Nociceptive mechanisms driving pain in a post-traumatic osteoarthritis mouse model

In osteoarthritis (OA), pain is the dominant clinical symptom, yet the therapeutic approaches remain inadequate. The knowledge of the nociceptive mechanisms in OA, which will allow to develop effective therapies for OA pain, is of utmost need. In this study, we investigated the nociceptive mechanisms involved in post-traumatic OA pain, using the destabilization of the medial meniscus (DMM) mouse model. Our results revealed the development of peripheral pain sensitization, reflected by augmented mechanical allodynia. Along with the development of pain behaviour, we observed an increase in the expression of calcitonin gene-related peptide (CGRP) in both the sensory nerve fibers of the periosteum and the dorsal root ganglia. Interestingly, we also observed that other nociceptive mechanisms commonly described in non-traumatic OA phenotypes, such as infiltration of the synovium by immune cells, neuropathic mechanisms and also central sensitization were not present. Overall, our results suggest that CGRP in the sensory nervous system is underlying the peripheral sensitization observed after traumatic knee injury in the DMM model, highlighting the CGRP as a putative therapeutic target to treat pain in post-traumatic OA. Moreover, our findings suggest that the nociceptive mechanisms involved in driving pain in post-traumatic OA are considerably different from those in non-traumatic OA.


Results
Pain-related behaviour in the OA post-traumatic DMM model. The development of pain in OA animal models is integral to its validation and utility as models for translational research 12 . Rodent OA models present weight-bearing asymmetry and mechanical allodynia, resembling OA patients. Alterations in innate behaviour and gait pattern have been also used as indicators of pain in these animal models 12 .
In this study, the Von Frey data analysis revealed that 8 weeks after DMM surgery mice presented increased mechanical allodynia when compared with the non-operated group (p = 0.026), and at week 10 and 12 when compared with non-operated and sham-operated mice (10 weeks: sham-operated p < 0.001 and non-operated p = 0.047; 12 weeks: sham-operated p = 0.004 and non-operated p = 0.042) (Fig. 1A). Moreover, DMM mice presented increased mechanical allodynia at week 8, 10 and 12 post-surgery when compared with week 4 (week 8 p = 0.008; week 10 p = 0.001: week 12 p = 0.015).
Instead, the analysis of the data from the open field test showed no differences, at each analysed time point, between the DMM and the control groups (non-operated and sham-operated mice) concerning (i) distance travelled, (ii) mean velocity and (iii) time spent immobile (Fig. 1B). These results indicate that, even at 12 weeks post-DMM surgery, the locomotor activity was not affected. Regarding gait pattern, no effects were observed in the evaluated measures: (i) right (affected)-left paw distance, (ii) angle between consecutive paw prints and (iii) relative step length step (Fig. 1C). However, the analysis of the footprint revealed that ITS was decreased in the DMM mice when compared with the sham-operated mice (p = 0.019) (Fig. 1C), indicating that mice foot placement was impaired. Mechanical allodynia (A), and alterations in locomotor activity (B) and gait pattern (C) were assessed 4, 6, 8, 10 and 12 weeks after the knee joint traumatic lesion. The Von Frey data analysis showed that DMM mice presented increased mechanical allodynia when compared with sham-operated, starting at week 8. No effects were observed in the locomotor activity. A decrease in the ITS was observed in the DMM mice. Results are presented as mean ± SEM, n = 5 in the control group and n = 8 in the sham-operated and DMM groups. *Indicates differences between sham-operated and DMM groups; # indicates differences when compared with non-operated group; & indicates differences when compared with week 4; *p < 0.05; **p < 0.01; ***p < 0.001; # p < 0.05; ## p < 0.01; & p < 0.05; && p < 0.01. ITS intermediate toe spread, PL print length, TS toe spread.

