# Organization of collagen fibers and tissue hardening: Markers of fibrotic scarring after spinal cord injury in mice revealed by multiphoton-atomic force microscopy imaging

Clara Manesco, Oscar Saavedra-Villanueva, Marta Martin, Joshua de Lizaraga, Béla Varga, Thierry Cloitre, Yannick Nicolas Gerber, Florence Evelyne Perrin, Csilla Gergely

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Clara Manesco, Oscar Saavedra-Villanueva, Marta Martin, Joshua de Lizaraga, Béla Varga, et al.. Organization of collagen fibers and tissue hardening: Markers of fibrotic scarring after spinal cord injury in mice revealed by multiphoton-atomic force microscopy imaging. Nanomedicine: Nanotechnology, Biology and Medicine, 2023, 53, pp.102699. 10.1016/j.nano.2023.102699. hal-04195286

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## I.1 Title

# Organization of collagen fibers and tissue hardening: markers of fibrotic scarring after spinal cord injury in mice revealed by multiphoton-atomic force microscopy imaging

Clara Manesco, MScª, Oscar Saavedra-Villanueva, PhDª, Marta Martin, PhDª, Joshua de Lizaraga, MScª, Béla Varga, PhDª, Thierry Cloitre PhD.ª, Yannick Nicolas Gerber, PhDᵇ, c, Florence Evelyne Perrin, Prof.ᵇ and Csilla Gergely, Prof.ª*

*Funding:* This work was publicly funded through ANR (the French National Research Agency) under the "Investissements d'avenir" program with the reference ANR-16-IDEX-0006 and by the CPER-BNIF Bioimaging Nanofoundry.

*Conflict of interest:* no conflict of interest reported.

Abstract word count: 142 (max 150)

Manuscript word count: 4173 + 709 (figures) + 65 (Author) = 4947 (max 5000)

<footnote>a L2C, Univ Montpellier, CNRS, Montpellier, France</footnote>

<footnote>b MMDN, Univ Montpellier, EPHE, INSERM, Montpellier, France</footnote>

<footnote>c IUF, Intitut Universitaire de France, Paris</footnote>

<footnote>* Corresponding author. Laboratoire Charles Coulomb (L2C), Université de Montpellier, CNRS, Bâtiment 21 – CC069 Place Eugène Bataillon, 34095 Montpellier cedex 5, France.</footnote>

<footnote>*E-mail address*: csilla.gergely@umontpellier.fr</footnote>

<footnote n="2">²Abbreviations</footnote>

<footnote>SCI: spinal cord injury; ECM: extra cellular matrix; MPM: multiphoton microscopy; SHG: second harmonic generation; 2PEF: two-photon excited fluorescence; AFM: atomic force microscopy; hpi: hours post injury; wpi: week(s) post injury</footnote>
Spinal cord injury is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After injury the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier. The scarring process involves microglia, astrocytes and extracellular matrix components, such as collagen, constructing the fibrotic component of the scar. To investigate the role of collagen, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopy. The second harmonic generation signal exhibited by *fibrillar collagen* enabled to specifically monitor it as a biomarker of the lesion. An increase in collagen density and the formation of more *tortuous fibers* over time after injury are observed. Nano-mechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers' formation. These observations indicate the concomitance of important structural and mechanical modifications during the fibrotic scar evolution.

**Key words:** spinal cord injury (SCI), collagen, second harmonic generation (SHG), atomic force microscopy (AFM), nano-mechanics.
Spinal cord injuries (SCI) are one of the most impactful pathologies in the central nervous system (CNS), implying dramatic effects for the patients¹,² and consequent costs for the health care system³. The pathological state depends on the severity of the injury and causes the alteration of the sensory/motor functions located caudal to the level of the affected spinal cord¹. The destructive and self-propagating biological changes in cells and tissues lead to their dysfunction or death over seconds to weeks after the initial damage, a mechanism known as secondary injury. It includes the formation of a glial scar, composed of two main structures: the “glial” part and the “fibrotic” part. The glial part is formed by activated microglial cells and astrocytes that proliferate and migrate to the injury site. The fibrotic part is constructed by fibroblasts associated with extracellular matrix (ECM) components, such as collagen and is supported by reactive astrocytes⁴. In the chronic stage, the fibrotic part represents the inner zone of the scarring⁴, surrounded by the glial part where astrocytes form the outer border⁵. The scar acts as a dense chemical and physical barrier with dual effects. It plays a major role for natural healing process and neuroprotection after injury while preventing the axonal regeneration and sustaining the neuroinflammation (reviewed in ⁵,⁶). No curative treatment is currently available. However, approaches have been developed in mice to modulate responses of astrocytes⁷ or microglia⁸. In SCI, total depletion of microglia through the inhibition of CFS1R has been shown to be detrimental⁹,¹⁰. Conversely, transient inhibition of microglia proliferation after SCI using GW2580 (a CSF1R inhibitor that specifically blocks the proliferation of microglia¹¹) showed positive outcome in mice and nonhuman primates¹². Indeed, in both species a transient post injury treatment promoted motor recovery and tissue sparing in close vicinity of the lesion¹² while, in mice, increasing the treatment duration worsened the symptoms¹³.

