Experimental procedures followed the European legislative, administrative and statutory measures for animal experimentation (86/609/EEC). The study was approved by the “Direction des Services Vétérinaires de l'Hérault” and the “Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, ethics committee N°36,” France (authorization number 34118).

Transgenic mice expressing enhanced green fluorescent protein (eGFP) in microglia/monocytes (CX3CR1^+/eGFP) (Jung et al., 2000) were obtained from Dr. Dan Littman, Howard Hughes Medical Institute, Skirball Institute, NYU Medical Centre, New York, USA and maintained on a C57BL/6 background (The Jackson Laboratory, Bar Harbor, ME, USA). Heterozygous eGFP^−/+ mice were used in the current study. All mice were housed in controlled conditions (hygrometry, temperature and 12-h light/dark cycle) with free access to food and water. Adult male mice (12 weeks of age) were anesthetized by inhalation of 1.5% isoflurane (Aerane, Baxter, Deerfield, IL, USA). The skin and muscles overlying the low thoracic segment of the rachis were cut, followed by a dorsal laminectomy of the thoracic 9 (T9) vertebra. Meninges were incised and transection of the spinal cord was carried out under a microscope using a micro-scalpel (FST, Heidelberg, Germany). Lesions were made at T9 level to obtain complete paraplegia whilst preserving full respiratory function. Following lesion, muscles and skin were sutured and animals were left to recover on a heated surface (not exceeding 38°C). Bladders were emptied manually twice daily and bodyweight was measured throughout the study period. Three time-points were chosen to reveal microglia/monocytes changes both at acute (72 hours) and chronic (4 and 6 weeks) stages after severe spinal cord lesion. Non-injured group served as control (n = 4 for each group).

Mice were deeply anesthetized via intraperitoneal injection of tribromoethanol (500 mg/kg) followed by transcardial perfusion using cold 0.1 M phosphate buffer saline (PBS) at pH 7.2 and 4% paraformaldehyde (PFA, Sigma Aldrich, Saint Louis, USA). The entire spinal cords were then dissected and post-fixed in the 4% PFA for an additional 2 h.

Spinal cords were positioned in a 5-mm-diameter glass tube filled with 0.1 M PBS surrounded by a custom-made ribbon solenoid coil (40 mm in length, 8 mm outer diameter and 4 turns, Supplementary Figure 1K) (Coillot et al., 2016) and imaged using 9.4 Tesla apparatus (Agilent Varian 9.4/160/ASR, Santa Clara, California, USA) associated with VnmrJ imaging acquisition system (Agilent, Palo Alto, California, USA). The use of the custom-made ribbon solenoid coil enhanced signal-to-noise ratio (SNR) and, drastically reduced acquisition time. Specifically, for this experiment, SNR was increased by 7 compared to a regular 43 mm quadrature volumic coil. We first acquired high resolution T2 weighed ¹H-MRI using a spin echo sequence to obtain MR images without diffusion-weighting (diffusion gradient: G = 0 G.cm⁻¹). The following acquisition parameters were used: delta = 6.88 ms, G = 0 G/cm⁻¹, separation = 15.05 ms, (TR) repetition time = 1,580 ms, (TE) echo time = 30.55 ms, AVG = 30, FOV = 10 × 10 mm, slices = 36, thickness = 1 mm without gap and acquisition matrix = 128 × 128. Scanning time: approximately 90 min. Diffusion-weighted MRI sequence was then used to acquire DWI and to calculate the ADC. ADC is a quantitative measurement of local water diffusion (mm².s⁻¹) on a given region and direction which is sensitive to the local structure and anisotropy of tissues. ADC results from the combination of acquisitions with (S at b = 500) and without (S0 at b = 0) diffusion gradient. To acquire axial images, the sensitivity of diffusion MRI was preliminary evaluated in three directions including the longitudinal axis of the spinal cord. Longitudinal DWI offered better signal-to-noise ratio and image contrast to discriminate between the intact and the damaged regions of the spinal cord. We thus acquired DWI only on the longitudinal axis with the following parameters: magnetic field gradient G = 20 G.cm⁻¹ during 6.88 ms, separation = 15.05 ms; (TR) repetition time = 1,580 ms, (TE) echo time = 30.55 ms, AVG = 30, FOV = 10 × 10 mm, slices = 36, thickness = 1 mm without gap and acquisition matrix = 128 × 128. Scanning time: approximately 180 min. Diffusion data were processed by using a MATLAB-based in-house toolbox. All MRI visualization and segmentation were done using Myrian software (Intrasense, Montpellier, France); intact (entire spinal cord, white and gray matters) and damaged tissues were manually surrounded (Figures 1B–D and Supplementary Figures 1A–J).