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| (2020) 10:15271 | https://doi.org/10.1038/s41598-020-72227-9 www.nature.com/scientificreports/ Joint morphological and histological alterations in the OA post-traumatic DMM model. Currently, it is well established that the pathophysiology of OA involves, in addition to cartilage lesion, damage in other joint tissues. Alterations in the subchondral bone and the synovial inflammation have been shown in both patients and animal models 13 . In the present study, the X-ray and μ-CT images acquired 12 weeks post-DMM surgery revealed the narrowing of the knee joint space and the formation of osteophytes ( Fig. 2A,B). Further quantitative μ-CT analyses uncovered the development of subchondral bone sclerosis in the tibia of DMM mice, as indicated by the increase in subchondral bone volume (p = 0.0039) and thickness (p = 0.0171) (Fig. 2B). The safranin O staining, a basic stain that binds cartilage proteoglycans, revealed cartilage damage ranging from intense fibrillation to thinning of the articular cartilage in the DMM mice (Fig. 1C). Moreover, the knee sections of DMM mice showed a lower intensity in the red staining of cartilage when compared with sham-operated mice, suggesting the loss of proteoglycan content (Fig. 1C). These alterations in the articular cartilage were traduced in significantly higher OARSI score in the DMM when compared with sham-operated mice (p = 0.038) (Fig. 1D).
Overall, the degradation of cartilage, the narrowing of the joint space, and the development of osteophytes and subchondral bone sclerosis support the induction of an OA phenotype at 12 weeks post-DMM surgery. As above mentioned, increasing evidence suggests that synovial membrane inflammation (characterized by the infiltration of mononuclear cells, thickening of the synovial lining layer and production of inflammatory cytokines) is an important mediator in the pathogenesis of both OA structural degeneration and OA pain 14 .
In this experiment, we analysed the synovium for the infiltration by immune cells previously described in synovitis 14 . Our results revealed no infiltration by immune cells, such as macrophages (Fig. 1E), T cells and B cells (data not shown) at 12 weeks after the knee joint traumatic injury. Positive-staining control images are provided as supplementary data (Fig. S1).
Alteration in the joint innervation in the OA post-traumatic DMM model. Contrary to the lack of innervation in the articular cartilage of healthy joints, sensory and sympathetic nerve fibers have been observed in the articular cartilage of mild and severe human OA stages 15 . It has been hypothesized that the changes in joint innervation might be related to pain severity 16 . Here, we evaluated the presence of nerve fibers positive for growth-associated protein (GAP)-43 (a marker of neurons growth and regeneration) in the knee joint structures. We observed no GAP-43 positive nerve fibers innervating the cartilage (data not shown) and no alterations in the GAP-43 innervation profile of the synovium of the DMM mice (Fig. 3A). Nevertheless, increased GAP-43 innervation was observed in the periosteum of these mice when compared with sham-operated mice (p = 0.0357).
Data from clinical and animal OA studies highlighted a crucial role of sensory innervation in OA pain [16][17][18][19] . In this study, we investigated changes in the sensory innervation through the evaluation of the CGRP innervation pattern of the joint. We found no CGRP-nerve fibers invading articular cartilage (data not shown) and no alterations in the CGRP innervation profile of the synovium of the DMM mice. However, as for the GAP-43, we observed an increase in the density of CGRP-nerve fibers innervating the periosteum of the DMM mice (p = 0.027) (Fig. 3B).
Alteration in peripheral nociceptive pathways in the OA post-traumatic DMM model. In addition to the structural and biochemical changes in the injured joint, alterations in the peripheral and central pain signalling pathways are key components of OA pain 10 . Both pre-clinical and clinical studies have implicated the nerve growth factor (NGF)/receptor Tyrosine Kinase A (TrkA) signalling pathway in the OA pain, and its antagonism was shown to result in OA pain reduction [19][20][21][22] . The NGF released by injured tissues activates TrkA in sensory neurons, up-regulating the neuropeptidergic signalling (by CGRP and SP) and transient receptor potential cation channel subfamily V member 1 (TRPV1), pain-related molecules involved in peripheral sensitization 23,24 . Here, we investigated alterations in the TrkA, CGRP, and TRPV1 expression in the DRG (where the cell bodies of sensory neurons are located). Our data showed increased expression of CGRP in the DRG of DMM mice (p = 0.0401) (Fig. 4A). No alterations were observed in the expression of TrkA and TRPV1 (Fig. 4A).
We also assessed the expression levels of neuropeptide Y (NPY), a marker of neuropathic pain that is expressed in DRG only in response to nerve injury 25,26 . As above described the innervation by GAP-43 is upregulated in the periosteum, which could suggest nerve damage in the DMM mice. However, we observed no detectable expression of NPY in the DRG at 12 weeks post-DMM surgery (Fig. 4B).