The implication of collagen in the fibrotic scar formation has raised poor attention compared to microglia or astrocyte's role in the glial part. In addition, collagen studies in SCI are mainly focused on collagen IV¹⁴,¹⁵ and not on fibrillar collagen despite of its major role in common scarring and fibrotic processes¹⁶. Fibrillar collagen undergo a supramolecular assembly into cross-linked fibrils that leads to unique optical properties. Fibrillar collagen is a bright source for second harmonic generation (SHG) due to its highly non-centrosymmetric molecular assembly and large first hyperpolarizability¹⁷. Moreover, as SHG is a coherent optical process, the SH signal intensity, collected in the far field, is sensitive to the spatial distribution of individual molecular scatterers in the focal volume, but also to their orientations with respect to the direction of the incident polarization¹⁸. As a result, polarization-resolved SHG (P-SHG) microscopy is gaining increased attention to obtain detailed information on structural organization of the fibrils forming collagen fibers. We used P-SHG microscopy to discriminate between the two different supramolecular fibrils assembly encountered in collagen fibers involved in
the scarring process after SCI. Polarization curves (SH signal intensity as a function of incident polarization angle) show two typical profiles. Fibers constituted from straight and aligned fibril bundles are associated with a cylindrical symmetry and their polarization curve exhibits a typical two-hump profile. On the other hand, more disordered bundles of wavy fibrils are characterized by a trigonal symmetry and a single-hump profile of their polarization curves¹⁹⁻²¹. SHG signal is generally collected in transmission mode in combination with epi-collected two photon excited fluorescence (2PEF) signal from intrinsic or extrinsic fluorescence emitters, with no special sample preparation and in a non-invasive way. We have also monitored the mechanical properties of the samples by nano-indentation measurements performed by atomic force microscopy (AFM) in force spectroscopy mode (AFM-FS). The deflection of a flexible cantilever due to the interaction forces between a sharp tip, located at the end of the cantilever, and the sample surface is measured with force resolution in the piconewton range²².

Our multimodal label-free imaging approach is aimed to study the scarring process in a mouse model of spinal cord injury at different time-points post injury providing a new insight on the collagen contribution correlated with the mechanical properties of the tissue. Besides, we also aimed to investigate the potential effect of a GW2580 treatment. To the best of our knowledge, no current study has ever combined SHG and AFM to assess the evolution of the fibrotic scar. SHG was previously employed to analyze collagen fibers orientation at two different time-points post-injury²³. AFM studies on SCI are also scarce, suggesting that the lesion area is softer compared to the spared parts of the spinal cord²⁴ with a hardening of the tissue that propagates away from the injury core²⁵.
## ANIMALS, LESION MODEL, CARES AND SAMPLE PREPARATION

### Ethics approval

Experimental work with animals followed the European legislative, administrative and statutory measures for animal experimentation (EU/Directive/2010/63 of the European Parliament and Council) and the ARRIVE guidelines. The study was approved by the local ethic committee (nº 36), by the Veterinary Services Department of Hérault, and by the French Ministry of National Education Higher Education and Research (authorization n° 34118).

