | multiple sclerosis B cells are uniquely positioned to mediate multiple aspects of central nervous system (CNS) autoimmunity by secreting both pro-and antiinflammatory cytokines, producing antigenspecific Ig, and efficiently capturing and presenting antigen to cluster of differentiation (CD4) T cells. The concept that B cells are integral to the pathogenesis of multiple sclerosis (MS) has been solidified by the recent success of B cell depletion therapy (BCDT) for both relapsing remitting (RRMS) and primary progressive (PPMS) forms of the disease (1)(2)(3)(4). Modeling of B cell involvement in MS using experimental autoimmune encephalomyelitis (EAE) has demonstrated the importance of B cell antigen presentation (5). Moreover, EAE studies have revealed the necessity of activation-induced cytidine deaminase (AID) expression and elevated antigen specificity by B cells, indicating a pathogenic role for B cell antigen processing and presentation (5)(6)(7).
In MS, the presence of oligoclonal bands (OCBs) within the cerebrospinal fluid (CSF) raises the important question of where cognate B:T cell interactions occur during disease. Ig isolated from the CSF of MS patients are often class-switched to complement-activating IgG subtypes and show signs of affinity maturation, indicative of germinal center (GC) reactions between CD4 T cells and B cells (8). Ongoing cognate interactions within the meninges are supported by the presence of ectopic lymphoid tissue (ELT) in a sizeable fraction of secondary progressive MS (SPMS) patients (9,10) as well as in mice with various forms of EAE (11)(12)(13). The association of both OCBs (14) and ELT (10,15,16) with more severe disability in MS patients suggests that antigen-specific B:T cell interactions oc-curring locally within the CNS compartment participate in the propagation of neuroinflammation. These features of MS also exemplify the importance of compartmental restrictions on B cell-mediated CD4 T cell activation in neuroinflammation and autoimmunity.
The genesis of ELT in autoimmune diseases within the specialized immune CNS compartment is poorly understood. Lehmann-Horn et al. recently found that B cell-specific deficiency in the α4 integrin subunit of very late antigen-4 (VLA-4) reduces susceptibility to active EAE induced by human myelin oligodendrocyte glycoprotein (hMOG) and decreases trafficking of both Th17 CD4 T cells and macrophages to the CNS (17). Fibroblastic reticular cells (FRCs) and chemokines such as CXC chemokine ligand (CXCL13) have been identified as retention factors within the meninges during EAE (11,15,18). However, the steps initially involved in creating a suitable meningeal space for B cell traf-Significance A distinct murine model of multiple sclerosis used to examine factors involved in ectopic lymphoid tissue formation during central nervous system autoimmunity reveals that infiltration and aggregation of B cells within the leptomeninges is dependent upon B cell expression of VLA-4 and is preceded by neutrophil migration. This finding establishes the early mechanisms involved in the establishment of chronic inflammatory changes within the meninges during autoimmune inflammation that promote the formation of ectopic lymphoid tissue associated with disease progression and disability in multiple sclerosis.
ficking and retention, critical for ELT organization within the specialized immune target of the CNS, have yet to be explored.
To examine the unique contributions of B cell antigenpresenting cell (APC) function to CD4 T cell encephalitogenicity, we developed an EAE model allowing for timed onset of disease mediated solely by B cell antigen presentation (6). In conjunction with rapid disease onset, we now describe the formation of large clusters of B cells within the spinal cord subarachnoid space resembling immature ELT. The accumulation of B cells in the spinal cord meninges was preceded by an increase in CD45 hi , CD11b hi , Ly6C-, Gr-1+ myeloid cells. These inflammatory myeloid cells promoted B cell trafficking to the CNS compartment and influenced the formation of pathogenic B cell clusters. Additionally, B cell expression of VLA-4 was necessary for B cell accumulation and the development of passive EAE. These data suggest that neutrophils enhance recruitment or retention of B cells in an anatomic compartment that facilitates B cell access to both antigens and autoreactive T cells.