After diffusion MRI acquisition, spinal cords were first rinsed in 0.1 M PBS and clarified using the 3-dimensional imaging of solvent cleared organs (3DISCO) procedure, as previously described (Erturk et al., 2012). Briefly, spinal cords were incubated in 50, 70, and 80% tetrahydrofuran (THF, Sigma Aldrich, Saint Louis, USA) followed by three incubations in 100% THF (30 min each). Spinal cords were then placed in dichloromethane (Sigma Aldrich, Saint Louis, USA) for 30 min and finally in dibenzyl ether (DBE, Sigma Aldrich, Saint Louis, USA) for 20–30 min, until the samples became transparent (Figure 2). All incubation steps were done in the dark; spinal cords were placed in glass vials on a rotator. Samples were then mounted on a glass slide in DBE and coversliped, then immediately imaged using a two-photon scanning microscope (Zeiss LSM 7MP) equipped with a femtosecond pulsed Ti:Sapphire laser (Ultra II, Coherent) at 950 nm. We scanned at a 1,024 × 1,024 pixel resolution and 5 μm z steps; full 3D scan took 20–30 min.

To quantify microglia/monocytes density (numerical density (N_v), number (#)/mm³) throughout the entire thickness of spinal cord, we chose 3 different zones located at different distances from the edge of the lesion site: 0–500 μm immediately adjacent to the injury site as well as 500-1,000 and 4,000–4,500 μm away from the lesion epicenter, both rostral and caudal to the injury site (Figure 3). For non-injured controls, we analyzed equivalent anatomical levels of the spinal cord. Tissue volumes and microglial/monocytes density were calculated using the 3D image processing software Imaris x64 7.2.2. (Bitplane AG, Zürich, Switzerland). Microglia/monocytes were converted into 3D objects for fully automated counting (Supplementary Video 1). To ensure accuracy of the counts, selected segments were rotated to different angles and assessed visually. Only a sub-population of microglia/monocytes with no overlapping processes were selected for morphological analysis of their somata. Microglial/monocytes somata morphology was measured in 190–300 cells per time-point and compared to those of the un-injured control. The original scan was also converted into 3D objects for automated morphological analysis. Given that microglial/monocyte activation is characterized by pronounced enlargement of their somata, morphological analysis was carried out using Imaris to determine somata surface area (μm²) and volume (μm³), as previously described (Rodriguez et al., 2015).

Un-paired t-tests were used to compare differences between 2 groups. The Wilcoxon signed-rank test was used to compare ADC values between groups along an 8 mm segment centered on the lesion site. A paired-t-test was used to compare ADC values between groups in the rostral and caudal segments. Significance was defined as p ≤ 0.05.

All data were analyzed using GraphPad Prism 4.0 (GraphPad Software, Inc., CA, USA). Data are shown as the mean ± standard error of the mean (SEM).

We acquired 36 axial diffusion MR images (1 mm thick) over a 3.6 cm segment centered on the lesion epicenter (Figure 1A). We then quantified the rostro-caudal extension of the lesion. Lesion segmentation was carried out manually by outlining the spared (Figures 1B,D, green: total; blue: gray matter) and injured (Figures 1C,D, red) tissues on axial images. The mean extension of the lesion at 72 hours was 4 ± 0.4 mm centered on the epicenter and decreased to 2.75 ± 0.25 and 3 ± 0 mm by 4 and 6 weeks, respectively, thus revealing a significant decrease in rostro-caudal extension of the lesion between 72 hours and 4 weeks post-injury, followed by a stabilization (Figure 1E). The mean lesion volume amounted to 5.3 ± 0.9, 4.8 ± 0.7, and 5.9 ± 1 mm³ at 72 hours, 4 and 6 weeks post-injury, respectively, and quantitative assessment showed no significant changes over time (Figure 1F).