### Animals - Mice

We analyzed the same animals as those used in a previous study¹². CX3CR1⁺/eGFP transgenic female mice were used, expressing enhanced green fluorescent protein (eGFP), downstream of the CX₃CR1 promoter²⁶. The promoter is expressed in resident CNS microglia and peripheral monocytes. Animals were maintained in a C57BL/6 background (The Jackson Laboratory, Bar Harbor, ME, USA) and placed in controlled conditions for hygrometry, temperature and a cycle light/dark of 12 hours. Only heterozygotes were used. Immunostaining was carried out on 2 animals per time-point after the injury, with 4 to 5 spinal cord sections for each animal. The multimodal imaging acquisitions were conducted on 3 animals per time-points, with 2 to 5 sections for each animal.

### Spinal cord injury and post-operative cares

The protocol has been described elsewhere¹². Shortly, mice were anaesthetized with 3-4% isoflurane (Vetflurane®, Virbac, France). Anesthesia was then maintained with a 1-2 % isoflurane mixed with 1L/min oxygen flow rate during the surgery and eye gel was applied to the cornea. The lesion model consisted in a lateral spinal cord hemisection (HS) at thoracic 9 level (T9) performed with a micro knife [10315-12, Fine Science Tools (FST)]. Monitoring of the mice was conducted during 1 hour after surgery. To overcome sphincter disability induced by the lesion, bladders were emptied manually twice a day until the full recovery of the sphincter control. Bodyweights were monitored before surgery, and then regularly until the spinal cord processing. Analyses were done at 24 hours post-injury (hpi), 72hpi, 1 week post injury (1wpi), 2wpi, 4wpi, 6wpi without treatment. Additional investigations were performed at 6wpi on a group receiving a GW2580 oral treatment (6wpiT) as previously described¹².
For details see supplementary data (SD).
## Histology and sample preparation

Animals received an intra-peritoneal injection of a lethal dose of tribromoethanol (500 mg/kg, Sigma-Aldrich Darmstadt, Germany). They were perfused intracardially with PBS followed by paraformaldehyde (PFA) 4% (Sigma Aldrich). The samples were postfixed in the same fixative then immersed in sucrose, embedded in Tissue-Tek O.C.T™ Compound and frozen at -80°C. Serial 22 µm and 30 µm thick longitudinal spinal cord cryosections (Microm HM550, Thermofisher Scientific, Waltham, USA) were collected on Superfrost Plus® slides using a cryostat (Microm HM550, Thermofisher Scientific, Waltham, MA, USA). When needed, our label-free imaging approach was complemented with peroxidase staining to identify type I and IV collagen (see SD).

# MULTIMODAL LABEL-FREE IMAGING ACQUISITIONS

## Multiphoton microscopy (MPM)

Images were acquired with a custom-made multiphoton microscope based on an upright *scanning microscope (SliceScope, Scientifica Ltd, UK)* equipped with a galvanometer scan head and a Nikon 16x water immersion objective (CFI75 LWD-16x-W, NA=0.8, Nikon, Japan). The samples were excited at 780nm by a femtosecond Ti:Sapphire laser with ~100 fs pulse duration at 80 MHz (Tsunami, *Spectra - Physics, USA*). The polarization of the beam was controlled by a combination of achromatic half-wave and quarter-wave plates mounted on rotary stages. The 2PEF was epi-collected through the objective, filtered by a 735 nm long-pass dichroic (FF735-Di02, Semrock, USA), a 750nm short-pass filter (FESH0750, ThorLabs Inc, USA), a 450nm long-pass filter (62-942, Edmund Optics Ltd, UK), and then detected by a photomultiplier (R928P, Hamamatsu, Japan). The SHG signal was detected in transmission through a 1.4-NA water-immersion condenser (U-AAC, Olympus, Japan), filtered by a 482 nm long-pass dichroic mirror (86-331, Edmund Optics, Ltd, UK), a 550nm short pass filter (84-708, Edmund Optics, Ltd, UK) and a 447 nm high-performance band-pass filter (NT 48-074, Edmund Optics, Ltd, UK). The SHG signal was detected by a high sensitivity GaAsP photomultiplier (H7422P, Hamamatsu, Japan). Z-stack images were recorded in a window of 432x432 µm², at 1024x1024 pixels resolution, with a 2 µm Z step. P-SHG polarization stacks were obtained with an incident polarization angle θ ranging from 0° to 180° with 20° step (figure 5-B). To estimate the collagen fibers spatial extension, large mosaic images were obtained by stitching tiled images. These mosaic images cover the whole samples section around the injury site until no SHG signal is detected when scanning the rostro-caudal axis, as illustrated in Figure 1-B.
## Atomic Force Microscopy (AFM)