Results
Meningeal Space Is a Niche for B Cells during EAE. To assess the location where B cells may participate as APCs within the CNS during EAE, we used flow cytometry to assess the frequency of B cells in various CNS tissues. Following immunization of B6 mice with MOG 35-55 , B cells were found in low frequency and detected primarily in the meninges rather than the CNS tissue parenchyma (Fig. 1A). A similar frequency and distribution of B cells was observed in the B cell-dependent EAE model involving active immunization of B6 mice with hMOG ( Fig. 1B), which is unlike an initial assessment of B cell numbers in these models (19). In contrast, passive transfer of MOG-specific encephalitogenic CD4 T cells into B6 mice or mice with selective major histocompatibility complex class II (MHCII) expression by B cells with an elevated precursor frequency of MOG-specific B cells led to a significantly greater frequency of B cells in the CNS. In these passive EAE models, B cells were found in the spinal cord meninges in preponderance to the CNS tissue parenchyma (Fig. 1 C and D).
These results indicate that the meningeal compartment is the primary location where B cells collect during EAE and suggest that, given the proximity to antigenic debris and cognate CD4 T cells, B cells could serve as potent APCs within the meninges.
We previously reported a novel EAE model, in which MHCII expression is induced in a cell-specific as well as temporal manner. B cells are capable of serving all antigen presentation functions during passive EAE as long as the repertoire of B cells  is narrowed on the T cell cognate antigen (MOG) resulting from IgH MOG expression (20). Using this system to examine the critical timing of cognate interactions during neuroinflammation, the onset of disease between Tam-WT APC and Tam-B APC mice can be synchronized by inducing MHCII expression upon oral gavage with Tam after encephalitogenic CD4 T cell transfer (20). We showed that Tam-WT APC mice develop passive EAE similar to WT mice, with or without IgH MOG transgene expression, while Tam-B APC mice can exhibit accelerated disease onset dependent on the timing of MHCII expression relative to encephalitogenic T cell transfer (20). As with other models of EAE, flow cytometric assessment of Tam-WT APC and Tam-B APC mice with EAE revealed infiltration of B cells predominantly within the spinal cord meninges ( Fig. 1 E and F). No significant difference in disease severity was observed between different models at day 3 postonset (Fig. 1G). Examination of spinal cord tissue for the presence of B cells in Tam 2B). Qualitatively, B cell clusters in the meninges of Tam-B APC spinal cords appeared denser than the diffuse B cell infiltration seen in Tam-WT APC mice (Movie S1). Quantifying the area and number of distinct areas of B cell staining in cleared spinal cord specimens, a statistically significant difference in mean voxel area per cluster between the spinal cords of Tam-WT APC (6857 ± 1236) and Tam-B APC mice (9675 ± 757) was observed (P = 0.05). Our findings suggest that this model of EAE offers an opportunity to explore the earliest steps involved in B cell organization leading to ELT formation. To gain insight into the resemblance of B cell clusters in the spinal cord meninges to ELT, we first isolated the spinal cord meninges of Tam-B APC mice at 3 d post EAE onset and used flow cytometry to analyze the composition of leukocytes. Splenocytes harvested from an hMOG-immunized mouse were used to develop a gating strategy to detect the expression of GC markers and determine B cell populations present in the meningeal clusters (Fig. 3A). Based on expression of IgM and IgD we found that the majority of B cells in the spinal cord meninges included IgM+ IgD− B cells and IgM− IgD− naive mature follicular B cells (Fig. 3B). While relatively few B cells existed in the CNS parenchyma, most expressed MHCII. In the spinal cord meninges, where B cells are plentiful, nearly all were MHCII+, indicating a capacity for cognate interactions with CD4 T cells (Fig. 3C). CD138+ plasmablasts and plasma cells were very rare in all tissues analyzed but were most frequent in the brain compared to the spinal cord and spinal cord meninges (Fig. 3C). The mean frequency of GL7+ GC B cells in the spinal cord meninges was low, although elevated compared to brain and spinal cord parenchyma, indicating that most B cells in the CNS were not undergoing GC-like reactions at this early stage of the disease (Fig. 3C). Spinal cord specimens stained with both B220 and the proliferation marker K i -67 revealed the absence of costaining (Fig. 3D), demonstrating the formation of B cell clusters just 3 d following the onset of EAE is likely due to a large infiltration of B cells rather than proliferation in situ. Some B cell clusters in the spinal cord meninges were heavily infiltrated by CD3+ T cells (Fig. 3 E, i, arrows), while T cells were sparse in other clusters ( Fig. 3 E, ii, arrows). In summary, the spinal cord meninges of Tam-B APC mice with EAE rapidly developed dense clusters of activated, class-switched, MHCII+ B cells, suggesting that this is a critical location for cognate interactions during EAE.
