PDGF-B, but not PDGF-D, prevents spinal cord injury-induced cavitation in rats via fibroblast recruitment and extracellular matrix remodeling

PDGF-B, but not PDGF-D, prevents spinal cord injury-induced cavitation in rats via fibroblast recruitment and extracellular matrix remodeling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PDGF-B, but not PDGF-D, prevents spinal cord injury-induced cavitation in rats via fibroblast recruitment and extracellular matrix remodeling Chao Liang, Hao Hu, Ningyuan Zhang, Zhaoming Xiao, Zhikun Bai, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8587175/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Spinal cord injury (SCI) induces substantial cell loss at the injury site, leading to the formation of cystic cavities, a major obstacle to neural repair in both humans and rats. The mechanisms driving cavity development remain elusive. Given the well-established role of platelet-derived growth factor-B (PDGF-B) in wound healing via activation of its receptor PDGFR-β, as demonstrated in mice, we propose that impaired PDGFR-β signaling may be a key contributor to cavity formation post-SCI in rats. Methods: First, the spatiotemporal expression patterns of PDGF-B, PDGF-D, PDGFR-β, and key extracellular matrix (ECM) components (Fibronectin, Laminin, Collagen I) were assessed in rat spinal cord following crush injury using immunofluorescence, and the process of cavity formation was observed. Subsequently, the therapeutic potential of immediate and delayed in situ delivery of PDGF-B, PDGF-D, or Fibronectin on post-traumatic cavitation were evaluated. Additionally, the necessity of fibroblast activation and recruitment was assessed by intraperitoneal administration of the PDGFR-β inhibitor SU16f. Results: Expression of PDGFR-β, Fibronectin, laminin, and collagen I peaked at 7 days post-injury (dpi) and subsequently regressed within GFAP-negative regions, coinciding with progressive cystic cavity formation by 28 and 56 dpi in the injured rat spinal cord. Endogenous PDGF-D peaked at 3 dpi, while PDGF-B peaked at 14 dpi, both exhibiting lower levels thereafter. Immediate or delayed in situ injection of exogenous PDGF-B, but not PDGF-D, specifically prevented and reversed cavitation, upon PDGFRβ-mediated fibroblast recruitment and ECM deposition, especially Fibronectin upregulation, thereby yielding marked neurological and motor function recovery following SCI in rats. Fibronectin injection recapitulated the benefits of PDGF-B, whereas systemic SU16f abolished the therapeutic effects of both PDGF-B and Fibronectin. Conclusion: Our findings demonstrate that PDGF-B specifically prevents SCI-induced cavitation in rats by orchestrating fibroblast recruitment and ECM remodeling, providing a translational framework for growth factor therapy after SCI. Spinal cord injury Cystic cavity Fibroblast recruitment PDGF-B Fibronectin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Spinal cord injury (SCI) triggers a cascade of deleterious events, including ischemic necrosis, inflammatory cell infiltration, and tissue degeneration, culminating in cystic cavity formation in both humans and rats [ 1 , 2 ]. These cystic cavities, characterized by cerebrospinal fluid accumulation, sparse connective tissue, and remnants of damaged vasculature and axons, pose a formidable obstacle to neuronal survival and axonal regeneration due to the absence of a supportive extracellular matrix (ECM) at the lesion site [ 3 , 4 ]. In rats, cystic cavities emerge within a week of SCI, with their volume stabilizing around ten weeks [ 5 ]. The lack of a fibrotic scar at the lesion core, in contrast to the response observed in mice, is thought to contribute to the development of cavities [ 6 – 8 ]. After SCI in mice, platelet-derived growth factor receptor beta positive (PDGFR-β + ) perivascular fibroblasts, along with ECM secretion, fill the injury core, potentially preventing cavity formation [ 7 ]. Our previous studies have demonstrated the pivotal role of the platelet-derived growth factor (PDGF)/PDGFR-β pathway in fibroblast recruitment and ECM remodeling post-SCI in mice. Moreover, in situ injection of PDGF-B has been observed to promote PDGFR-β + fibrosis in the uninjured spinal cord, indicating its potential role in tissue repair mechanisms [ 9 ]. PDGF-B and PDGF-D, the member of the PDGF family, activate PDGFR-β to exert fibrotic effects after tissue trauma [ 10 ]. Compared to PDGF-D, the PDGF-B molecule features a C-terminal tail enriched in basic amino acids, notably arginine and lysine. This positively charged basic amino acid cluster confers the ability to bind specifically and with high affinity to negatively charged heparan sulfate proteoglycans within ECM [ 11 , 12 ]. This specific interaction is critical for promoting cellular recruitment and proliferation during developmental processes, such as angiogenesis, and in tissue repair [ 13 ]. During the early stages of tissue injury, PDGF-B is released from platelet α granules, initiating and accelerating tissue repair processes [ 14 ]. The expression of PDGF-B has been found to increase after SCI in rats treated with erythropoietin via intraperitoneal injection, suggesting its neuroprotective effects and importance in neurological recovery [ 15 ]. However, the expression dynamics and activation of the PDGF-B/PDGFR-β pathway post-SCI in rats, as well as its impact on cavity formation, have not been fully explored. To address this gap, we constructed a rat SCI model and administered exogenous PDGF-B via in situ injection in this study. Our results indicate that PDGF-B, but not PDGF-D, specifically prevents and resolves SCI-induced cavitation in rats by recruiting PDGFR-β + fibroblasts and restoring ECM integrity, thereby facilitating long-term neuroprotection and functional recovery. Materials and Methods Animals All animal experiments were approved by the Ethics Committee of Anhui Medical University (Approval NO. LLSC20230809). Adult female C57BL/6 mice (18–20 g) and Sprague-Dawley (SD) rats (200–250 g) were used in this study and were provided by the Animal Experimentation Center of Anhui Medical University. All experimental animals were randomly grouped and housed in an environment with controlled temperature and humidity, a 12-hour light/dark cycle, and free access to food and water. Surgical procedures A clinically relevant model of spinal cord compression injury, as described by Ashley McDonough et al [ 16 ] was employed. Animals were anesthetized with isoflurane (induction dose 5%, maintenance dose 2%) and underwent dorsal laminectomy at the level of the 8th thoracic vertebra to expose the dorsal surface of the T10 spinal cord. Subsequently, a calibrated Dumont No. 2 clamp (1122320, Fine Science Tools, Germany) was utilized to apply compression for 10 s. In the situ injection experiment, injections were administered at two time points: immediately after SCI and on 14 days post-injury (dpi), following determination of the injury area under bright light irradiation, the animals were secured on a stereotaxic instrument, and 2 µl of 100 ng/µl recombinant human PDGF-B (HZ-1308, Proteintech, China), 2 µl of 1 µg/µl recombinant human Fibronectin (#11051407001, Sigma-Aldrich, United States), 2 µl of 100 ng/µl recombinant human PDGF-D (1159-SB/CF, R&D Systems, United States) or 2 µl of 0.01 M Phosphate Buffered Saline (PBS) solution (BL302A, biosharp, China) was injected using a 5-µl Hamilton microinjector (7634-01, Hamilton, Switzerland) at a rate of 0.5 µl/min, targeting the right ventral gray matter of the injured area with the following coordinates: posterior median vein as midline, 0.5 mm lateral to the midline, and a depth of 1.3 mm [ 17 ]. The syringe was left in place for 1 min after injection to prevent reflux of the injected PDGF-B, Fibronectin, PDGF-D or PBS solution through the injection site. The wound was gently irrigated with saline (1–2 ml) after the needle was removed evenly and slowly, and the muscle layer and skin were sutured. The rats were then placed in a warming thermostat until fully awakened. On the first postoperative day, animals were carefully evaluated, and only those exhibiting complete hind limb paralysis were included in the study. Bladder care was performed twice daily postoperatively until spontaneous urination returned. Tissue processing Animals were sacrificed prior injury and at 3, 7, 14, 28 and 56 dpi for histologic evaluation. Following deep isoflurane anesthesia, animals underwent transcardiac perfusion with 0.1 M PBS (BL302A, biosharp, China) and 4% paraformaldehyde (BL539A, biosharp, China). The spinal cord was then dissected, and 5-mm segments containing the injured core was immersed in 4% paraformaldehyde for 24 h, followed by preservation through dehydration and refrigeration in a 30% sucrose solution for 48 h. The spinal cord was cut horizontally into 16-µm-thick serial frozen sections using a cryostat (NX50, Thermo Fisher Scientific, USA). Markings were made to ensure consistency, and three samples were collected from each group. Hematoxylin-eosin staining To evaluate the morphology of the lesioned cystic cavity, serial spinal cord sections were stained with hematoxylin and eosin (HE) (BL700b, biosharp, China). Horizontal sections were immersed in 200 ml hematoxylin staining solution for 5 min, then rinsed with tap water for 5 min, followed by hydrochloric acid-ethanol solution for differentiation. Subsequently, the sections were immersed in 200 ml of eosin staining solution for 2 min. Finally, gradient ethanol dehydration, transparency treatment and neutral resin sealing of the sections were performed. Immunofluorescence staining For tissue immunofluorescence staining, spinal cord tissue sections was blocked in PBS (BL302A, biosharp, China) containing 0.3% Triton X-100 (T8200, Solarbio, China) and 10% donkey serum albumin (DSA) (SL050, Solarbio, China) for 1 h at room temperature, and then the primary antibodies were incubated overnight at 4°C: rabbit anti-PDGF-B (1:100; #NBP1-58279, Novus, USA), rabbit anti-PDGF-D (1:100; 40-2100, ThermoFisher, USA), rabbit anti-Fibronectin (1:500; 15613-1-AP, Sigma-Aldrich, USA), rabbit anti-Laminin (1:500; L9393, Sigma-Aldrich, USA), rabbit anti-Collagen I (1:50; 2150 − 1908, Bio-rad, USA), goat anti-PDGFR-β (1:100; #AF1042-SP, R&D Systems, USA), rabbit anti-p-PDGFR-β (1:100; YA3062 MCE, China), chicken anti-GFAP (1: 100; #16825-1-AP, Aveslabs, USA), goat anti-Iba-1 (1:500; NB100-1028, Novus, USA), goat anti-CD68 (1:200; Sc-70761, Santa Cruz, USA) ,goat anti-CD31 (1:200; AF3628, R&D Systems, USA), rabbit anti-Ki67 (1:200; Ab15580, abcam, USA), goat anti-5-HT (1:200; #20079, Immunostar, USA), and rabbit anti-NeuN (1:300; #ab177487 