In this study, we also investigate the infiltration of DRG by macrophages. This mechanism has been highlighted as playing an important role in the initiation and maintenance of peripheral neuron sensitization in neuropathic and inflammatory pain models 27 . However, here we found no macrophages infiltrating the DRG of DMM mice (Fig. 4B).
Markers of central sensitization in the OA post-traumatic DMM model. It was reported that up to 80% of the OA patients achieve pain relief with peripheral analgesia, supporting a prevalence of peripheral mechanisms driving OA pain 28,29 . However, centrally mediated pain sensitization has also been reported in some OA patients 30 . Central sensitization results from the excessive activity of peripheral nociceptive pathways, that leads to increased release of excitatory molecules, including the CGRP, into the spinal cord dorsal horn contributing to the sensitization of the second-order pain transmission neurons 31 . In this study, we investigated whether the observed increase in the CGRP levels in the DRG of DMM mice could have contributed to the establishment of central sensitization. To assess this, the protein levels of c-Fos and phospho-extracellular signal-regulated kinases 1/2 (p-ERK1/2), markers of neuronal activation, and the levels of ionized calcium-binding adapter molecule 1(IBA-1) and glial fibrillary acidic protein (GFAP), known to be upregulated in activated microglia and Scientific RepoRtS | (2020) 10:15271 | https://doi.org/10.1038/s41598-020-72227-9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ astrocytes 32,33 , were quantified in the spinal cord. DMM mice presented no alterations in the expression levels of these markers (Fig. 5).

Discussion
In the present study, we demonstrated that the expression of CGRP is augmented both in the sensory nerve fibers of the periosteum and in the DRG, at 12 weeks after the traumatic injury of the knee joint in the DMM model. This increased CGRP expression might be underlying the peripheral pain sensitization, reflected by an increase www.nature.com/scientificreports/ in mechanical allodynia. Additionally, at this time point of OA progression, our observations revealed that the infiltration of the synovium by immune cells, and neuropathic and central sensitization mechanisms did not contribute to the displayed pain behaviour. The marked cartilage damage, the narrowing of the joint space, and the presence of osteophytes and subchondral bone sclerosis support the development of an OA phenotype at 12 weeks post-DMM surgery. However, the inflammation of the synovial membrane, another common feature of OA 34 , seems not to have a critical role on the pathophysiology of this preclinical model at this stage of the disease progression. In fact, we did not observe infiltration of the synovium by immune cells. Using the DMM model, Driscoll et al. also reported no increase in joint inflammation 12 weeks post-surgery 35 . Together, these results corroborate previous studies describing an initial intense inflammatory response to the traumatic joint injury, which is followed by lower inflammation levels in later phases 36 . www.nature.com/scientificreports/ Along with the observed morphological and histological alterations, mice developed mechanical allodynia. This supports previous studies reporting the presence of mechanical allodynia in this OA model 9 and indicates the development of pain sensitization 37 .
Decreased locomotor activity and altered gait pattern are also a common consequence of OA pain. The critical time point for alteration in the locomotor activity in post-traumatic OA models is not clearly defined. Sambamurthy et al. reported altered locomotor activity at week 8, when comparing DMM animals with non-operated controls, but not when comparing with sham-operated animals 38 . On the other hand, Miller et al. described decreased locomotor activity in DMM mice at week 8, but at week 16 this effect was no longer observed 39 . In this study, we observed no differences in the locomotor activity between DMM and the control groups (non-operated and sham-operated mice), indicating that, until the week 12 post-DMM surgery, pain is not affecting the locomotor activity. Changes in gait parameters, when using the CatWalk system, have been reported to emerge between 10 and 12 weeks post-DMM surgery 40,41 . Here, we evaluated putative changes in the gait pattern following the method described by Boettger et al. 42 , and none of the assessed measures were different between DMM and the non-operated and sham-operated controls. However, we observed a decrease in the ITS in the affected paw of DMM mice, suggesting that pain was interfering with the foot placement.
Overall, the OA post-traumatic DMM model is characterized, at 12 weeks progression stage, by a marked knee joint damage that was associated with mechanical allodynia but not associated with alterations in locomotor activity and gait pattern.