We then quantified the diffusion of the water in the tissues using diffusion MRI and evaluated changes in ADC values. At the lesion site, no clear distinction between the gray and white matter was possible due to complete tissue damage (Figure 1C). ADC was measured on an 8 mm segment centered on the lesion site. Overall, non-injured spinal cords showed an ADC of 6.78 × 10⁻⁴ mm²/s (Figure 1G). The mean ADC value at the lesion epicenter reached 8.73, 10.78, and 12.22 × 10⁻⁴ mm²/s at 72 hours, 4 and 6 weeks, respectively (Figures 1C,G). Moreover, an overall increase in the ADC contiguous to the lesion site, both rostral and caudal, was observed from 72 hours up to 6 weeks post-injury (Figure 1G). For all post-injury time points, the ADC was higher in the caudal than in the rostral segments 4 mm away from the lesion site. Moreover, there was an increased ADC 6 weeks post-injury in the rostral segment, whereas the ADC in the caudal segment remained relatively stable after injury (Figure 1G). All injured groups displayed a significant increase in ADC compared to non-injured animals; however, no significant differences between post-injury time points were observed (Figure 1G, Table 1). A Wilcoxon signed-rank test showed a significant increase in ADC between 72 hours and 6 weeks post-injury (Figure 1G, Table 1) compared to non-injured animals. To provide a more in-depth analysis, we analyzed separately the rostral and caudal segments. A paired t-test revealed a significant increase in ADC 72 hours and 6 weeks post-injury in the caudal segment when compared to non-injured animals; conversely, the injury did not modify the ADC in the rostral segment (Table 1).

Together, these data show that, in contrast to the quantification of the extension and the volume of the lesion, the quantification of water diffusion allows identification of time-dependent modifications. Moreover, we highlight differences in water diffusion between segments located symmetrically rostral and caudal to the lesion site.

We used tetrahydrofuran (THF)-based clearing to render the adult spinal cord transparent while preserving eGFP signal in microglia/monocytes (Figure 2). While un-cleared tissue can only be imaged at a depth of <100 μm using two-photon microscope (Figures 2A,C), clearing of the spinal cord permitted imaging throughout its entire thickness (Figures 2B,D).

Using two-photon image analysis, we determined microglia/monocytes density in samples that previously underwent ex vivo diffusion MRI at acute (72 hours) and chronic stages (4 and 6 weeks) after complete SCI and compared to that of un-injured controls. Microglial/monocytes density was evaluated in CX3CR1^+/eGFP mice at distances of 0–500, 500–1,000, and 4,000–4,500 μm from the lesion site (Figure 3). The increase in microglia/monocytes density was particularly evident at acute compared to more chronic stages (Figure 3). In addition, as expected, areas adjacent to the lesion site displayed a greater increase in microglia/monocytes density than more distal regions (Figure 3). Quantitative analysis revealed significant increases in microglia/monocytes density at 72 hours and 4 weeks post-lesion compared to non-injured controls (Figure 3A). By 6 weeks post-injury, microglia/monocytes density decreased considerably, retuning to non-injured control level at 4 mm distal to the lesion site (Figure 3A). Overall, microglia/monocytes density was similarly elevated both rostral and caudal to the lesion site (Figures 3B,C).

Together, these data show (1) there is a greater increase in microglial/monocytes density contiguous to the lesion compared to more distal regions, (2) the highest microglial/monocytes density is observed at 72 hours post-injury, followed by a decrease at chronic stages after SCI, and (3) there is generally a similar increase in microglial/monocytes density after injury rostral and caudal to the lesion site.