Abcam, USA). After three washes, the slides were incubated with secondary antibodies conjugated to appropriate Alexa fluorescent dyes: donkey anti-Chicken Alexa Fluor 488, donkey anti-goat Alexa Fluor 555, donkey anti-rabbit Alexa Fluor 555 and donkey anti-goat Alexa Fluor 647 (1:500; A-78948, A-21432, A-21428, A-21447, Thermo Fisher Scientific, USA) at room temperature for 1 h. Finally, nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI) (P0130, Beyotime Biotechnology, China). Images were captured using a fluorescence microscope (Zeiss, Germany). Quantitative image analysis In this experiment, three sections spaced 160 µm apart were selected from the middle of each spinal cord tissue for immunofluorescence staining. After processing the fluorescence-stained images using ImageJ [ 18 ], the average of the quantified results from the three sections was taken as the data for one spinal cord sample. Each experimental group included three spinal cord tissue samples. For fibrotic scar analysis, the percentage of PDGFR-β, Fibronectin, Laminin, Collagen I, and GFAP positive area was quantified as the proportion of their positive areas relative to the spinal cord area within a 4 × microscopic field. The GFAP-negative area percentage was calculated as the proportion of its negative area relative to the spinal cord area in a 4 × microscopic field. The number of neurons was quantified by counting NeuN + cells in the Z1 (0–250 µm), Z2 (250–500 µm), Z3 (1000–1250 µm), and Z4 (2000–2250 µm) regions after SCI [ 19 ]. The percentage of positive-serotonergic (5-HT + ) areas was measured as its positive area relative to the field of view in 20 × microscopic fields at 0 µm and 500 µm from the injury core. The density of Ki67 + PDGFR-β + cells was calculated as the ratio of the number of Ki67 + PDGFR-β + cells to the area of the GFAP − region. Laser Speckle Contrast Blood Flow Imaging System After SCI, rats received in situ injections of PDGF-B, PDGF-D, and Fibronectin were maintained for 28 days. Following the original modeling procedures, the injured spinal cord region was re-exposed. The Laser Speckle Contrast Blood Flow Imaging System (RFLSI ZW, China ) was used to measure the average blood flow through the lesion core over a 10 s, which was recorded as the blood perfusion volume through the lesion core per unit time for each sample. Three rat samples were analysed per experimental group. Behavioral assessment Recovery of motor function after SCI in rats was assessed using the Basso, Beattie & Bresnahan locomotor rating scale (BBB) open field locomotor test as reported [ 20 ]. All rats used BBB to assess that locomotor function is normal before SCI and received BBB after surgery to confirm that the SCI model is successful. To ensure blinded assessment of behavioral recovery, animals were evaluated at 0, 7, 14, 21and 28 dpi by two investigators not involved in animal surgery. Animals were allowed to run freely in an open field and motor function scores were measured after 4 min of observation. We used footprint analysis to further assess improvement in motor function of animals 28 dpi followed by published articles [ 21 ]. Briefly, all evaluated animals had their front paws dipped in green dye and hind paws dipped in blue dye. The tracks of animals with intact spinal cords were assigned to the Sham group. Each animal was run four times without significant interference as a valid run. Three gait parameters were then calculated. Stride length was the distance from the starting point to the end point of the hind paws in one gait cycle. Stride width is the distance between the left and right hind paws. Paw rotation is defined as the angle of the hind paw axis with respect to the stride axis. An increase in the angle of rotation indicates external rotation of the hind paws. All assessments were made in three consecutive gait cycles. Values were averaged for both hind paws. The footprints of the hind paws tend to overlap the footprints of the forepaws during gait in animals with an uninjured spinal cord, whereas animals with a SCI typically lose this coordination between n the hind and forepaws. Statistical analysis Statistical analysis was performed using GraphPad Prism software (version 8.0). All data are presented as mean ± standard error of the mean (SEM). The two-tailed Student's t-test was used to compare the means of two groups. One-way analysis of variance (ANOVA) and Two-way analysis of variance followed by Tukey's multiple comparisons were used to compare the means of three or more groups. P < 0.05 values were considered statistically significant. Results Cystic cavities form at the injury site following SCI in rats, accompanied by insufficient secretion of PDGF-B and PDGF-D. Given the pivotal role of the PDGF/PDGFR-β signaling pathway in post-SCI wound repair, we postulated that cystic cavitation in the injured rat spinal cord may result from inadequate activation of this pathway. To test this hypothesis, we first compared the spatiotemporal expression patterns of PDGF/PDGFR-β components within the lesion core between rats and mice during the chronic phase of SCI. Immunofluorescence analysis revealed that at 28 days post-injury (dpi), the lesion core in mice was densely infiltrated with PDGFR-β⁺ fibroblasts, while the expression levels of PDGF-B and PDGF-D were sustainably high. In stark contrast, at the same time point in rats, well-delineated cystic cavities had developed within the lesion, coinciding with markedly reduced recruitment of PDGFR-β⁺ fibroblasts and scarcely detectable expression of PDGF-B and PDGF-D (Fig. 1 A). To systematically characterize post-SCI cavitation in rats and its relationship to fibrotic scarring, we analysed the temporal dynamics of fibroblast and its ECM components within the spinal cord lesion. PDGFR-β expression was initially increased at 3 dpi, peaked at 7 dpi, and progressively declined from 14 to 56 dpi in GFAP − area (Fig. 1 B, D). ECM including Fibronectin, Laminin, Collagen I within the lesion was deposited paralleled with PDGFR-β + fibroblasts recruitment after SCI (Fig. 1 B, E, F). Cystic cavities emerged by 14 dpi and gradually expanded into large DAPI − area, cell-free regions within the lesion epicentre by 28–56 dpi (Fig. 1 B, C). These observations imply an inverse correlation between residual PDGFR-β or ECM density and cavity volume after SCI in rats. Importantly, PDGF-D peaked at 3 dpi, while PDGF-B reached its maximum at 14 dpi; both subsequently decreased to low baselines (Fig. 1 E, G). Immunostaining for phosphorylated PDGFR-β (p-PDGFR-β at Tyr 740) confirmed sustained activation in mice at 7–14 dpi, whereas rat tissue displayed only a transient 7-dpi peak (Figure S1A, B) . These results indicate that insufficient activation of the PDGFR-β pathway in rats may contribute to cavity expansion. PDGF-B, but not PDGF-D, specially abolishes post-SCI cavitation in rats by recruiting fibroblasts and amplifying Fibronectin deposition. Given the established role of PDGF-B in wound repair and its deficient endogenous expression, we administered recombinant PDGF-B or PDGF-D via immediate in situ injection after SCI to evaluate their effects on cystic cavity formation (Fig. 2 A). Successful delivery and localized protein retention were confirmed at 7 dpi through immunofluorescence, which showed elevated levels of PDGF-B and PDGF-D within the lesion core compared to controls (Fig. 2 B, D-F). HE staining revealed near-complete prevention of cavitation in the PDGF-B group, with lesion site filled densely with cellular tissue, contrasting sharply with the extensive cavities observed in both Control and PDGF-D groups at 28 dpi (Fig. 2 C, G). These findings suggest that exogenous PDGF-B effectively mitigates cavity formation during the subacute stage of SCI in rats. To identify the composition of the preserved tissue filling the cavity, we evaluated the expression of the fibroblast marker PDGFR-β and key ECM components following PDGF-B or PDGF-D administration. PDGF-B treatment resulted in markedly enhanced fibroblast recruitment and denser deposition of ECM components, particularly Fibronectin, relative to the Control group, whereas PDGF-D group showed no benefit (Fig. 2 H-I). These results indicate that PDGF-B specifically triggers fibroblast recruitment and Fibronectin-rich ECM remodeling. Given the prominent upregulation of Fibronectin, we next assessed its therapeutic potential by in situ injection of exogenous Fibronectin protein after SCI. The results demonstrated that direct Fibronectin administration reproduced the anti-cavitary outcome (Fig. 2 B-C, G-I). To further investigate whether the role of PDGF-B is associated with the phosphorylation of PDGFR-β, we conducted phosphorylation assays on tissues injected in situ with PDGF-D, PDGF-B, and Fibronectin. The results revealed that the PDGF-B group exhibited increased phosphorylation levels of PDGFR-β compared to other groups, indicating that PDGF-B functions via activation of PDGFR-β phosphorylation (Figure S1C, D) . Treatment with PDGF-B or Fibronectin, but not PDGF-D, enhances neurological and motor functional recovery after SCI in rats. We next evaluated neuronal preservation and axonal regrowth by NeuN and 5-HT staining. At 28 dpi, both PDGF-B and Fibronectin groups significantly increased the number of preserved NeuN + neurons in the peri-lesion (Z2) regions and enhanced 5-HT + axon density within the lesion core compared to the Control and PDGF-D groups (Fig. 3 A-D). Locomotor function assessed by BBB scoring showed significant improvement in PDGF-B and Fibronectin groups at 28 dpi (Fig. 3 E). Footprint analysis also revealed improved coordination, stride length, and rotation angle in these groups (Fig. 3 F, G-I). Immunofluorescence analysis of Iba-1 + and CD68 + microglia/macrophages revealed a persistent inflammatory response in the Control and PDGF-D groups at 28 dpi ( Fig. 4 A ) . In contrast, both PDGF-B and Fibronectin groups significantly reduced the density of these immune cells within the lesion core (Fig. 4 A-C). Given the critical role of vascular recovery in tissue repair, we examined angiogenesis via CD31 + endothelial cell staining and blood flow perfusion. PDGF-B and Fibronectin treatment resulted in significantly higher vascular density and improved perfusion within the lesion site compared to the Control and PDGF-D groups (Fig. 4 D-F, H, J). These results confirm that PDGF-B and Fibronectin not only mitigate chronic inflammation but also improve vascular recovery, further supporting their reparative functions after SCI in rats. Ki67 labeling confirmed that PDGF-B and Fibronectin, but not PDGF-D, drove markedly fibroblast proliferation (Fig. 4 