The normally aneural articular cartilage of the healthy joints, is described to become innervated by sensory and sympathetic nerve fibers in mild and severe human OA stages 15 . It has been hypothesized that the changes in joint innervation might be related to pain severity 16 . Contrary, a reduction of the sensory innervation in the synovium was reported in human samples, and was shown to be closely related with inflammation 43 . Decrease synovial sensory innervation was also described in inflammatory collagenase-induced OA mouse model 44 . In the present study, 12 weeks after the DMM surgery we observed no sensory nerve fibers in the cartilage and no differences in the sensory innervation of the synovium. The lack of differences in the synovial sensory innervation might be related with the low inflammatory nature of the DMM model. However, we observed alterations in the innervation pattern of the periosteum.
The increased expression of neuronal GAP-43 in the periosteum that we observed in the DMM mice indicates nerve sprouting in this area. We hypothesized that these growing nerve fibers were probably mainly sensory, as we also observed an increase in the number of CGRP-positive nerve fibers in the periosteum. CGRP, a neuropeptide found primarily in the C and Aδ sensory fibers, is well known to modulate neuronal sensitization and increase pain. To our knowledge, we are the first reporting sprouting of sensory innervation in the periosteum in a post-traumatic OA model. The periosteal sensory innervation has been highlighted as a critical player in bone pain. The increase in the density of CGRP nerve fibers in periosteum has been reported associated to pain in bone pathological conditions such as non-healed fracture 45 and bone cancer 46,47 . The increase in the periosteum innervation by CGRP-positive nerve fibers was also previously reported in Complete Freund's adjuvant (CFA)induced arthritis in mice 48 and in and the Monoiodoacetate (MIA) rat model 49 . Importantly, the blockade of the sprouting of the periosteal sensory innervation, by the administration of an anti-NGF antibody, is associated with the inhibition of the pain-related behavior in bone cancer 46,47 . Together with our results, these data support the sensory innervation of the periosteum as an important potential source of pain in post-traumatic OA. www.nature.com/scientificreports/ The CGRP expressing neurons are responsive to NGF through its action on TrkA 31 . The activation of the NGF/TrkA signalling pathway induces the up-regulation of CGRP and other molecules such as TRPV1, both known to be involved in pain sensitization 23,24 . Importantly, both pre-clinical and clinical studies show that the blockage of NGF signalling produces analgesia in OA pain [19][20][21] . The up-regulation of CGRP, TRPV1, and TrkA in DRG was previously described in MIA-induced OA model 50,51 . Here, we report an increase in the DRG expression of CGRP, but no alterations in the expression of TrkA and TRPV1. The different observations between MIA-induced and DMM models may be related to the different prevalence of the inflammatory mechanisms between these two models.
In this study, we hypothesized that the increased expression of CGRP in the sensory nervous system is involved in the mechanisms underlying the mechanical allodynia presented by the DMM mice. Our hypothesis is in line with previous data showing that exogenous administration of CGRP to the knee mimics the peripheral sensitization of arthritic joints, which is decreased by CGRP antagonisms 52 . In fact, the targeting of CGRP signalling in arthritic joints has been highlighted, in animal studies, as a putative strategy to treat pain 52,53 .
Further studies are needed to unveil the mechanisms underlying the increase in the CGRP expression and other related mechanisms of peripheral sensitization, from the receptors expressed on the nociceptors membrane, to the activated intracellular pathways (e.g. PKC, PKA, PI3K, the MAP kinases ERK and p38, and JNK) and the downstream effectors of peripheral sensitization (e.g. the phosphorylation of TRP and voltage-gated channels, such as TRPV1, TRPA1 VGSCs, Nav1.8, Nav1.7, and Nav1.9).
In addition to pain sensitization, neuropathic pain mechanisms were also described in some chemicallyinduced OA models, for instance in the MIA model 54 . Neuropathic pain is defined as the pain caused by lesion or disease of the somatosensory system 55 . Although we had observed an increase in the GAP-43 positive-nerve fibers in the DMM mice, which could suggest nerve damage, we found no DRG expression of NPY, which is a marker of neuropathic pain expressed in DRG only in response to nerve injury 25,26 . Therefore, our results suggest that, at this stage of OA progression post-DMM surgery, the neuropathic mechanisms were not a significant component of OA pain. The low contribution of a neuropathic component is further supported by the observed absence of macrophages invading the DRG. Macrophages are typically found in DRG after nerve injury, as observed in the constriction or transection of the sciatic nerve models 56,57 . In inflammatory joint models, macrophages are also observed invading DRG [58][59][60] . Here, the absence of macrophages in DRG is in line with our observation of a putative low inflammatory response. Overall, the neuropathic and the inflammatory components did not have a significant role in the pain displayed at 12 weeks post-DMM surgery.