To define the specific anatomical site of B cell infiltration within the meninges during EAE, we analyzed decalcified spinal cords harvested from Tam-B APC mice 3 d after EAE onset using immunofluorescent staining. We detected B cell extravasation from the cerebrovasculature which formed clusters surrounding vascular cell adhesion molecule-1 (VCAM-1)+ activated endothelial cells (Fig. 4 A and B). B cell clusters were excluded from the spinal cord parenchyma, identified by glial fibrillary acidic protein (GFAP) staining (Fig. 4B), and pia mater, identified by laminin (Fig. 4C). Rather, B cell clusters were associated with the arachnoid mater as seen by Podoplanin-1 staining (Fig. 4 B and C). Overlays of these immunofluorescent stains revealed that B cell clusters were specifically in the subarachnoid space, an anatomical compartment with access to both antigens and cognate CD4 T cells.   5B). Strikingly, we observed early and sustained infiltration of CD45 hi CD11b hi Gr-1 hi inflammatory, granulocytic myeloid cells in the spinal cord meninges of Tam-B APC mice but not Tam-WT APC mice, a difference that was statistically significant at 24 h post Tam, 72 h post Tam, and 3 d post EAE onset (Fig. 5C). We reasoned that these CD45 hi CD11b hi Gr-1 hi inflammatory cells were most likely polymorphonuclear neutrophils (PMNs), as this cell type is rapidly recruited to sites of inflammation and infection and has been detected during initial phases of EAE (21,22). Imaging of decalcified spinal cords from Tam-B APC mice with EAE revealed the proximity of B cell clusters with Ly6G+ PMNs (SI Appendix, Fig. S1), consistent with a coordinated influx of PMNs promoting an environment conducive to B cell antigen presentation and the rapid establishment of B cell clusters following EAE onset. The mean concentrations of analytes observed in naive WT mice were similar to the levels detected in CSF collected from Tam-B APC mice harvested 3 wk post CD4 T cell transfer but prior to Tam treatment ( Fig. 6 A-E), indicating that encephalitogenic CD4 T cells did not induce inflammatory responses in the absence of MHCII expression. Tam-WT APC (red squares) exhibited higher concentration of IFNγ at 72 h post Tam compared to Tam-B APC mice (P = 0.006). At the day of EAE onset, the concentration in Tam-WT APC mice was still significantly upregulated compared to Tam-B APC mice (P < 0.0001) (Fig. 6A). Other mediators of inflammation, including CC chemokine ligand (CCL)5 and CCL19 within the CSF CCL5 were significantly different between Tam-WT APC and Tam-B APC mice (Fig. 6 B and  C). Drastically different concentrations of the CXC glutamateleucine-arginine (ELR)+ chemokines CXCL5, CXCL2, and CXCL1 were identified in the CSF of Tam-WT APC and Tam-B APC mice at the same stages of disease. Tam-WT APC mice tended to rapidly develop much higher concentrations of these chemokines than Tam-B APC mice (Fig. 6D).
Given the significance of CXCL13 for B cell trafficking and GC reactions, differences in the concentration of this chemokine between Tam-WT APC and Tam-B APC mice could provide insight into the progression of B cell cluster formation. Although a substantial increase in concentration was observed in both groups, the only difference was identified, at 3 d post EAE onset, with Tam-WT APC mice exhibiting significantly lower CXCL13 concentrations compared to Tam-B APC mice (Fig. 6E).