G, I). Thus, PDGF-B specially orchestrates fibroblast-mediated ECM remodeling, chiefly via Fibronectin, to prevent SCI-induced cavitation in rats (Fig. 4 G, I). Delayed administration of PDGF-B or Fibronectin also attenuates cavitation and facilitates functional recovery after SCI in rats. To evaluate the therapeutic potential of delayed intervention, we performed in situ injection of exogenous PDGF-B or Fibronectin in rats at 14 dpi (Fig. 5 A). Immunofluorescence analysis at 28 dpi revealed that delayed treatment with either agent markedly reduced cavitation and enhanced fibroblast recruitment and ECM deposition—evidenced by increased expression of PDGFR-β, Fibronectin, Laminin and Collagen I—compared to the Control group (Fig. 5 B-E ) . However, it is noteworthy that delayed administration resulted in an expansion of the GFAP − area (Fig. 5 F ) . Delayed injection of exogenous PDGF-B or Fibronectin can also promote serotonergic axon regrowth and increase neuronal preservation in both Z1 and Z2 regions (Fig. 5 G-J). Consistent with these anatomical improvements, treated animals exhibited substantial functional recovery, as indicated by higher BBB scores (Fig. 5 K) and improved gait parameters ( Figure S2A-D ). Furthermore, delayed PDGF-B or Fibronectin administration attenuated inflammation, indicated by reduced Iba-1 + immunoreactivity, and increased vascular regeneration, as shown by enhanced CD31 + vessel density ( Figure S2E-H ). These results demonstrate that even delayed injection of PDGF-B or Fibronectin mitigates cavitation and promotes functional recovery after SCI in rats. SU16f abrogates the therapeutic benefits of PDGF-B and Fibronectin by inhibiting fibroblast recruitment and ECM remodeling. To confirm the specificity of PDGF-B/PDGFR-β signaling, we administered SU16f, a PDGFR-β inhibitor, systemically following PDGF-B injection after SCI (Fig. 6 A). SU16f treatment significantly reversed the PDGF-B-induced benefits, reducing fibroblast recruitment, ECM deposition (Fibronectin, Laminin, Collagen I), and leading to cavity re-formation (Fig. 6 B-D). Similarly, SU16f compromised the therapeutic effects of Fibronectin, indicating that its benefits partially depend on PDGFR-β pathway activation ( Figure S3A-D ). SU16f also negated the beneficial effects of PDGF-B on neuronal retention and axon regrowth (Fig. 6 E-H). Functionally, SU16f injection reduced BBB scores, reduced hindlimb step length and rotation angle, and increased step widths ( Fig. 6 I-M S3E-I ) . These findings underscore the essential role of PDGF-B/PDGFR-β signaling in fibroblast recruitment, ECM remodeling, cavity prevention, and functional recovery post-SCI. Discussion Our study demonstrates that insufficient PDGF-B expression following SCI in rats compromises its reparative function and contributes to post-traumatic cystic cavity formation. By single intraparenchymal injections of exogenous PDGF-B or its downstream effector Fibronectin, both during early and delayed phases, we successfully mitigated cavity formation in a clinically relevant rat model of compressive SCI through enhanced fibroblast recruitment and ECM remodeling. This intervention yielded multifaceted benefits, including attenuated neuroinflammation, restored vascular perfusion, promoted axonal regrowth, increased neuronal survival, and significant functional recovery. Conversely, PDGFR-β inhibition via SU16f abolished these therapeutic effects and re-induced cavitation. These findings identify PDGF-B, but not PDGF-D, as a critical regulator of fibrotic scarring and tissue repair post-SCI, highlighting its potential as a therapeutic target. Previous studies have reported that exogenous matrix implants facilitate axonal growth but fail to prevent cystic cavities in SCI models [ 22 , 23 ]. Similarly, cell transplantation and biomaterial-based treatments shown limited functional improvement despite some anatomical repair [ 24 – 26 ]. Although injectable hydrogels—such as those functionalized with imidazole-poly (organophosphazenes) or Fibronectin-derived cell-adhesive RGD peptides—had been developed to reduce cavitation and promote motor outcomes in rats [ 27 , 28 ], their clinical translation remains challenging due to unresolved issues regarding biodegradation, mechanical compatibility, and immunogenicity [ 29 ]. In contrast, our findings indicate that PDGF-B and Fibronectin administration nearly completely resolves cystic cavities and supports functional recovery, suggesting a potent and clinically feasible alternative. Fibrotic scarring is a conserved response that supports wound sealing and tissue integrity. In the central nervous system (CNS), PDGFR-β + cells serve as a major source of fibroblasts and ECM components after SCI [ 8 , 30 – 33 ]. Goritz et al. demonstrated that PDGFR-β + cells form a fibrotic scar that prevents cavitation in mice; ablation of these cells led to large cystic cavities and loss of tissue integrity [ 8 ]. Similarly, Shearer et al. reported that fibroblast-driven deposition of Fibronectin, Collagen Ⅰ and Laminin precludes cyst formation in SCI mice [ 34 ]. In rats, however, insufficient fibrotic scarring at the injury epicenter results in prominent cystic cavities [ 6 , 7 , 35 ]. Our data show that exogenous PDGF-B potently stimulates PDGFR-β + fibroblast recruitment and ECM remodeling, effectively sealing cystic cavities and restoring tissue continuity. To our knowledge, this is among the first studies to achieve successful cystic repair using a growth factor after SCI, rather than cell transplantation or biomaterial treatment. Moreover, this agent is clinically safe for humans and holds promise as a novel therapeutic strategy. The mechanisms underlying PDGF-B-mediated repair are likely multifactorial, involving not only fibrotic scar formation and ECM reorganization, but also modulation of angiogenesis and inflammation [ 36 , 37 ]. Sun et al. showed that local administration of PDGF-B can induce Fibronectin fibril alignment and extension, effectively converting adult pericytes into a permissive substrate for axon growth in SCI mice [ 38 ], consistent with our results. We have confirmed that PDGF-B, but not PDGF-D, specifically upregulates Fibronectin and recruits fibroblasts to fill cystic cavities, thereby facilitating functional recovery post-SCI in rats. Structurally, PDGF-B contains a C-terminal domain rich in basic amino acids such as arginine and lysine, enabling high-affinity binding to heparan sulfate proteoglycans in the ECM, while its N-terminal region binds to cell-surface receptors to promote fibroblast recruitment and proliferation during development and repair [ 11 – 13 ]. Whether this structural specificity underlies the differential effects of PDGF-B and PDGF-D remains unclear and warrants further investigation. Although dense fibrotic scarring has been suggested to inhibit regeneration in mice [ 9 ], we found that in rats, PDGFR-β + fibroblast recruitment and ECM remodeling are essential for cavity repair and functional recovery. Axonal staining indicated that PDGF-B treatment supports axonal extension across previously cavitied areas. Fibronectin administration mimicked these benefits, aligning with reports that Fibronectin-rich matrices form bridges that ligate the severed ends of the spinal cord and promote axonal regrowth [ 27 , 39 ]. Moreover, Fibronectin knockdown worsened functional outcomes, increased cavity size, and increased neuronal apoptosis in CNS injury models [ 40 , 41 ]. Notably, SU16f inhibition abrogated the benefits of both PDGF-B and Fibronectin, suggesting that Fibronectin’s effects are partly mediated through PDGFR-β signaling and that a positive feedback loop may exist between them ( Fig. 7 ) . Further studies are needed to elucidate how Fibronectin influences fibroblast proliferation and PDGFR-β activation. In summary, we demonstrate that PDGF-B, but not PDGF-D, specifically prevents cavitation and facilitates recovery after SCI in rats through fibroblast-dependent ECM remodeling. Its dual role in modulating inflammation and promoting angiogenesis positions PDGF-B as s promising therapeutic candidate. These findings underscore the PDGF-B/PDGFR-β axis as a critical diver of cavity repair and a potential target for clinical translation in SCI. Abbreviations PDGF-B platelet-derived growth factor B PDGF-D platelet-derived growth factor D PBS phosphate buffer saline dpi day post-injury HE hematoxylin-eosin IF Immunofluorescence staining SCI spinal cord injury dpi days post-injury PDGFR-β platelet-derived growth factor receptor beta NeuN neuronal nuclei 5-HT 5-Hydroxytryptamine BBB Basso, Beattie & Bresnahan GFAP glial fibrillary acidic protein Iba-1 ionized calcium-binding adaptor molecule-1 ROI region of interest. Declarations Acknowledgements We are grateful for the support of the Scientific Research and Experiment Center of the Second Affiliated Hospital of Anhui Medical University. Author contributions Study design: MGZ, DST and ZYL; experimental implementation: CL, HH, NYZ, ZMX, ZKB, RL, XW, FLS, and JNY; data analysis and figure preparation: CL, ZMX, SKZ, CPM, ZDM and SSY; paper writing: CL and SSY; study supervising and paper reviewing: MGZ, DST and ZYL. All authors approved the final version of the paper. Funding This study was funded by the National Natural Science Foundation of China (Grant Nos. 82372506 to MGZ, 82301563 to ZYL, 82401616 to SSY), the Anhui Provincial Natural Science Foundation (Grant Nos. 2308085MH257 to MGZ, 2508085MH203 to DST, 2308085QH266 to ZYL, 2408085QH262 to SSY); the Clinical Medical Research Transformation Project of Anhui Province (Grant Nos. 202304295107020014 to MGZ, 202304295107020011 to DST); and the Natural Science Research Project of Anhui Educational Committee (Grant Nos. 2022AH050746 to MGZ, 2022AH050713 to ZYL, KJ2021A0316 to SSY). The Graphical abstract in this article was created using BioRender.com, and the author sincerely acknowledges BioRender (BioRender.com) for the powerful drawing support provided for this work. Availability of data and materials All data generated or analyzed during the current study are contained within this published article. Ethics approval and consent to participate All animal procedures were carried out with the approval of the Ethics Committee of the Anhui Medical University (Approval No. LLSC20230809). C onsent for publication Not applicable. Competing Interest The authors declare that they have no conflicts of interest. Author details a Department of Orthopaedics, The Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China b Institute of Orthopaedics, Research Center for Translational Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China Supplementary Data Supplementary figures and tables. References Zrzavy T, Schwaiger C, Wimmer I, Berger T, Bauer J, Butovsky O, et al. Acute and non-resolving inflammation associate with oxidative injury after human spinal cord injury. Brain 2021, 144:144-161. Fedorova J, Kellerova E, Bimbova K, Pavel J. 