AFM force spectroscopy experiments were performed using an Asylum MFP-3D head coupled to a Molecular Force Probe 3D controller (Asylum Research, Santa Barbara, CA, USA). Spherical colloid built-in probe cantilevers with a nominal spring constant of 200 pN/nm, radius of 2 µm (Biosphere B2000-CONT, NanoTools GmbH) were used. The spring constant of the cantilevers was determined using the thermal noise method available within the MFP-3D software. Force measurements were performed, in PBS buffer, at room temperature and with a maximum loading force of 25 nN corresponding to a maximal indentation depth of 1 µm. The force-volume experiments were performed on a grid of 20x20 points over a region of 60x60 µm² (Figure 1-C) in different areas: injury site, grey matter (GM) and white matter (WM) with 3 to 10 experiments per area type. The recorded AFM-FS data (Figure 1-D) were used to determine the Young's modulus (E) of the tissue (Figure 1-E) using the Hertz contact model²⁷. Histograms of the distribution of Young's moduli were fitted with a Gaussian function (Figure 1-D) to obtain the mean E value of the probed area in different locations on the spinal cord (Figure 1-A). Young's modulus values were subsequently globally normalized to bring all values into the range [0,1] before comparison.

## DATA ANALYSIS

Collagen fibers characterization was performed using a home-made MATLAB code based on the fingerprint enhancement method²⁸. Briefly, the SHG images are processed to extract a skeletonized map of collagen fibers from which relevant metrics are then extracted. These metrics are defined below, and the image processing is detailed in the supplementary data.

### Polarization curves

To investigate the supramolecular assembly of collagen fibrils we analyzed the polarization curves following the work of Mercatelli et al.¹⁹. For each region imaged, we manually selected a set of regions of interest (ROI) delineating individual fibers (or bundles of fibers) exhibiting a high contrast in the maximum projection image of the P-SHG stacks. The mean SHG intensity was then calculated for each ROI to obtain a polarization curve. The profile of the polarization curves allowed us to discriminate between two types of fibers either consisting of an assembly of straight/aligned fibrils or of wavy/disordered fibrils and to evaluate the relative abundance of each fiber type.

### Metrics definition

All metrics are evaluated from the skeletonized map of collagen fibers obtained from processed SHG images (see SD). The parameters deduced are the fibers' density, tortuosity, local alignment map of the fibers and the statistical entropy.
## Fibers' density ($\phi$)

To quantify collagen in the SC tissue we have first calculated, from the skeletonized map the total length of the collagen fibers, that was then normalized to the surface covered by the tissue extracted from the 2PEF image. Herein, we name this parameter shortly as fibers' density ($\phi$):

$$ \phi = \frac{F_l}{S_t} \qquad (1) $$

with $F_l$ the sum of fiber's length detected across the tissue and $S_t$ the tissue area. $S_t$ is determined according to the morphological thresholding of the corresponding 2PEF image as depicted in yellow in Figure 6-C.

## Tortuosity

To quantify the straightness of individual fibers, we have calculated the tortuosity defined as the ratio of the fiber length over the fiber end-to-end distance²⁹. Lowest is the tortuosity straighter is the fiber.

## Statistical entropy (S)

To evaluate the fibers' organization within the tissue the statistical entropy $S$ of the orientation distribution at pixel level was computed from the skeletonized SHG image following Ducourthial et al.³⁰:

$$ S = \frac{-1}{\ln(NB)} \sum_{\beta=-90^{\circ}}^{90^{\circ}} p(\beta) \ln(p(\beta)) \qquad (4) $$

Where $NB$ is the number of bins, $\beta$ is the angular bin used, $p(\beta)$ is the normalized number of pixels with the orientation $\beta$ in the local orientation map as explained in the SD. The entropy provides the degree of orientation disorder at tissue level: $S = 1$ means a fully isotropic orientation distribution, whereas small $S$ values indicate that the orientation distribution is narrower.