The variability in chemokine concentrations in the CSF between Tam-WT APC mice and Tam-B APC mice, especially at 72 h post Tam, could explain our observation that these 2 genotypes differ in CD45 hi CD11b hi Gr-1 hi inflammatory PMN invasion of the spinal cord meninges (Fig. 5C). These ELR+ CXC chemokines all share the receptor CXC chemokine receptor (CXCR)2, which is predominantly expressed by PMNs. In Tam-B APC mice 72 h post Tam, CXCR2 expression was detected in splenic CD45 hi CD11b hi Gr-1 hi inflammatory PMN cells, but not on PMNs isolated from the meninges (Fig. 6F). However, 3 d post EAE onset, PMN expression of CXCR2 in the spinal cord meninges was notably higher (Fig. 6F). B cells did not express CXCR2 in any tissue at any time point examined (Fig. 6F). Further flow cytometric analysis of the phenotype of CD45 hi CD11b hi Gr-1 hi inflammatory PMNs in the spinal cord meninges of Tam-B APC mice confirmed minimal contamination with Ly6C+ monocytes (SI Appendix, Fig. S2).
Between 72 h post Tam and 3 d post EAE onset, PMNs isolated from the spinal cord meninges exhibited signs of activation with a reduction in surface expression of CD62L coinciding with in-creased CXCR2 (SI Appendix, Fig. S2). These data are corroborated by reports that transmigration of the blood-brain barrier (BBB) induces enhanced inflammatory phenotypes in PMNs (23  of PMNs to the CNS was functionally relevant to EAE dependent on B cell antigen presentation, we neutralized CXCR2 in Tam-B APC mice. Anti-CXCR2 serum was administered after encephalitogenic CD4 T cells were transferred into Tam-B APC mice but prior to MHCII induction 3 wk later. The disease severity as represented by total area under the curve of mean disease scores was similar for mice receiving control serum and untreated control mice (Fig. 7A). Mice treated with anti-CXCR2 blocking antibody did not develop EAE as quickly or as severely as the control groups (Fig. 7A). Examination of immunofluorescence images indicates that B cell clusters were present in the spinal cord meninges of both control groups, but were not detected in mice treated with anti-CXCR2 serum (Fig. 7B), suggesting that meningeal B cell cluster formation is pathogenic for EAE dependent on B cell antigen presentation. By flow cytometry, donor T cells in the spinal cord meninges of anti-CXCR2-treated mice compared to untreated or control serum groups were similar (SI Appendix, Fig. S3  A and B). As well, B cells and MHCII+ B cells were found in similar numbers in the spleen of anti-CXCR2 mice compared to control groups (SI Appendix, Fig. S3C). However, in accordance with the absence of meningeal B cell clusters observed 3 d post EAE onset, there were very few B cells in the spinal cord meninges of anti-CXCR2-treated mice (SI Appendix, Fig. S3D (Fig. 7C). WT and Cre − VLA-4 f/f mice with WT VLA-4 expression levels were both susceptible to EAE (SI Appendix, Table S1) with a similar day of onset and disease severity (Fig. 7 D, Top). CD19-B MHCII xIgH MOG mice and CD19-B MHCII xIgH MOG xVLA-4 f/+ mice were susceptible to EAE, but CD19-B MHCII xIgH MOG xVLA-4 f/f mice were completely protected (Fig. 7 D, Center). We repeated the passive EAE experiment with Tam

Discussion
While cognate interactions between B cells and CD4 T cells are known to be important for autoantibody formation in MS, the nature and location of these pathogenic interactions in EAE are unclear. Similarly, the genesis of ELT within the meninges of patients with progressive MS is thought to be clinically relevant, but the mechanisms leading to the formation of these structures remain unknown. We sought to determine the sequence of proinflammatory events that fosters the formation of antigen-specific B and T cell interactions within the CNS intrinsic to the clinicopathologic features of MS. We have found that the subarachnoid space of the spinal cord meninges serves as a niche for B cell cluster formation during EAE. This is corroborated by other reports of ELT in EAE, including Dang et al. (26), who found nonclassically activated B cells in the spinal cord meninges of TCR MOG xIgH MOG mice with spontaneous EAE. In our model, we found a high frequency of MHCII+ B cells that frequently exhibited surface markers such as IgM+ IgD− B cells (27) and naive follicular B cells. The low frequency of CD138+ plasma cells or plasmablasts and few GL7+ GC B cells we observed in the spinal cord meninges can be accounted for by the early time points used in this study. One caveat to these results is that CD138 staining can be challenging based on the lack of stability of this surface marker during processing for flow cytometry, leading to a potential underestimation of the number of plasma cells/plasmablasts. Also, it is important to note that the encephalitogenic donor CD4 T cell lines used to induce passive EAE in our model are very strongly Th1-skewed and thus distinct from models exhibiting ELT in B6 or SJL (Swiss Jim Lambert) mice involving IL-17skewed CD4 T cells (12,18). This is relevant, given that prior work has shown that Th17 cells are capable of trafficking to the CNS during EAE without expression of VLA-4 (28). Additionally, donor T cells driving disease in our model may not be capable of functioning as efficient T follicular helper cells to sustain GC reactions. These explanations are not mutually exclusive and deserve further exploration to elucidate the critical mechanisms involved in transitions from immature to established ELT structures in EAE.