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15:27:19","extension":"xml","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131371,"visible":true,"origin":"","legend":"","description":"","filename":"30882370467e49cbafa32c35eebf91231structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/ed4a2e9a4f51a5df57d071b5.xml"},{"id":100904068,"identity":"625a70a4-b3c6-454a-bb48-b9c579fb8faa","added_by":"auto","created_at":"2026-01-22 15:27:20","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142121,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/e3a5a75710b2833b227ab0e3.html"},{"id":100904037,"identity":"04e34539-c8ad-4044-a452-048e19789df3","added_by":"auto","created_at":"2026-01-22 15:27:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2581459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCavity formation at the lesion site correlates with reduced secretion of PDGF-B and PDGF-D in the chronic phase of rat SCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of immunofluorescence staining for PDGFR-β (Magenta), GFAP (Green), and PDGF-B (White), PDGF-D (Red) at 28 days post-injury (dpi) in SCI of mouse and rats. (B) Representative images of HE staining and immunofluorescence staining for PDGFR-β (Red), GFAP (Green), and Nuclei (DAPI) at sham, 3, 7, 14, 28, and 56 dpi in SCI rats. The cystic cavity formed after SCI is outlined by dotted lines. The region of interest (ROI) indicates the field of view at high magnification in the left square area. (C, D) Quantification of the area of cystic cavities (DAPI-negative regions) (C) and the percentage of PDGFR-β\u003csup\u003e+\u003c/sup\u003e area relative to the GFAP\u003csup\u003e-\u003c/sup\u003e area (D) at 3, 7, 14, 28, and 56 dpi. \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. (E) Representative images of immunofluorescence staining for Fibronectin (Red), Collagen I (Cyan), Laminin (Yellow), PDGF-B (Green), and PDGF-D (Gray) at sham, 3, 7, 14, 28, and 56 dpi. (F) Quantification of the positive area index for Fibronectin, Collagen I and Laminin at 3, 7, 14, 28, and 56 dpi. \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e###\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to 7 dpi for Fibronectin. \u003csup\u003e\u003cem\u003e@@@@\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to others for Collagen I. \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to 7 dpi for Laminin. Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. (G) Quantification of the fluorescence intensity of PDGF-B and PDGF-D at 3, 7, 14, 28, and 56 dpi. \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to others for PDGF-B. \u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to others for PDGF-D. Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Data are presented as mean ± SEM. n = 5 independent rats per time point in A, B and E. Scale bars, 200 μm in B, E and rat of A, 100 μm in mouse of A. 20 μm in ROI.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/dcc68b0caff5654265a5b3cc.png"},{"id":100904041,"identity":"b59c277c-a37b-4eb7-82da-b9c123a06d41","added_by":"auto","created_at":"2026-01-22 15:27:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3827894,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDGF-B, but not PDGF-D, specifically prevents SCI-induced cavitation in rats via promoting fibroblast enrichment and fibronectin deposition.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental timeline of in situ injection,\u003cbr\u003e\nhistological evaluation, and behavioral assessment after SCI in rats. (B) Representative images of immunofluorescence staining for PDGF-D (Cyan), PDGF-B (Gray), and Fibronectin (Red) at 7 dpi. (C) HE staining depicting cystic cavities in the Control, PDGF-D, PDGF-B, and Fibronectin treatment groups at 28 dpi. The dotted lines outline the cystic cavity following SCI. (D-F) Quantification of the fluorescence intensity of PDGF-D (D), PDGF-B (E), and the positive area index of Fibronectin (F) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 7 dpi. \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Student’s two-tailed unpaired t-test, n = 5 independent rats per group. (G) Quantification of the cystic cavity area in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi.\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (H) Representative images of immunofluorescence staining for PDGFR-β (Magenta), Fibronectin (Red), Collagen I (Cyan), and Laminin (Gray) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. The dotted lines represent the cystic cavity following SCI. (I) Quantification of the positive area index for PDGFR-β, Collagen I, Fibronectin, and Laminin in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. \u003csup\u003e\u003cem\u003e\u0026amp;\u0026amp;\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to Control group for PDGFR-β. \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to Control group for Collagen I. \u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to Control group for Fibronectin. \u003csup\u003e\u003cem\u003e@@@\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e\u003cem\u003e@@@@\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, compared to Control group for Laminin. Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Data are presented as mean ± SEM. n = 5 independent rats per group. \u0026nbsp;Scale bar, 200 μm in B, C, and H.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/47e49ac4f96b1559511cc4c3.png"},{"id":100950901,"identity":"639c6704-0740-490e-8503-35d00ad2cea0","added_by":"auto","created_at":"2026-01-23 07:09:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1844379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment with PDGF-B or Fibronectin, but not PDGF-D, improves neurological repair and locomotor function recovery after SCI in rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of immunofluorescence staining for NeuN (Gray) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. LC indicates the lesion core. (D) Representative images of immunofluorescence staining for 5-HT (Magenta) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. ROI indicates the field of view at high magnification in the left square area. (B, C) Quantification of the number of NeuN\u003csup\u003e+\u003c/sup\u003e cells in Z1, Z2, Z3, and Z4 regions (B) and the percentage of 5-HT\u003csup\u003e+\u003c/sup\u003e area (C) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats. (E) Footprint analysis of the rats in Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. Hind paws (Green) and fore paws (Blue). (F) BBB scores for locomotor function assessment of mice in the Control, PDGF-D, PDGF-B, Fibronectin, and Sham groups at 0, 7, 14, 21, and 28 dpi. \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. (G-I) Quantification of stride length (G), stride width (H), and rotation angle (I) from the footprint data in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Data are presented as mean ± SEM. n = 6 independent rats in E, F, G, H and I. Scale bars, 1 mm in A, 100 μm in D and 20 μm in ROI.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/42fa1e4cf4151568efd29172.png"},{"id":100904043,"identity":"d28f2e6f-b5c6-4e0f-8e09-006d2974114a","added_by":"auto","created_at":"2026-01-22 15:27:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3211801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment with PDGF-B or Fibronectin, but not PDGF-D, suppresses inflammation, stimulates fibroblasts proliferation, and promotes vascular regeneration after SCI in rats.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(A) Representative images of immunofluorescence staining for GFAP (Green), Iba-1 (Red), CD68 (Red), and Nuclei (Blue) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. The dotted lines outline the cystic cavity following SCI. ROI indicates the field of view at high magnification. (B-C) Quantification of the fluorescence intensity of CD68 (B) and Iba-1 (C) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (D) Representative images of immunofluorescence staining for CD31 (Gray), Collagen I (Red), and GFAP (Green) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. The dotted lines outline the cystic cavity formed following SCI. ROI indicates the field of view at high magnification. (E-F) Quantification of CD31\u003csup\u003e+\u003c/sup\u003e area index and GFAP\u003csup\u003e-\u003c/sup\u003e area index in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi.\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (G) Representative images of immunofluorescence staining for Ki67 (Green) and PDGFR-β (Red) in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 7 dpi. ROI indicates the field of view at high magnification. (I) Quantification of the Ki67\u003csup\u003e+ \u003c/sup\u003ePDGFR-β\u003csup\u003e+\u003c/sup\u003e cell density in the lesion core in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 7 dpi. \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group.\u0026nbsp; (H) Blood flow imaging of the injured spinal cord in the Control, PDGF-D, PDGF-B, and Fibronectin groups at 28 dpi. (J) Quantification of blood flow in the injured spinal cord.\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. Data are presented as mean ± SEM. Scale bars, 200 μm in A, D, 20 μm in G and 10 μm in ROI, 300 μm in H.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/6eddd02cc60b113ea7aaaceb.png"},{"id":100904045,"identity":"0ce11977-39bf-44c3-b437-c8c8b80f21e7","added_by":"auto","created_at":"2026-01-22 15:27:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2313365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDelayed injection of PDGF-B or Fibronectin also prevents cavitation and improves neurological repair and locomotor function recovery after SCI in rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental timeline of in situ injection, histological evaluation, and behavioral assessment after SCI in rats. (B, C) Representative images of immunofluorescence staining for PDGFR-β (Magenta), GFAP (Green), Collagen I (Cyan), Fibronectin (Red), and Laminin (Gray) in the Control, PDGF-B, and Fibronectin groups at 28 dpi. The dotted lines outline the cystic cavity following SCI. (D) Quantification of the area of cystic cavities in the Control, PDGF-B, and Fibronectin groups at 28 dpi. \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (E) Quantification of PDGFR-β, Collagen I, Fibronectin, and Laminin positive area index in Control, PDGF-B, and Fibronectin groups at 28 dpi. Compared to Control group for PDGFR-β,\u003csup\u003e\u003cem\u003e ####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Compared to Control group for Collagen I, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Compared to Control group for Fibronectin,\u003csup\u003e\u003cem\u003e \u0026amp;\u0026amp;\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Compared to Control group for Laminin,\u003csup\u003e\u003cem\u003e @@@@\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (F) Quantification of GFAP\u003csup\u003e-\u003c/sup\u003e area index in Control, PDGF-B, and Fibronectin groups at 28 dpi. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01,\u003csup\u003e\u003cem\u003e ***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (G) Representative images of immunofluorescence staining for NeuN (Gray) in the Control, PDGF-B, and Fibronectin groups at 28 dpi. (I) Representative images of immunofluorescence staining for 5-HT (Magenta) in the Control, PDGF-B, and Fibronectin groups at 28 dpi. ROI indicates the field of view at high magnification. (H, J) Quantification of the number of NeuN\u003csup\u003e+\u003c/sup\u003e cells and in Z1, Z2, Z3 and Z4 regions (H) and the percentage of 5-HT\u003csup\u003e+\u003c/sup\u003e area (J) in the Control, PDGF-B, and Fibronectin groups at 28 dpi. n = 5 independent rats. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats for the Control/PDGF-B/Fibronectin groups. (K) BBB scores of the rats in the Control, PDGF-B, Fibronectin, and Sham groups at 0, 7, 14, 21, and 28 dpi. \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001, Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 6 independent rats per group. Data are presented as mean ± SEM. Scale bars, 200 μm in B, 200 μm in I, 1 mm in G and 20 μm in ROI.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/2814bc0e6aab26e34148288f.png"},{"id":100904066,"identity":"183b738c-dadb-4eda-a5a9-e4793a5e0c2a","added_by":"auto","created_at":"2026-01-22 15:27:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2110108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSU16f reverses the effects of exogenous PDGF-B on fibroblast recruitment and ECM deposition, thereby re-inducing cavity formation and hindering locomotor function improvement.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental timeline of in situ injection, intraperitoneal injection, histological evaluation, and behavioral assessment after SCI in rats. (B) Representative images of immunofluorescence staining for PDGFR-β (Magenta), GFAP (Green), Collagen I (Cyan), Fibronectin (Red), and Laminin (Gray) in the Vehicle, and SU16f groups at 28 dpi. The dotted lines outline the cystic cavity following SCI. (C) Quantification of PDGFR-β, Collagen I, Fibronectin, and Laminin positive area index in the Control and SU16f groups at 28 dpi. Vehicle vs. SU16f group: PDGFR-β, \u003csup\u003e\u003cem\u003e####\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Vehicle vs SU16f group: Collagen I, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Vehicle vs SU16f group: Fibronectin,\u003csup\u003e\u003cem\u003e \u0026amp;\u0026amp;\u0026amp;\u0026amp;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Vehicle vs SU16f group: Laminin,\u003csup\u003e\u003cem\u003e @@@@\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (D) Quantification of the area of cystic cavities in the Vehicle and SU16f group at 28 dpi.\u003csup\u003e\u003cem\u003e ****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Student’s two-tailed unpaired t-test, n = 5 independent rats per group. (E) Representative images of immunofluorescence staining for NeuN (Gray) in the in the Vehicle and SU16f group at 28 dpi. (G) Representative images of immunofluorescence staining for 5-HT (Magenta) in the Vehicle and SU16f groups at 28 dpi. ROI indicates the field of view at high magnification. (F, H) Quantification of the number of NeuN\u003csup\u003e+\u003c/sup\u003e cells and in Z1, Z2, Z3, and Z4 regions (F) and the percentage of 5-HT\u003csup\u003e+\u003c/sup\u003e area (H) in the Vehicle and SU16f group at 28 dpi. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 5 independent rats per group. (I) BBB scores of the rats in the Control and SU16f groups at 0, 7, 14, 21, and 28 dpi. \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Two-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. n = 6 independent rats per group. (J) Footprint analysis of the Control and SU16f groups at 28 dpi. Hind paws (Green) and Forepaw (Blue). (K-M) Quantification of stride length (K), stride width (L), and rotation angle (M) in the Control and SU16f groups at 28 dpi. \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e****\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001, Student’s two-tailed unpaired t-test, n = 6 independent rats per group. Data are presented as mean ± SEM. Scale bars, 200 μm in B, 100 μm in G, 1 mm in E, 20 μm at ROI.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/8ea2fb21b42b6f15c28459d6.png"},{"id":100904051,"identity":"8e9012bf-fa2a-48e3-b1c8-8e890714201c","added_by":"auto","created_at":"2026-01-22 15:27:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2774047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism of PDGF-B-mediated prevention of cavitation after SCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Following SCI in rats, neither PBS nor PDGF-D administration activates PDGFR-β signaling. The lack of pathway activation results in impaired fibroblast recruitment and insufficient extracellular matrix deposition, leading to progressive cavitation and persistent motor deficits. (B) In situ injection of PDGF-B activates PDGFR-β and induces its phosphorylation, triggering fibroblast recruitment and Fibronectin upregulation in the lesion core. These cellular and molecular changes collectively contribute to the cavity repair and functional recovery. (C) Exogenous Fibronectin bypasses PDGF-B, independently stimulates fibroblast recruitment and matrix assembly, and likewise closes the cavity and improves locomotion. (D) The PDGFR-β blocker SU16f abolishes both PDGF-B- and Fibronectin-induced fibroblast responses, leading to cavity re-formation and loss of motor recovery. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/9c95d5c1cdb04861f4991919.png"},{"id":101754016,"identity":"d612febe-3da2-40fa-91ac-2cd41a0f6461","added_by":"auto","created_at":"2026-02-03 10:41:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20115408,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/25fc482e-140a-4e82-ba69-fe623446c4c1.pdf"},{"id":100950864,"identity":"fda72673-b703-45b6-9979-240e32216315","added_by":"auto","created_at":"2026-01-23 07:09:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":84965362,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8587175/v1/e92057ca693bcebf2782ce17.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"PDGF-B, but not PDGF-D, prevents spinal cord injury-induced cavitation in rats via fibroblast recruitment and extracellular matrix remodeling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) triggers a cascade of deleterious events, including ischemic necrosis, inflammatory cell infiltration, and tissue degeneration, culminating in cystic cavity formation in both humans and rats [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These cystic cavities, characterized by cerebrospinal fluid accumulation, sparse connective tissue, and remnants of damaged vasculature and axons, pose a formidable obstacle to neuronal survival and axonal regeneration due to the absence of a supportive extracellular matrix (ECM) at the lesion site [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn rats, cystic cavities emerge within a week of SCI, with their volume stabilizing around ten weeks [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The lack of a fibrotic scar at the lesion core, in contrast to the response observed in mice, is thought to contribute to the development of cavities [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. After SCI in mice, platelet-derived growth factor receptor beta positive (PDGFR-β\u003csup\u003e+\u003c/sup\u003e) perivascular fibroblasts, along with ECM secretion, fill the injury core, potentially preventing cavity formation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Our previous studies have demonstrated the pivotal role of the platelet-derived growth factor (PDGF)/PDGFR-β pathway in fibroblast recruitment and ECM remodeling post-SCI in mice. Moreover, in situ injection of PDGF-B has been observed to promote PDGFR-β\u003csup\u003e+\u003c/sup\u003e fibrosis in the uninjured spinal cord, indicating its potential role in tissue repair mechanisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePDGF-B and PDGF-D, the member of the PDGF family, activate PDGFR-β to exert fibrotic effects after tissue trauma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Compared to PDGF-D, the PDGF-B molecule features a C-terminal tail enriched in basic amino acids, notably arginine and lysine. This positively charged basic amino acid cluster confers the ability to bind specifically and with high affinity to negatively charged heparan sulfate proteoglycans within ECM [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This specific interaction is critical for promoting cellular recruitment and proliferation during developmental processes, such as angiogenesis, and in tissue repair [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. During the early stages of tissue injury, PDGF-B is released from platelet α granules, initiating and accelerating tissue repair processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The expression of PDGF-B has been found to increase after SCI in rats treated with erythropoietin via intraperitoneal injection, suggesting its neuroprotective effects and importance in neurological recovery [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the expression dynamics and activation of the PDGF-B/PDGFR-β pathway post-SCI in rats, as well as its impact on cavity formation, have not been fully explored.\u003c/p\u003e \u003cp\u003eTo address this gap, we constructed a rat SCI model and administered exogenous PDGF-B via in situ injection in this study. Our results indicate that PDGF-B, but not PDGF-D, specifically prevents and resolves SCI-induced cavitation in rats by recruiting PDGFR-β\u003csup\u003e+\u003c/sup\u003e fibroblasts and restoring ECM integrity, thereby facilitating long-term neuroprotection and functional recovery.