Centrally mediated pain sensitization has been reported in OA patients 30,61 and animal models 62,63 . But, to our knowledge, this mechanism was not previously studied in the DMM model. Peripheral sensitization leads to the increased release of excitatory molecules by the sensory neurons, including the CGRP, into the spinal dorsal horn, contributing to the sensitization of the second-order pain transmission neurons 31 . In the MIA-induced OA model, pain development is correlated with an increase in the c-Fos and p-ERK1/2 (markers of neuronal sensitization 64,65 ) in neurons of the spinal cord dorsal horn 66,67 . Moreover, the intrathecal injection of the ERK1/2 signalling pathway inhibitor PD98059 reduces the pain and the p-ERK1/2 induction in this OA model 66 . In the CIA-induced arthritis model the analgesia achieved by the intrathecal administration of tramadol also show the reduction in the p-ERK1/2 increase 68 . The spinal microglia and astrocytes were shown to play a critical role in development of the pain hypersensitivity, as they can dynamic modulate the activity of spinal neurons 69,70 . This has been demonstrated namely in models of arthritis, such as collagenase-induced 71 and CFA-induced arthritis 72 . The involvement of microglia and astrocytes is strongly supported by the data showing that their inhibition decreases nociception 71,72 . Here, we observed that the spinal levels of c-Fos and p-ERK1/2, as well as, IBA-1 and GFAP (upregulated in activated microglia and astrocytes, respectively) were not affected. These results indicate no neuronal sensitization or activation of microglia and astrocytes, and therefore, no development of mechanisms of central sensitization.
Our data revealed that there was an increased expression of CGRP in the sensory nervous system 12 weeks after the traumatic knee joint injury, which we hypothesized to be underlying the observed peripheral pain sensitization. Features previously described in other OA models, such as the infiltration of synovial membrane by immune cells, development of neuropathic mechanisms and central pain sensitization were not observed in the OA post-traumatic DMM model. Therefore, our results suggest that the nociceptive mechanisms involved in driving pain in post-traumatic OA are considerably different from those in non-traumatic OA. These findings highlight an important demand to adjust the therapeutic approaches to the nociceptive mechanisms driving pain in post-traumatic OA and uncover the modulation of the CGRP expression as a putative therapeutic approach.

DMM surgery.
Mice were subjected to volatile anaesthesia with isoflurane, and under the stereomicroscope, a longitudinal incision medial to the patellar ligament was performed in the right leg and the joint capsule was opened. The meniscotibial ligament, anchoring medial meniscus to the tibial plateau was transected, resulting in the destabilization of the medial meniscus, as previously described 8 . The joint capsule and subcutaneous layer were sutured, and the skin was closed by intradermal suture. The sham-operated mice underwent the same surgical procedure but the meniscotibial ligament was left intact. Analgesia was provided by the administration of 0.08 μg/g body weight of buprenorphine (Bupaq ® , Richer Pharma AG, Wels, Austria) on the surgery day and the following 3 days. At 12 weeks post-surgery, mice were euthanized with an overdose of anaesthetic and tissues were collected (hind limbs, ipsilateral lumbar L2-L5 dorsal root ganglia (DRG) and spinal cord). X-ray. Animals were subjected to volatile anaesthesia with isoflurane and X-ray images were obtained at 12 weeks post-DMM surgery using the X-Ray Owandy system (Croissy-Beaubourg, France).

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| (2020) 10:15271 | https://doi.org/10.1038/s41598-020-72227-9 www.nature.com/scientificreports/ Pain-related behaviour, locomotor activity, and gait pattern. Mechanical allodynia. The Von Frey test was used to evaluate mechanical allodynia at 4, 6, 8, 10 and 12 weeks post-surgery in sham-operated and DMM mice. Data from a non-control group was also obtained. The mice were placed individually in small cages over a hire mesh platform and allowed to acclimate for 30 min. Whenever the exploratory behaviour was not reduced during this period, the animals were allowed to acclimate for an extra period until the activity was reduced. The filaments were applied perpendicularly to the plantar surface of the ipsilateral hind paw until it bowed and then held for 4 s, following the up-down method 73 . Briefly, a series of Von Frey filaments (Ugo Basile, Varese, Italy) were applied with increasing bending forces (ranging from 0.04 to 1.4 g), beginning with the 0.04 g filament and until paw withdrawal response (positive response) was observed. The withdrawal threshold was recorded to the smallest filament to evoke at least 3 responses out of 5 repeated applications. When a positive withdrawal response was obtained with 0.04 g filament, the paw was retested starting with next descending Von Frey hair until no response occurred.