Microglia/monocytes activation is characterized by a pronounced modification of their morphology. We thus quantified the surface area (μm²) and volume (μm³) of the somata of at least 190 individual cells per time-point. Only microglia/monocytes without overlapping somata with their neighboring cells were selected for morphological analysis (Figure 4, pink objects). Analyses were done at 500 μm rostral to the lesion site as well as 4 mm both rostral and caudal to the lesion epicenter (Figure 5). The increase in microglia/monocytes somata surface area and volume was particularly evident in the 500 μm segment rostral to the lesion than in more distant regions on both the rostral and caudal sides (Figure 5). Quantitative analysis of microglia/monocyte soma surface area and volume in proximity to the lesion site revealed a significant increase from 72 hours to 6 weeks post-injury (Figures 5B,E). Rostral to the lesion site, the size of microglia/monocytes somata displayed a time-dependent increase (Figures 5A,D), while caudal to the lesion site there was a continuous decrease in somata surface area and volume (Figures 5C,F). Thus, time course analysis of microglia/monocytes somata morphology revealed opposite responses rostral (Figures 5A,D) and caudal (Figures 5C,F) to the lesion site.

Together, these findings demonstrated that (1) microglia/monocytes display over a 5-fold increase in average somata size adjacent to the lesion site that remains elevated up to 6 weeks post-lesion, (2) microglia/monocytes rostral to the lesion site display a time-dependent increase in somata size, and (3) microglia/monocytes caudal to the lesion site display a time-dependent decrease in somata size. We thus highlight variation between segments located symmetrically rostral and caudal to the lesion site.

We then investigated the potential link between injury-induced tissue re-organization and neuroinflammation in the injured spinal cord by examining putative correlation between ex vivo diffusion MRI and microglia/monocytes density. The increase in lesion extension, which was greatest at 72 hours post-injury amongst all the time points analyzed, coincided with the upsurge of microglia/monocytes density (Figures 1E, 3A and Supplementary Figure 2).

Lastly, we compared ADC values with the density and morphology of microglia/monocytes somata (Supplementary Figure 2). Rostral to the lesion site, the ADC at all time-points post-injury was similar to that of the non-injured control (Figure 1G). At the lesion site, ADC increased in a time-dependent manner (Figure 1G). All injured groups, except at 4 weeks post-injury (Table 1), displayed a higher ADC caudal to the lesion than the non-injured control. Unlike ADC, microglia/monocytes somata showed changes in surface area and volume both rostral and caudal to the epicenter (Figures 1G, 5 and Supplementary Figure 2). Thus, ADC does not seem to be correlated with microglia/monocytes somata morphology.

Despite the established presence of neuroinflammation in SCI pathophysiology, only a few studies have investigated post-injury microglia/monocytes density in mice, and findings are contradictory (Schnell et al., 1999; Kigerl et al., 2006; Stirling and Yong, 2008). A peak increase in microglia/monocytes density had been shown between 7 and 14 days post-injury (Schnell et al., 1999; Kigerl et al., 2006) but has also been reported as early as 2 days post-lesion (Stirling and Yong, 2008). In addition to the different techniques used to quantify microglia/monocyte density, differences in mouse strains may also account for these discrepancies (Basso et al., 2006; Lapointe et al., 2006). Using tissue clearing, Erturk and colleagues more recently reported a pronounced increase in microglia/monocytes density 10 days after spinal cord hemisection in mice (Erturk et al., 2012). Our quantitative analysis in severely injured spinal cords expands previous reports and provides a comprehensive analysis of microglia/monocytes density not only at acute and chronic stages after SCI but also at different distances from the lesion epicenter. Specifically, we observed that microglia/monocytes density is increased at 72 hours post-injury, then significantly reduced by 4 and 6 weeks with no major differences between the regions rostral and caudal to the lesion site. Although the pronounced increase in microglia/monocytes density at 72 hours post-injury may be attributed to infiltrating monocytes, CNS resident microglia can proliferate in the absence of peripheral monocytes (Elmore et al., 2014). Recent molecular study from our laboratory also confirmed that microglia are primarily involved in proliferation at 72 hours post-lesion (Noristani et al., 2017). In fact, resident microglia display greater proliferation compared to infiltrating monocytes after SCI (Greenhalgh and David, 2014). The increase in microglia/monocyte density at 72 hours occurred concomitantly with the greatest lesion extension observed by diffusion MRI. Modifications in diffusion MRI represent changes in the displacement of water molecules within different tissue compartments of the spinal cord. Thus, the positive association between microglia/monocytes density and lesion extension most likely corresponds to the early vasogenic edema resulting from plasma leakage and concomitant monocyte infiltration/microglia proliferation. Given that CX3CR1 is expressed both in infiltrating monocytes and resident microglia, in the current study, we cannot distinguish the exact proportion of these cells after SCI.