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Ethics Committee of Anhui Medical University (Approval NO. LLSC20230809). Adult female C57BL/6 mice (18\u0026ndash;20 g) and Sprague-Dawley (SD) rats (200\u0026ndash;250 g) were used in this study and were provided by the Animal Experimentation Center of Anhui Medical University. All experimental animals were randomly grouped and housed in an environment with controlled temperature and humidity, a 12-hour light/dark cycle, and free access to food and water.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSurgical procedures\u003c/h3\u003e\n\u003cp\u003eA clinically relevant model of spinal cord compression injury, as described by Ashley McDonough et al [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] was employed. Animals were anesthetized with isoflurane (induction dose 5%, maintenance dose 2%) and underwent dorsal laminectomy at the level of the 8th thoracic vertebra to expose the dorsal surface of the T10 spinal cord. Subsequently, a calibrated Dumont No. 2 clamp (1122320, Fine Science Tools, Germany) was utilized to apply compression for 10 s. In the situ injection experiment, injections were administered at two time points: immediately after SCI and on 14 days post-injury (dpi), following determination of the injury area under bright light irradiation, the animals were secured on a stereotaxic instrument, and 2 \u0026micro;l of 100 ng/\u0026micro;l recombinant human PDGF-B (HZ-1308, Proteintech, China), 2 \u0026micro;l of 1 \u0026micro;g/\u0026micro;l recombinant human Fibronectin (#11051407001, Sigma-Aldrich, United States), 2 \u0026micro;l of 100 ng/\u0026micro;l recombinant human PDGF-D (1159-SB/CF, R\u0026amp;D Systems, United States) or 2 \u0026micro;l of 0.01 M Phosphate Buffered Saline (PBS) solution (BL302A, biosharp, China) was injected using a 5-\u0026micro;l Hamilton microinjector (7634-01, Hamilton, Switzerland) at a rate of 0.5 \u0026micro;l/min, targeting the right ventral gray matter of the injured area with the following coordinates: posterior median vein as midline, 0.5 mm lateral to the midline, and a depth of 1.3 mm [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The syringe was left in place for 1 min after injection to prevent reflux of the injected PDGF-B, Fibronectin, PDGF-D or PBS solution through the injection site. The wound was gently irrigated with saline (1\u0026ndash;2 ml) after the needle was removed evenly and slowly, and the muscle layer and skin were sutured. The rats were then placed in a warming thermostat until fully awakened. On the first postoperative day, animals were carefully evaluated, and only those exhibiting complete hind limb paralysis were included in the study. Bladder care was performed twice daily postoperatively until spontaneous urination returned.\u003c/p\u003e\n\u003ch3\u003eTissue processing\u003c/h3\u003e\n\u003cp\u003eAnimals were sacrificed prior injury and at 3, 7, 14, 28 and 56 dpi for histologic evaluation. Following deep isoflurane anesthesia, animals underwent transcardiac perfusion with 0.1 M PBS (BL302A, biosharp, China) and 4% paraformaldehyde (BL539A, biosharp, China). The spinal cord was then dissected, and 5-mm segments containing the injured core was immersed in 4% paraformaldehyde for 24 h, followed by preservation through dehydration and refrigeration in a 30% sucrose solution for 48 h. The spinal cord was cut horizontally into 16-\u0026micro;m-thick serial frozen sections using a cryostat (NX50, Thermo Fisher Scientific, USA). Markings were made to ensure consistency, and three samples were collected from each group.\u003c/p\u003e\n\u003ch3\u003eHematoxylin-eosin staining\u003c/h3\u003e\n\u003cp\u003eTo evaluate the morphology of the lesioned cystic cavity, serial spinal cord sections were stained with hematoxylin and eosin (HE) (BL700b, biosharp, China). Horizontal sections were immersed in 200 ml hematoxylin staining solution for 5 min, then rinsed with tap water for 5 min, followed by hydrochloric acid-ethanol solution for differentiation. Subsequently, the sections were immersed in 200 ml of eosin staining solution for 2 min. Finally, gradient ethanol dehydration, transparency treatment and neutral resin sealing of the sections were performed.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eFor tissue immunofluorescence staining, spinal cord tissue sections was blocked in PBS (BL302A, biosharp, China) containing 0.3% Triton X-100 (T8200, Solarbio, China) and 10% donkey serum albumin (DSA) (SL050, Solarbio, China) for 1 h at room temperature, and then the primary antibodies were incubated overnight at 4\u0026deg;C: rabbit anti-PDGF-B (1:100; #NBP1-58279, Novus, USA), rabbit anti-PDGF-D (1:100; 40-2100, ThermoFisher, USA), rabbit anti-Fibronectin (1:500; 15613-1-AP, Sigma-Aldrich, USA), rabbit anti-Laminin (1:500; L9393, Sigma-Aldrich, USA), rabbit anti-Collagen I (1:50; 2150\u0026thinsp;\u0026minus;\u0026thinsp;1908, Bio-rad, USA), goat anti-PDGFR-β (1:100; #AF1042-SP, R\u0026amp;D Systems, USA), rabbit anti-p-PDGFR-β (1:100; YA3062 MCE, China), chicken anti-GFAP (1: 100; #16825-1-AP, Aveslabs, USA), goat anti-Iba-1 (1:500; NB100-1028, Novus, USA), goat anti-CD68 (1:200; Sc-70761, Santa Cruz, USA) ,goat anti-CD31 (1:200; AF3628, R\u0026amp;D Systems, USA), rabbit anti-Ki67 (1:200; Ab15580, abcam, USA), goat anti-5-HT (1:200; #20079, Immunostar, USA), and rabbit anti-NeuN (1:300; #ab177487 Abcam, USA). After three washes, the slides were incubated with secondary antibodies conjugated to appropriate Alexa fluorescent dyes: donkey anti-Chicken Alexa Fluor 488, donkey anti-goat Alexa Fluor 555, donkey anti-rabbit Alexa Fluor 555 and donkey anti-goat Alexa Fluor 647 (1:500; A-78948, A-21432, A-21428, A-21447, Thermo Fisher Scientific, USA) at room temperature for 1 h. Finally, nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI) (P0130, Beyotime Biotechnology, China). Images were captured using a fluorescence microscope (Zeiss, Germany).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative image analysis\u003c/h2\u003e \u003cp\u003eIn this experiment, three sections spaced 160 \u0026micro;m apart were selected from the middle of each spinal cord tissue for immunofluorescence staining. After processing the fluorescence-stained images using ImageJ [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the average of the quantified results from the three sections was taken as the data for one spinal cord sample. Each experimental group included three spinal cord tissue samples. For fibrotic scar analysis, the percentage of PDGFR-β, Fibronectin, Laminin, Collagen I, and GFAP positive area was quantified as the proportion of their positive areas relative to the spinal cord area within a 4 \u0026times; microscopic field. The GFAP-negative area percentage was calculated as the proportion of its negative area relative to the spinal cord area in a 4 \u0026times; microscopic field. The number of neurons was quantified by counting NeuN\u003csup\u003e+\u003c/sup\u003e cells in the Z1 (0\u0026ndash;250 \u0026micro;m), Z2 (250\u0026ndash;500 \u0026micro;m), Z3 (1000\u0026ndash;1250 \u0026micro;m), and Z4 (2000\u0026ndash;2250 \u0026micro;m) regions after SCI [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The percentage of positive-serotonergic (5-HT\u003csup\u003e+\u003c/sup\u003e) areas was measured as its positive area relative to the field of view in 20 \u0026times; microscopic fields at 0 \u0026micro;m and 500 \u0026micro;m from the injury core. The density of Ki67\u003csup\u003e+\u003c/sup\u003ePDGFR-β\u003csup\u003e+\u003c/sup\u003e cells was calculated as the ratio of the number of Ki67\u003csup\u003e+\u003c/sup\u003ePDGFR-β\u003csup\u003e+\u003c/sup\u003e cells to the area of the GFAP\u003csup\u003e\u0026minus;\u003c/sup\u003e region.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLaser Speckle Contrast Blood Flow Imaging System\u003c/h3\u003e\n\u003cp\u003eAfter SCI, rats received in situ injections of PDGF-B, PDGF-D, and Fibronectin were maintained for 28 days. Following the original modeling procedures, the injured spinal cord region was re-exposed. The Laser Speckle Contrast Blood Flow Imaging System (RFLSI ZW, China\u003cb\u003e)\u003c/b\u003e was used to measure the average blood flow through the lesion core over a 10 s, which was recorded as the blood perfusion volume through the lesion core per unit time for each sample. Three rat samples were analysed per experimental group.\u003c/p\u003e\n\u003ch3\u003eBehavioral assessment\u003c/h3\u003e\n\u003cp\u003eRecovery of motor function after SCI in rats was assessed using the Basso, Beattie \u0026amp; Bresnahan locomotor rating scale (BBB) open field locomotor test as reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All rats used BBB to assess that locomotor function is normal before SCI and received BBB after surgery to confirm that the SCI model is successful. To ensure blinded assessment of behavioral recovery, animals were evaluated at 0, 7, 14, 21and 28 dpi by two investigators not involved in animal surgery. Animals were allowed to run freely in an open field and motor function scores were measured after 4 min of observation.\u003c/p\u003e \u003cp\u003eWe used footprint analysis to further assess improvement in motor function of animals 28 dpi followed by published articles [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, all evaluated animals had their front paws dipped in green dye and hind paws dipped in blue dye. The tracks of animals with intact spinal cords were assigned to the Sham group. Each animal was run four times without significant interference as a valid run. Three gait parameters were then calculated. Stride length was the distance from the starting point to the end point of the hind paws in one gait cycle. Stride width is the distance between the left and right hind paws. Paw rotation is defined as the angle of the hind paw axis with respect to the stride axis. An increase in the angle of rotation indicates external rotation of the hind paws. All assessments were made in three consecutive gait cycles. Values were averaged for both hind paws. The footprints of the hind paws tend to overlap the footprints of the forepaws during gait in animals with an uninjured spinal cord, whereas animals with a SCI typically lose this coordination between n the hind and forepaws.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad Prism software (version 8.0). All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The two-tailed Student's t-test was used to compare the means of two groups. One-way analysis of variance (ANOVA) and Two-way analysis of variance followed by Tukey's multiple comparisons were used to compare the means of three or more groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 values were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCystic cavities form at the injury site following SCI in rats, accompanied by insufficient secretion of PDGF-B and PDGF-D.