To explore the factors recruiting and retaining B cells to the subarachnoid space to the exclusion of other CNS tissues we investigated the kinetics of innate and adaptive immune cell trafficking at several stages of EAE progression. Prior to EAE onset, CD45 hi CD11b hi Gr-1 hi PMNs are the most common cell type observed in the spinal cord meninges of Tam-WT APC and Tam-B APC mice. This proinflammatory cell type is often a major component of early inflammatory infiltrates in EAE (22,29,30), and it is especially elevated in Tam-B APC mice compared to Tam-WT APC mice. PMNs may be playing a unique role in the early stages of B cell-dependent EAE in which they prime the CNS microenvironment for B cell cluster formation through activating the endothelium or compromising the integrity of the BBB (29,31). It is possible that PMN transmigration of the BBB results in the up-regulation of VCAM-1 or CXCL13 to promote B cell extravasation into the subarachnoid space. Indeed, peripheral blood isolated from MS patients is enriched for neutrophil extracellular traps (NETs), which are a potent source of antigens and activation signals for autoreactive B cells in systemic lupus erythematosus (SLE) (32)(33)(34). Utilizing our unique ability to sample murine CSF, we decided to target CXCR2 as a means of testing our hypothesis that PMN trafficking is essential for B cell recruitment to the spinal cord meninges and initial ELT formation. CXCR2 expression was restricted to PMNs in Tam-B APC mice, and over time, PMNs harvested from the spinal cord meninges of these mice exhibited surface markers indicative of increased activation. We have found that treating Tam-B APC with anti-CXCR2 serum delays the day of EAE onset and resulted in less severe EAE symptoms, consistent with previous studies showing antagonism of CXCR2 signaling protects from neuroinflammation (24,25,35). Ameliorated EAE in anti-CXCR2 recipients was associated with complete ablation of B cell clusters in the subarachnoid space as well as changes in the tissue distribution of PMNs. The involvement of PMNs in neuroinflammation, both in MS as well as MS-related diseases, is complex. In our model of B cell-dependent EAE, PMN migration to the CNS is required for B cell antigen presentation function and the development of clinical disease. This does not necessarily indicate that CXCR2-dependent PMN migration is critical to the development of EAE in WT mice due to B cell antigen presentation. Rather, when APC function is isolated to B cells, our data indicate that the process by which B cells accumulate within the meninges is PMN-dependent. Overall, inhibiting the ability of PMNs to respond to timely expression of CXCR2 ligands in our model impedes formation of B cell clusters, potentially due to reductions in PMN-secreted proinflammatory cytokines, less PMN-induced damage to the BBB, and/or inhibited induction of other chemokines that promote the recruitment of other immune cells.