Although numerous studies have reported microglia/monocytes activation after injury (David and Kroner, 2011), no study to our knowledge has carried out quantitative analysis of injury-induced alteration of microglia/monocytes morphology. The most notable morphological alterations include a more than 3-fold increase in somata surface area and a more than 6-fold increase in somata volume that is most evident adjacent to the lesion site. The increase in somata volume induced by SCI is considerably higher than that reported in microglia following physiological stimulation, such as voluntary wheel running (Rodriguez et al., 2015). Interestingly, microglial/monocytes somata morphology distal to the lesion epicenter showed a clear difference in the regions rostral and caudal to the injury site (see Figure 6). Rostral to the lesion site, microglia/monocytes displayed a time-dependent increase in their reactivity that continued up to 6 weeks post-injury. In contrast, the greatest microglia/monocytes reactivity was observed caudal to the lesion site at 72 hours and decreased progressively between 4 and 6 weeks post-lesion. Measurements of ADC rostral and caudal to the lesion site also showed strong differences. Specifically, the ADC rostral to the lesion remained similar to that of un-injured controls throughout the 6 weeks following spinal cord trauma. In contrast, the ADC caudal to the lesion was significantly higher in all injured groups than the non-injured control. In the spinal cord, water diffuses in the white matter along the fibrous myelinated axons, which are orientated in a rostro-caudal direction. Diffusion MRI parameters, such as ADC are very sensitive to the changes in axonal integrity that occur during demyelination and inflammation (Stroman et al., 2014). Thus, an elevated ADC caudal to the lesion site might reflect demyelination. Indeed, even though limited and/or inadequate re-myelination may occur caudal to the lesion site after incomplete SCI (Alizadeh et al., 2015), this does not occur after a transection of the spinal cord. Microglia are predominantly involved in phagocytosis of damaged myelin sheaths and clearance of debris of axons caudal to the injury site that undergo Wallerian degeneration. The time-dependent decrease in microglia/monocytes reactivity caudal to the lesion site might thus reflect reduced myelin/debris clearance that is almost completed by 6 weeks post-injury. Rostral to the lesion site, the time-dependent augmentation in microglia/monocytes reactivity up to 6 weeks post-injury may correspond to a dying-back phenomenon and/or abortive regeneration. Indeed, while the distal segment of a cut axon undergoes Wallerian degeneration, the proximal segment dies back over a period of few days or weeks. At the tip of the axon rostral to the lesion site, “retraction balls” form and are physically disconnected from the axon. Moreover, the edge of the cut axon presents a structure that resembles a growth cone but is “dystrophic” (Steward, 2014), suggesting that there is a persistent regeneration attempt by the injured axon. However, since injured axons do not regrow to their targets, this regenerative process had been named “abortive regeneration” by Ramón y Cajal (1907). We thus hypothesize that the activated microglia/monocytes we observed rostral to the lesion site at 4 and 6 weeks post-lesion correspond to a delayed clearance of debris resulting from dying back and/or abortive regeneration. In addition, we cannot exclude the possibility that there may also be a greater infiltration of monocytes from the periphery caudal to the lesion site compared to rostral.

In this study we show that the greatest lesion extension observed following spinal cord transection, quantified using ex vivo diffusion MRI, occurs at 72 hours post-injury and coincides with changes in microglia/monocytes density but not with somata morphology. Using cleared spinal cords and two-photon imaging we also observed differences over time in microglia/monocytes somata morphology which, combined with diffusion MRI results, is compatible with time-dependent phagocytosis of debris by microglia/monocytes rostral and caudal to the lesion site.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.