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the pivotal role of the PDGF/PDGFR-β signaling pathway in post-SCI wound repair, we postulated that cystic cavitation in the injured rat spinal cord may result from inadequate activation of this pathway. To test this hypothesis, we first compared the spatiotemporal expression patterns of PDGF/PDGFR-β components within the lesion core between rats and mice during the chronic phase of SCI. Immunofluorescence analysis revealed that at 28 days post-injury (dpi), the lesion core in mice was densely infiltrated with PDGFR-β⁺ fibroblasts, while the expression levels of PDGF-B and PDGF-D were sustainably high. In stark contrast, at the same time point in rats, well-delineated cystic cavities had developed within the lesion, coinciding with markedly reduced recruitment of PDGFR-β⁺ fibroblasts and scarcely detectable expression of PDGF-B and PDGF-D (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To systematically characterize post-SCI cavitation in rats and its relationship to fibrotic scarring, we analysed the temporal dynamics of fibroblast and its ECM components within the spinal cord lesion. PDGFR-β expression was initially increased at 3 dpi, peaked at 7 dpi, and progressively declined from 14 to 56 dpi in GFAP\u003csup\u003e\u0026minus;\u003c/sup\u003e area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, D). ECM including Fibronectin, Laminin, Collagen I within the lesion was deposited paralleled with PDGFR-β\u003csup\u003e+\u003c/sup\u003e fibroblasts recruitment after SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, E, F). Cystic cavities emerged by 14 dpi and gradually expanded into large DAPI\u003csup\u003e\u0026minus;\u003c/sup\u003e area, cell-free regions within the lesion epicentre by 28\u0026ndash;56 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). These observations imply an inverse correlation between residual PDGFR-β or ECM density and cavity volume after SCI in rats. Importantly, PDGF-D peaked at 3 dpi, while PDGF-B reached its maximum at 14 dpi; both subsequently decreased to low baselines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, G). Immunostaining for phosphorylated PDGFR-β (p-PDGFR-β at Tyr 740) confirmed sustained activation in mice at 7\u0026ndash;14 dpi, whereas rat tissue displayed only a transient 7-dpi peak \u003cb\u003e(Figure S1A, B)\u003c/b\u003e. These results indicate that insufficient activation of the PDGFR-β pathway in rats may contribute to cavity expansion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePDGF-B, but not PDGF-D, specially abolishes post-SCI cavitation in rats by recruiting fibroblasts and amplifying Fibronectin deposition.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the established role of PDGF-B in wound repair and its deficient endogenous expression, we administered recombinant PDGF-B or PDGF-D via immediate in situ injection after SCI to evaluate their effects on cystic cavity formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Successful delivery and localized protein retention were confirmed at 7 dpi through immunofluorescence, which showed elevated levels of PDGF-B and PDGF-D within the lesion core compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D-F). HE staining revealed near-complete prevention of cavitation in the PDGF-B group, with lesion site filled densely with cellular tissue, contrasting sharply with the extensive cavities observed in both Control and PDGF-D groups at 28 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, G). These findings suggest that exogenous PDGF-B effectively mitigates cavity formation during the subacute stage of SCI in rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the composition of the preserved tissue filling the cavity, we evaluated the expression of the fibroblast marker PDGFR-β and key ECM components following PDGF-B or PDGF-D administration. PDGF-B treatment resulted in markedly enhanced fibroblast recruitment and denser deposition of ECM components, particularly Fibronectin, relative to the Control group, whereas PDGF-D group showed no benefit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I). These results indicate that PDGF-B specifically triggers fibroblast recruitment and Fibronectin-rich ECM remodeling.\u003c/p\u003e \u003cp\u003eGiven the prominent upregulation of Fibronectin, we next assessed its therapeutic potential by in situ injection of exogenous Fibronectin protein after SCI. The results demonstrated that direct Fibronectin administration reproduced the anti-cavitary outcome (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C, G-I). To further investigate whether the role of PDGF-B is associated with the phosphorylation of PDGFR-β, we conducted phosphorylation assays on tissues injected in situ with PDGF-D, PDGF-B, and Fibronectin. The results revealed that the PDGF-B group exhibited increased phosphorylation levels of PDGFR-β compared to other groups, indicating that PDGF-B functions via activation of PDGFR-β phosphorylation \u003cb\u003e(Figure S1C, D)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTreatment with PDGF-B or Fibronectin, but not PDGF-D, enhances neurological and motor functional recovery after SCI in rats.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe next evaluated neuronal preservation and axonal regrowth by NeuN and 5-HT staining. At 28 dpi, both PDGF-B and Fibronectin groups significantly increased the number of preserved NeuN\u003csup\u003e+\u003c/sup\u003e neurons in the peri-lesion (Z2) regions and enhanced 5-HT\u003csup\u003e+\u003c/sup\u003e axon density within the lesion core compared to the Control and PDGF-D groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). Locomotor function assessed by BBB scoring showed significant improvement in PDGF-B and Fibronectin groups at 28 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Footprint analysis also revealed improved coordination, stride length, and rotation angle in these groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence analysis of Iba-1\u003csup\u003e+\u003c/sup\u003e and CD68\u003csup\u003e+\u003c/sup\u003e microglia/macrophages revealed a persistent inflammatory response in the Control and PDGF-D groups at 28 dpi \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. In contrast, both PDGF-B and Fibronectin groups significantly reduced the density of these immune cells within the lesion core (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Given the critical role of vascular recovery in tissue repair, we examined angiogenesis via CD31\u003csup\u003e+\u003c/sup\u003e endothelial cell staining and blood flow perfusion. PDGF-B and Fibronectin treatment resulted in significantly higher vascular density and improved perfusion within the lesion site compared to the Control and PDGF-D groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F, H, J). These results confirm that PDGF-B and Fibronectin not only mitigate chronic inflammation but also improve vascular recovery, further supporting their reparative functions after SCI in rats. Ki67 labeling confirmed that PDGF-B and Fibronectin, but not PDGF-D, drove markedly fibroblast proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, I). Thus, PDGF-B specially orchestrates fibroblast-mediated ECM remodeling, chiefly via Fibronectin, to prevent SCI-induced cavitation in rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDelayed administration of PDGF-B or Fibronectin also attenuates cavitation and facilitates functional recovery after SCI in rats.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the therapeutic potential of delayed intervention, we performed in situ injection of exogenous PDGF-B or Fibronectin in rats at 14 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Immunofluorescence analysis at 28 dpi revealed that delayed treatment with either agent markedly reduced cavitation and enhanced fibroblast recruitment and ECM deposition\u0026mdash;evidenced by increased expression of PDGFR-β, Fibronectin, Laminin and Collagen I\u0026mdash;compared to the Control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-E\u003cb\u003e)\u003c/b\u003e. However, it is noteworthy that delayed administration resulted in an expansion of the GFAP\u003csup\u003e\u0026minus;\u003c/sup\u003e area (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. Delayed injection of exogenous PDGF-B or Fibronectin can also promote serotonergic axon regrowth and increase neuronal preservation in both Z1 and Z2 regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). Consistent with these anatomical improvements, treated animals exhibited substantial functional recovery, as indicated by higher BBB scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK) and improved gait parameters (\u003cb\u003eFigure S2A-D\u003c/b\u003e). Furthermore, delayed PDGF-B or Fibronectin administration attenuated inflammation, indicated by reduced Iba-1\u003csup\u003e+\u003c/sup\u003e immunoreactivity, and increased vascular regeneration, as shown by enhanced CD31\u003csup\u003e+\u003c/sup\u003e vessel density (\u003cb\u003eFigure S2E-H\u003c/b\u003e). These results demonstrate that even delayed injection of PDGF-B or Fibronectin mitigates cavitation and promotes functional recovery after SCI in rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSU16f abrogates the therapeutic benefits of PDGF-B and Fibronectin by inhibiting fibroblast recruitment and ECM remodeling.