## Local alignment of fibers, variance map

To assess fiber's alignment, we have performed a circular statistical analysis for different local orientations with $\theta \in [-90^{\circ}, 90^{\circ})$. By computing the variance³¹ we evaluate the fibers local organization:

$$ Var(\theta) = \frac{1}{N} \sum_{j=1}^{N} (1 - \cos(2\theta_j - 2\bar{\theta})) $$

with $\theta_j$ the orientation of a pixel associated to a skeleton fiber and $\bar{\theta}$ the mean orientation which is computed as follows:
$$ \bar{\theta} = \arg\left\{\sum_{j=1}^{N} e^{i2\theta_j}\right\} / 2 $$

Var(θ)=0 for perfectly aligned fibers, and Var(θ)=1 for random (not aligned) fibers. The method to obtain the local alignment map is described in the SD. The degree of fibers' alignment was compared at different time-points by segmenting the Var(θ) in four groups, as described in the SD.

## Statistics

1-way repeated measurements ANOVA Tukey's multiple comparisons, mixed effect model was assessed for comparison of collagen fibers metrics and Young's modulus values. Repeated measurements and the mixed effect model were necessary as the compared samples were paired and not always of the same size. Tukey's multiple comparisons were employed after performing normality tests for comparison of every mean with every other mean. A paired t-test with Welch's correction was used for comparing the treated group with the control one at 6wpi. 3 mice were analyzed for each condition; the number of acquisition areas are indicated in the plots. Statistical significance was set at $p \le 0.05$.
# Results

## Peroxidase staining and multiphoton microscopy imaging

Peroxidase staining was performed to reveal the presence of collagen types I and IV through a brown contrast (Figure 2). From 24hpi to 6wpi expression of collagen I and IV were observed with an overall increasing up to 2wpi that most likely stabilize after 4wpi onwards. Moreover, the expression of both collagen types seems to become restricted over time to the lesion area and to decrease following transient pharmacological depletion of microglia proliferation.

Tissue disorganization in the spinal cord evidenced in bright field images allowed to discriminate the injury site (Figure 3-A) from spared grey and white matter areas. 2PEF images revealed microglia and peripheral monocytes around the injury site (Figure 3-B). SHG image (Figure 3-C) evidenced fibrillar collagen specifically located at the injury side, (Figure 3-D). No fibrillar collagen was detected before 1wpi, then the *density of collagen* increased gradually with time from 1wpi to 6wpi (Figure 3-E). The group supplied with the treatment exhibited a weaker signal than the control one at 6wpi. The mean rostro-caudal *extension of fibrosis* detected by SHG was measured on mosaic images (Figure 4) and found to be 870±300, 1200±400, 1800±200 and 950±250 µm at 1wpi, 2wpi, 6wpi and 6wpiT, respectively. The transverse extension of the SHG signal is essentially limited to 2/3 of the spinal cord section at the lesion side. The variation of the SHG intensity, exhibited by fibrillar collagen, with the incident polarization orientation is exemplified in Figure 5. The polarization curve profiles showed that both straight (two-hump) and wavy-like fibrils bundles (one hump) profiles could be encountered in the SC tissues (Figure 5-D).

## Quantifying SHG signal to characterize collagen at injury site

Quantification of collagen fibers at macroscopic level was conducted with metrics calculated from the SHG signal and presented in Figure 6. The calculated *tortuosity* (Figure 6-B) was found to decrease at 6wpi (1.9±0.2), compared to 1wpi (1.6±0.2) and 2wpi (1.8±0.1), suggesting that fibers became more *tortuous* at later time-point. A statistical decrease was observed between the 6wpi and the treated group (6wpiT). A significant increase in the *density of fibers* (Φ) (Figure 6-D) was noticed over time: 0.06±0.03 µm⁻¹ at 1wpi, 0.12±0.01 µm⁻¹ at 2wpi and 0.13±0.02 µm⁻¹ at 6wpi. Collagen fibers in the treated group appeared to have a reduced Φ (0.06±0.03 µm⁻¹) compared to the control one. *The local alignment of fibers* (Var(θ)) (Figure 6-E), exhibited low values, between 0.3 and 0.5, suggesting that the fibers are rather well aligned *with each other* (Figure 6-F). However, no significant difference was observed between the different samples. *The partition of the local alignment of fibers in four groups* (Fig. S4) reveals that a majority of highly aligned (Var(θ) : [0, 0.5]) fibers are observed from 1wpi to 6wpi, but they become less aligned for the treated group. The distribution of fibers orientation on a
whole image was addressed through the calculated statistical entropy. The entropy values were globally high (between 0.90 and 0.95) and similar for the various time-points, meaning that no preferential orientation was detected at tissue level (Figure 6-G).