In conclusion, our research indicates that B cell clusters in the spinal cord subarachnoid space are aggregates of immune cells engaging in pathogenic, antigen-specific interactions. Formation of these rudimentary meningeal ELT in Tam-B APC mice is essential for B cell antigen presentation to support neuroinflammation. Prior to the onset of EAE and well before B cell cluster formation, PMNs are recruited to the CNS by ELR+ CXC chemokines. Inhibition of CXCR2-mediated PMN trafficking to the CNS reduces pathogenic B cell clusters, suggesting that PMN recruitment primes the CNS compartment for B cell cluster formation. Therapeutic targeting of CXCR2 signaling could be effective for MS, and more research is needed to investigate the possibility of direct or indirect interactions between PMNs and B cells. In accordance with successful therapies that eliminate B cells (BCDTs) or prevent immune cell trafficking to the CNS (Natalizumab or Fingolimod), B cell-restricted VLA-4 deficiency inhibits the development of EAE symptoms when B cells are the only APCs. Coordinated trafficking of innate and adaptive immune cells may promote EAE by supporting VLA-4dependent B cell accumulation within the meninges during neuroinflammation. Future experiments aimed at determining whether B cell access to the CNS is necessary for antigen acquisition or presentation to CD4 T cells will be essential in refining cellular immune therapeutic targeting with distinct CNS compartments in mind.

Materials and Methods
Mice. Mice were housed in a single pathogen-free housing facility and fed mouse chow and water ad libitum. Male and female mice between 6 and 10 wk of age were used for all experiments with the exception of the anti-CXCR2 treatment experiments that utilized female mice exclusively. Further information regarding strains and breeding is available in SI Appendix.
EAE. Passive EAE was induced as previously reported (6) and is described in more detail in SI Appendix. For induction of MHCII in vivo, Tam-WT APC , Tam-B APC , and Tam-B APC xVLA-4 f/f or Tam-B APC xVLA-4 f/+ mice were treated with 5 μg Tam (Sigma-Aldrich) in 50 μL corn oil (Sigma-Aldrich) by oral gavage 3 wk after CD4 T cell transfer as described (20). MHCII and VLA-4 expression was verified by flow cytometric analysis of peripheral blood. The Animal Studies Committee/Institutional Animal Care and Use Committee of Washington University in St. Louis approved all animal experiments.
In Vivo CXCR2 Neutralization. Rabbit anti-mouse CXCR2 serum was generated according to previous reports (24) and 0.2-μm vacuum-filtered. Control polyclonal rabbit serum (R4505, Sigma-Aldrich) was sterilized with 0.45-μm syringe filters (TPP). Age-matched female Tam-B APC mice were injected intraperitoneally with 200 μL control or anti-CXCR2 serum on days −2, 0, 2, and 4 relative to Tam treatment by oral gavage (at day 0) 3 wk after CD4 T cell transfer. Mice were scored daily for clinical disease and killed 3 d post EAE onset or 13 to 15 d post Tam.
Flow Cytometry. Isolation and staining of single cells from various tissues for flow cytometric analysis was preformed according to prior reports (6) and is detailed in SI Appendix.
CSF Collection and Analysis. Mice were avertin-anesthetized prior to CSF collection from the cisterna magna. CSF was stored in MAXYMum Recovery, low-protein-binding PCR tubes and immediately spun down and frozen. Concentration of cytokines and chemokines was assessed using the BioRad 33-plex kit analyzed on a Luminex. Concentrations of CSF were transformed logarithmically for graphing and statistical analysis purposes.
Imaging. Immunofluorescence staining for CD3 and B220 was performed as previously reported (6). Intermittent background labeling from anti-CD3 staining was noted, but consistently within the gray matter and not in a pattern confounding for cellular labeling of T cells. Background immunofluorescence was a result of anti-CD3, as control staining with secondary antibody only never resulted in excessive background signal. Staining for additional markers, including VCAM-1, Ly-6G, podoplanin, and K i -67 is detailed in SI Appendix. Optical clearing of spinal cords was initiated by perfusing mice with 25 mL ice-cold phosphate buffered saline followed by 20 mL 4% paraformaldehyde (Sigma-Aldrich). CNS tissue was extracted while still encased in the skull and vertebral column, then fixed in 4% paraformaldehyde for more than 12 h. Spinal cord vertebrae were decalcified in 6% tricholoroacetic acid (Sigma-Aldrich) for 5 d and washed with PBS prior to optical clearing using iDISCO protocol (36). Additional details including confocal imaging are provided in SI Appendix.