Locomotor activity. Sham-operated and DMM mice were subjected to the open field test at 4, 6, 8, 10 and 12 weeks post-surgery, and spontaneous locomotor activity was analysed. Data from a non-operated group was also obtained. The apparatus consisted of an empty square arena (40 × 40 × 40 cm), illuminated by indirect white lighting (100 lx). Each mouse was placed in the centre area and allowed to walk freely for 10 min. The following behaviours were automatically recorded by the Smart Video Tracking Software (Panlab) version 3.0: (i) total distance travelled (ii) mean velocity (excluding the immobility time) and (iii) immobility time.
Gait pattern. Mice hind paws were coloured with ink, and animals were immediately allowed to cross a tunnel (10 cm wide and 60 cm long, that ended in a dark chamber to entice the mice) leaving their paw prints on a white paper. Data was obtained at 4, 6, 8, 10 and 12 weeks post-surgery from the non-operated, sham-operated and DMM mice. The following measures were collected as previously described by Boettger et al. 42 : the distance between a print from the right paw and a consecutive print from the right and left paw (right-right and right-left distance, respectively) and the angle between consecutive paw prints (i.e. rotation of paws). To exclude the mice speed as a confounding factor, the distance between the right (affected) paw print and the consecutive left one was normalized to a complete walking cycle (right-to-right-distance), and this parameter is referred to as "relative step length" 42 . Additionally, footprint analysis was used to study paw posture, and parameters such as toe spread (TS), intermediate toe spread (ITS) and print length (PL) were also collected 74 .
μ-CT analysis. The right knee joints were scanned at 70 kV, 200 mA with a 0.5 mm aluminum filter and an isotropic resolution of 8 mm, using a Skyscan 1276 System (Brucker, Belgium). The obtained projection images were reconstructed with NRecon software, followed by bone alignment along the sagittal axis using DataViewer software. Morphological analysis was carried out at the medial and lateral tibial condyle (at 50 slices for each side) with CTAnalyser software. Following the recommended guidelines 75 , the parameters calculated included: mineral density, total volume, and thickness of the subchondral bone. Representative three-dimensional images were generated using CTVol software.
Histological analysis. After fixed in 4% neutral buffered formalin for 24 h, limbs were decalcified in 10% formic acid for 1 week at 4 °C on a constant shaker, and processed for paraffin embedding. Serial 3 μm thick sections from 5 animals in each group were obtained in the microtome (RM2255, Leica Biosystems) and stained with Safranin O to evaluate cartilage integrity. After deparaffinising, the sections were rehydrated with descending concentrations of alcohol and stained with haematoxylin for 2 min, followed by staining in Fast green for 5 min, washed with 1% acetic acid and counterstained with 1.5% Safranin O for 30 min. Sections were then dehydrated in absolute alcohol, cleared in xylene and mounted under coverslips using DPX mounting medium (VWR, UK). Images were acquired with an Olympus CX31 light microscope equipped with a DP-25 camera (Imaging Software CellB, Olympus, PA, USA). The Osteoarthritis Research Society International (OARSI) scoring system was used to evaluate histological changes of articular cartilage in the medial femoral condyle and tibial plateau 76 . For each knee, the maximum OA score was determined by the lesion with the highest score. www.nature.com/scientificreports/ In all experiments, for signal detection, tissue sections were incubated for 1 h at RT with anti-rabbit Alexa Fluor 568 antibody, or anti-mouse Alexa Fluor 488 antibody or anti-rat Alexa Fluor 488 antibody at 1:1,000 (Life Technologies, USA). Nuclei were stained with DAPI and tissue sections mounted with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, USA). Images were acquired on the confocal Leica TCS SP5 microscope (Leica Microsystems, Germany).