## Spinal cord tissue mechanics via AFM force measurements

The mechanical properties of tissues were addressed via AFM force spectroscopy measurements. The combined height and elasticity color maps obtained from the calculated Young's moduli (E) for the GM, WM and injury are exemplified in Figure S1. The statistical comparison of the normalized E moduli indicate that GM is stiffer than WM at all time-points (Figure S2). Measurements performed on healthy spinal cord (Figure S3), confirm this tendency. Furthermore, the lesion area exhibited larger normalized elasticity variations between different time-points compared to the spared GM and WM areas (Figure S2). The comparison of E values supported these observations, showing that the elasticity of GM (Figure 7-A) and WM (Figure 7-B) varies very little at different time-points post-injury. Contrary, for the injury area, a noticeable stiffness increase was observed (Figure 7-C) after 72hpi (7±4 kPa; 0.04 normalized), stabilizing at 2wpi (54±32 kPa; 0.32 normalized). The initially soft injured tissue gets even stiffer than the WM at already 1wpi reaching the elasticity value of GM at 2wpi (Figure S4). No significant difference was reported between the elasticity of the treated and control group at 6wpi (Figure-7D).
**Fibrillar collagen as a biomarker of the injury**

Immunohistochemistry and SHG imaging evidence that: i) both collagen type IV and *fibrillar collagen* are present in the lesion area, ii) the SHG signal specifically reveals the presence of fibrillar collagen as a biomarker of the scarring starting from 1wpi, and iii) the extension of the area occupied by fibrillar collagen increases along the rostro-caudal axis at the injury site from 1wpi to 6wpi. Collagen IV has already been evidenced by immunohistochemistry as a major component of the scar fibrotic process in rodents<sup>15,32-34</sup>, and 1wpi has been reported to be a key time-point in collagen IV matrix formation<sup>14</sup>. Less attention was yet dedicated to identification of fibrillar collagen using labelling techniques, even if some studies evidenced its presence mainly after 2wpi<sup>35-37</sup>. Anik et al. work<sup>34</sup> is one of the few examples identifying both collagen I and IV together. While it has been widely investigated in common scarring<sup>16</sup>, label-free SHG imaging of collagen I has only started recently in the SCI context<sup>38</sup>, and has been reported at 2wpi after a transection in rats<sup>39</sup> and in a crush model in mice<sup>40</sup>. Collagen amount was assessed at 4wpi and 22wpi in a hemisection model in rats<sup>23</sup>, showing massive formation but without significant difference between the two time-points. In another work collagen III has been revealed by staining at the SCI injury site<sup>41</sup>.

**Collagen fibers tortuosity and density of fibers increase in time**

Quantification of the SHG signal provided two metrics suitable to monitor the fibrotic scarring process. At fiber-level, i) the tortuosity, reveals less straight fibers at 6wpi, and ii) the fibers' density increases with time and decreases in the treated group. The variations of SHG signal in function of polarization, monitoring the nature of collagen fibrils at a supramolecular level indicates that both straight and wavy fibrils were found. Even if no direct link can be evidenced between the tortuosity of fibers and the nature (straight or wavy) of fibrils which constitute them, this may bring complementary information about the collagen organization. The different configurations of fibrils have been assessed in cancer<sup>19</sup> showing that collagen fibers associated with tumor had a high straightness while those in peri-lesional tissue exhibited high waviness. In our case, there is no collagen initially present in the spinal cord but it appears after 24h, as revealed by immunochemistry and forms fiber bundles at 1wpi, as indicated by their SHG signal, gradually invading the injury site and exhibiting both straight and wavy fibrils with no evidence of spatial separation between these two configurations. However, we observed that straight aligned fibrils are more represented at 1wpi, while wavy-like collagen structures became dominant at 2wpi and 6wpi. The specificity of the SHG signal exhibited by collagen fibers allowed to quantify their extension along the injury over time. After GW2580 treatment targeting specifically the microglial proliferation also affecting macrophages in the scar, the collagen extension appeared to be reduced.
Gerber et al.¹¹ and Poulen et al.¹² described that GW2580 efficiently decreased the proliferative microglia, but no effect on the lesion extension was reported. The decrease of collagen fibers' density and their apparent reduced extension in the treated group suggests inhibition of microglia proliferation directly or indirectly modulating collagen expression in the scar⁴. The relative orientation of fibers did not exhibit any variation up to 6wpi. Similarly, no preferential direction was reported in collagen organization in rats at 4wpi or 22wpi²³. Collagen fibers orientation has yet been defined as an important marker in fibrotic disease or cancers, mainly associated with highly aligned collagen bundles compared to normal state⁴²,⁴³.