Dorsal root ganglia. The expression of peripheral sensitization-related molecules such as CGRP, TRPV1, NGF, TrkA, and of the NPY was evaluated in DRG sections, from 5 animals in each group, by immunohistochemistry analysis. After deparaffinization, rehydration, antigen retrieval and quenching of endogenous fluorescence, histological sections were blocked at RT, and then incubated with rabbit anti-CGRP (1:6,000, Sigma-Aldrich, USA), or rabbit anti-TRPV1 (1:200, Antibodies-online.com, Aachen, Germany), or rabbit anti-TrkA (1:100, Abcam, UK) or rabbit anti-NPY (1:1,000, Sigma-Aldrich, Germany) overnight at 4 °C. For signal detection, tissue sections were incubated for 1 h at RT with anti-rabbit Alexa Fluor 568 antibody (1:1,000, Life Technologies, USA), incubated with DAPI for the nuclei staining and then mounted with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, USA). Images were acquired on the confocal Leica TCS SP5 microscope (Leica Microsystems, Germany). The staining intensity was quantified using NIH ImageJ software.
Western blot analyses. Spinal cord samples were collected and frozen in 2-methylbutane cooled over dry ice and stored at − 80 °C until processing. The protein levels of c-Fos, p-ERK1/2, GFAP and IBA-1 were measured by western blot in the spinal cord from 5 animals in each group. The tissue samples were homogenized in lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton-100, 0.5% NP-40 Alternative) containing Protease Inhibitor Cocktail (1:100, Thermo Fisher Scientific) and Phosphatase Inhibitor Cocktail (1:100, Sigma-Aldrich, USA). The homogenate was incubated on ice during 1 h and was then centrifuged at 16,000×g for 10 min at 4 °C. Twenty-five microgram of protein supernatant were separated by acrylamide gel electrophoresis (Run Bolt Mini Gels; Bolt 8% Bis-Tris Plus, Invitrogen), at constant voltage 100 mV for 1 h and then electrotransferred using Invitrogen iBlot 2 Transfer Stacks (iBlot 2 NC Mini Stacks, Invitrogen) at 25 V for 7 min. Non-specific protein binding was prevented by membranes blocking for 1 h with 5% of BSA in TBS-T. Membranes were incubated overnight at 4 °C with mouse anti-c-Fos (1:500, Santa Cruz Biotechnology, USA), rabbit anti-pERK1/2 (1:1,000, Cell Signaling, MA, USA), mouse anti-ERK1/2 (1:1,000, BD Biosciences, CA, USA), rabbit anti-GFAP (1:10 000, Abcam, UK), rabbit anti-IBA1 (1:1,000, Wako, USA) and mouse anti-GAPDH (1:20 000, HyTest, Turku, Finland). After washing, membranes were incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies (both at 1:10,000, Santa Cruz Biotechnology, Texas, USA). Immunoreactive proteins were revealed using the enhanced chemiluminescence method (Amersham, ECL Prime Western Blotting Detection Reagent, GE Healthcare, UK) as recommended by manufactured instructions. The bands quantification was performed in Image J software version 2.0. Protein expression is presented as the ratio between the protein of interest and reference protein band densities (GAPDH or ERK1/2). Complete and unprocessed blots can be found in Supplementary Fig. S2.
Statistic analyses. Data from pain behaviour tests were analysed by a 2-way Mixed Anova analysis followed by Tukey's multiple comparisons test and data from μ-CT were analysed by 2-way ANOVA followed by Sidak's multiple comparisons test. All other data were assessed for normal distribution and non-parametric Mann-Whitney analyses were performed whenever normal distribution was not followed. Differences were considered at the significant level of p < 0.05. All data are expressed as mean ± SEM. Statistical analyses were performed using the software Prism 6 (GraphPad software, San Diego, CA, USA).
Animals. All animal procedures were approved by the i3S animal ethics committee and by the Portuguese Agency for Animal Welfare (Direção-Geral de Alimentação e Veterinária), in compliance with the European Community Council Directive of September 22, 2010 (2010/63/UE). Experiments were conducted by FELASA C and B graded researchers and all efforts were made to minimize the number of animals used and their suffering.
Three-months old C57BL/6 male mice were provided by the i3S Animal Facilities. Mice were kept under controlled conditions (20-22 °C, 60% humidity and 12:12 h light/dark cycle). Water and appropriate food were supplied ad libitum. Animals were randomized into the experimental groups (n = 5).