## The injury site is softer than healthy grey and white matter at 72hpi and hardens from 1wpi

AFM measurements demonstrated that: i) GM is globally stiffer than WM, ii) the injured tissue is softer than the spared parts at 72hpi but hardens from 1wpi and it stabilizes at 2wpi. Mechanical properties of spinal cord tissue have been evaluated in various studies with controversial conclusions about GM being stiffer than WM in both healthy⁴⁴ and injured samples²⁴ or not different in uninjured samples⁴⁵. Moeendarbary et al.²⁴ worked on fresh SC injured tissues in a crush injury model in rats, and found healthy GM to be stiffer than WM. They additionally observed a tissue softening in the lesion area at 1.5wpi and 3wpi that was correlated with gliosis markers, the presence of glial intermediate filaments or ECM protein markers like collagen type IV, however fibrillar collagen was not investigated. Tissues at chronic stage after spinal cord hemisection in rats (15-18-wpi) were also used²⁵ to evaluate the stiffness evolution near and far from the lesion, showing a small Young's modulus increasing at distance from the lesion core. Overall, the results reported on the lesion mechanical behavior seem to depend on the tissue preparation and on the injury model used. In our work, the significant increase in Young's moduli, the stiffening of the injury from 1wpi can be correlated with the formation of collagen fibers observed at the same time-point with SHG imaging. A recent work reported that more helicoidal fibers (wavy fibers) are associated with a stiffness increase of collagen in cornea⁴⁶. It could be thus interesting to perform further quantitative assessments on fibers waviness to evaluate whether this parameter plays a role in the tissue hardening of the injured zone.

To conclude, our imaging approaches enable to gather valuable data at micro/nano resolution on excised spinal cord tissues. Relevant markers can be obtained, sensitive enough to assess structural and mechanical modifications of the fibrotic scar formation over time in a hemisection SCI mouse model. We report and characterize the formation of fibrillar collagen that gradually invade the injury site from one to six-weeks post-injury. Further investigations are necessary to better understand the origin of the fibrotic process sustained by the hardening of the injured spinal cord tissue. Axonal regeneration following SCI is hampered by the modified tissue composition and the resulting glial and
fibrotic scar. This study being focused on monitoring collagen spreading at the injury site, further work
should address the accompanying modifications in the glial and astrocyte cells reorganization leading
to formation of the glial scar. Other non-linear technics like broadband CARS microscopy could provide
valuable information on the biological state of the cells and the concomitant metabolite modifications.
Alterations in the ECM after injury may contribute to the observed modifications in tissue stiffness.
Also, additional studies in other models of SCI will permit to investigate whether collagen formation
depends on the type of lesion. Our study provides novel insights into the fibrotic scar as a possible
treatment target for SCI.
<figure><img src="image_2.png" /><figcaption>**Figure 1:** Mosaic acquisition on longitudinal spinal cord tissue section and AFM force spectroscopy measurements and data analysis. A. Bright field image of a spinal cord (longitudinal section) where the injury area is located at the right side of highlighted by the black square. B. The enlarged area within the black square. The injury is on the right edge. For SHG mosaic acquisitions, tills (black squares) of 432x432µm² were measured. C. Bright field image of the sample showing the AFM tip and the acquisition area (square of 60x60 µm²) at the injury area. D. Typical force-distance curve fitted with the Hertz model. Inset: the histogram of Young's modulus is fitted with a Gaussian function to obtain the main value. E. Elasticity map of a scanned area (20x20 points). Scale bars are 500 µm (B), 80 µm (C), 15 µm (E).</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2: **Collagen types I and IV at the injury site via immunohistochemistry.** Optical (NanoZoomer) images of longitudinal spinal cord sections with a hemisection at thoracic 9 (T9) level (schema, orange square: size 1.2x1.2 mm²), immunohistochemistry was done at different time-points (24hpi, 72hpi, 1wpi, 2wpi, 4wpi, 6wpi and 12wpi) and with a treatment condition at 6wpi (6wpiT). Immunohistochemistry reveals a higher contrast at the injury site for both collagens I and IV. Scale bar is 500 µm.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 3: Multiphoton microscopy (MPM) of injured spinal cord.** A. Bright field image of a mouse spinal cord at 1wpi, lesion site is on the right; the black rectangle shows the MPM acquisition area. B. 2PEF image, C. SHG image, D. 2PEF-SHG merged images (scale bar is 200 µm). The SHG signal is visible only on the lesion side. Two enlarged area, corresponding to the yellow box close to the injury and the orange box far from the injury area, are shown. Scale bars are 10 µm. E. SHG images focused on the lesion area at 72hpi, 1wpi, 2wpi, 6wpi and 6wpi with treatment. Scale bar is 100 µm.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4: **Extension of fibrillar collagen around the lesion site.** Mosaic reconstruction of SHG images, showing the fibrotic extension at: A. 1wpi, B. 2wpi, C. 6wpi and D. 6wpiT. Insets are merged 2PEF (green) - SHG (red) zoomed images, centered at the lesion site as indicated by the yellow rounded squares (432x432 µm²). The mean rostro-caudal extension of the injury was found to be 870±300, 1200±400, 1800±200 and 950±250 µm for at 1wpi, 2wpi, 6wpi and 6wpiT, respectively. Scale bar is 400 µm.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5:** Polarization-resolved Second Harmonic Generation (P-SHG) of collagen fibers. A. P-SHG images at 3 different polarization angles: 0°-blue, 40°-yellow, 100°-red, all - composite image. Scale bar is 100 µm. B. Illustration of fiber's orientation, with Θ, the polarization angle of the laser. C. SHG image at the injury site at 1wpi, with zoomed areas (corresponding to the blue and red squares, 50x50 µm), showing a bundle of wavy-like fibrils (red cursor) and straight fibrils (blue cursor). D. The corresponding polarization curves are shown with the same colour code. E. Wavy-like fibrils become dominant at 2wpi and 6wpi with and without treatment. Scale bars are 50 and 20 µm, respectively.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 6:** Collagen fibers' metrics at different time-points. A. Fibers' skeleton map with individual fibers (zoomed area). B. Tortuosity C. The fibers' density was determined with the surface occupied by the tissue (inset). D. Fibers' density (Φ) E. Local alignment map. F. The variance of the local orientation θ. G. Statistical entropy (S). With * p<0,05, **p < 0.025. Scale bar is 50 µm.**</figcaption></figure>
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<figure><img src="image_9.png" /></figure>

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<figure><img src="image_11.png" /><figcaption>**Figure 7:** Statistics of normalized Young's modulus for the three spinal cord area categories. Evolution of normalized Young's modulus values with the post-injury time-points in **A.** grey matter, **B.** white matter, **C.** injury area. **D.** Normalized E for these 3 areas at 6wpi without and with treatment supplying. Results suggest that the mechanical properties of the injury are more varying in time compared to those of grey and white matters, which are quite stable considering the samples variability. Globally, grey matter is stiffer than white matter and the injury appears to become stiffer from 1wpi. No difference is observed in the treated group compared to the control one at 6wpi (with * p < 0.05, **p< 0.025, *** p < 0.001, **** p < 0.0001).</figcaption></figure>
CRediT author statement

Clara Manesco: Investigation, writing original draft, writing review and editing, data curation, methodology

Oscar Saavedra-Villanueva: Investigation, software, data curation, writing review & editing

Marta Martin: Investigation, formal analysis

Joshua de Lizaraga: Investigation

Béla Varga: Investigation

Thierry Cloitre: Conceptualization, writing review & editing, supervision, methodology

Yannick Gerber: Resources

Florence Perrin: Conceptualization, resources, methodology

Csilla Gergely: Conceptualization, writing review & editing, funding acquisition, project administration, supervision, validation
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T9

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Treatment