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm the specificity of PDGF-B/PDGFR-β signaling, we administered SU16f, a PDGFR-β inhibitor, systemically following PDGF-B injection after SCI (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). SU16f treatment significantly reversed the PDGF-B-induced benefits, reducing fibroblast recruitment, ECM deposition (Fibronectin, Laminin, Collagen I), and leading to cavity re-formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). Similarly, SU16f compromised the therapeutic effects of Fibronectin, indicating that its benefits partially depend on PDGFR-β pathway activation (\u003cb\u003eFigure S3A-D\u003c/b\u003e). SU16f also negated the beneficial effects of PDGF-B on neuronal retention and axon regrowth (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H). Functionally, SU16f injection reduced BBB scores, reduced hindlimb step length and rotation angle, and increased step widths \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-M S3E-I\u003cb\u003e)\u003c/b\u003e. These findings underscore the essential role of PDGF-B/PDGFR-β signaling in fibroblast recruitment, ECM remodeling, cavity prevention, and functional recovery post-SCI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrates that insufficient PDGF-B expression following SCI in rats compromises its reparative function and contributes to post-traumatic cystic cavity formation. By single intraparenchymal injections of exogenous PDGF-B or its downstream effector Fibronectin, both during early and delayed phases, we successfully mitigated cavity formation in a clinically relevant rat model of compressive SCI through enhanced fibroblast recruitment and ECM remodeling. This intervention yielded multifaceted benefits, including attenuated neuroinflammation, restored vascular perfusion, promoted axonal regrowth, increased neuronal survival, and significant functional recovery. Conversely, PDGFR-β inhibition via SU16f abolished these therapeutic effects and re-induced cavitation. These findings identify PDGF-B, but not PDGF-D, as a critical regulator of fibrotic scarring and tissue repair post-SCI, highlighting its potential as a therapeutic target.\u003c/p\u003e \u003cp\u003ePrevious studies have reported that exogenous matrix implants facilitate axonal growth but fail to prevent cystic cavities in SCI models [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Similarly, cell transplantation and biomaterial-based treatments shown limited functional improvement despite some anatomical repair [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although injectable hydrogels\u0026mdash;such as those functionalized with imidazole-poly (organophosphazenes) or Fibronectin-derived cell-adhesive RGD peptides\u0026mdash;had been developed to reduce cavitation and promote motor outcomes in rats [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], their clinical translation remains challenging due to unresolved issues regarding biodegradation, mechanical compatibility, and immunogenicity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In contrast, our findings indicate that PDGF-B and Fibronectin administration nearly completely resolves cystic cavities and supports functional recovery, suggesting a potent and clinically feasible alternative.\u003c/p\u003e \u003cp\u003eFibrotic scarring is a conserved response that supports wound sealing and tissue integrity. In the central nervous system (CNS), PDGFR-β\u003csup\u003e+\u003c/sup\u003e cells serve as a major source of fibroblasts and ECM components after SCI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Goritz et al. demonstrated that PDGFR-β\u003csup\u003e+\u003c/sup\u003e cells form a fibrotic scar that prevents cavitation in mice; ablation of these cells led to large cystic cavities and loss of tissue integrity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, Shearer et al. reported that fibroblast-driven deposition of Fibronectin, Collagen Ⅰ and Laminin precludes cyst formation in SCI mice [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In rats, however, insufficient fibrotic scarring at the injury epicenter results in prominent cystic cavities [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Our data show that exogenous PDGF-B potently stimulates PDGFR-β\u003csup\u003e+\u003c/sup\u003e fibroblast recruitment and ECM remodeling, effectively sealing cystic cavities and restoring tissue continuity. To our knowledge, this is among the first studies to achieve successful cystic repair using a growth factor after SCI, rather than cell transplantation or biomaterial treatment. Moreover, this agent is clinically safe for humans and holds promise as a novel therapeutic strategy.\u003c/p\u003e \u003cp\u003eThe mechanisms underlying PDGF-B-mediated repair are likely multifactorial, involving not only fibrotic scar formation and ECM reorganization, but also modulation of angiogenesis and inflammation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Sun et al. showed that local administration of PDGF-B can induce Fibronectin fibril alignment and extension, effectively converting adult pericytes into a permissive substrate for axon growth in SCI mice [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], consistent with our results. We have confirmed that PDGF-B, but not PDGF-D, specifically upregulates Fibronectin and recruits fibroblasts to fill cystic cavities, thereby facilitating functional recovery post-SCI in rats. Structurally, PDGF-B contains a C-terminal domain rich in basic amino acids such as arginine and lysine, enabling high-affinity binding to heparan sulfate proteoglycans in the ECM, while its N-terminal region binds to cell-surface receptors to promote fibroblast recruitment and proliferation during development and repair [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Whether this structural specificity underlies the differential effects of PDGF-B and PDGF-D remains unclear and warrants further investigation.\u003c/p\u003e \u003cp\u003eAlthough dense fibrotic scarring has been suggested to inhibit regeneration in mice [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], we found that in rats, PDGFR-β\u003csup\u003e+\u003c/sup\u003e fibroblast recruitment and ECM remodeling are essential for cavity repair and functional recovery. Axonal staining indicated that PDGF-B treatment supports axonal extension across previously cavitied areas. Fibronectin administration mimicked these benefits, aligning with reports that Fibronectin-rich matrices form bridges that ligate the severed ends of the spinal cord and promote axonal regrowth [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, Fibronectin knockdown worsened functional outcomes, increased cavity size, and increased neuronal apoptosis in CNS injury models [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Notably, SU16f inhibition abrogated the benefits of both PDGF-B and Fibronectin, suggesting that Fibronectin\u0026rsquo;s effects are partly mediated through PDGFR-β signaling and that a positive feedback loop may exist between them \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Further studies are needed to elucidate how Fibronectin influences fibroblast proliferation and PDGFR-β activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, we demonstrate that PDGF-B, but not PDGF-D, specifically prevents cavitation and facilitates recovery after SCI in rats through fibroblast-dependent ECM remodeling. Its dual role in modulating inflammation and promoting angiogenesis positions PDGF-B as s promising therapeutic candidate. These findings underscore the PDGF-B/PDGFR-β axis as a critical diver of cavity repair and a potential target for clinical translation in SCI.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDGF-B\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplatelet-derived growth factor B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDGF-D\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplatelet-derived growth factor D\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate buffer saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003edpi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eday post-injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehematoxylin-eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunofluorescence staining\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003espinal cord injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003edpi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edays post-injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDGFR-β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplatelet-derived growth factor receptor beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNeuN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneuronal nuclei\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e5-HT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e5-Hydroxytryptamine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBBB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBasso, Beattie \u0026amp; Bresnahan\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglial fibrillary acidic protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIba-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eionized calcium-binding adaptor molecule-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eregion of interest.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful for the support of the Scientific Research and Experiment Center of the Second Affiliated Hospital of Anhui Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy design: MGZ, DST and ZYL; experimental implementation: CL, HH, NYZ, ZMX, ZKB, RL, XW, FLS, and JNY; data analysis and figure preparation: CL, ZMX, SKZ, CPM, ZDM and SSY; paper writing: CL and SSY; study supervising and paper reviewing: MGZ, DST and ZYL. All authors approved the final version of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (Grant Nos. 82372506 to MGZ, 82301563 to ZYL, 82401616 to SSY), the Anhui Provincial Natural Science Foundation (Grant Nos. 2308085MH257 to MGZ, 2508085MH203 to DST, 2308085QH266 to ZYL, 2408085QH262 to SSY); the Clinical Medical Research Transformation Project of Anhui Province (Grant Nos. 202304295107020014 to MGZ, 202304295107020011 to DST); and the Natural Science Research Project of Anhui Educational Committee (Grant Nos. 2022AH050746 to MGZ, 2022AH050713 to ZYL, KJ2021A0316 to SSY). The Graphical abstract in this article was created using BioRender.com, and the author sincerely acknowledges BioRender (BioRender.com) for the powerful drawing support provided for this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during the current study are contained within this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were carried out with the approval of the Ethics Committee of the Anhui Medical University (Approval No. LLSC20230809).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eonsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eDepartment of Orthopaedics, The Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eInstitute of Orthopaedics, Research Center for Translational Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary figures and tables.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZrzavy T, Schwaiger C, Wimmer I, Berger T, Bauer J, Butovsky O, et al. 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J Neurosci 2013, 33:12870-12886.\u003c/li\u003e\n\u003cli\u003eBasso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995, 12:1-21.\u003c/li\u003e\n\u003cli\u003eYokota K, Kobayakawa K, Saito T, Hara M, Kijima K, Ohkawa Y, et al. Periostin promotes scar formation through the interaction between pericytes and infiltrating monocytes/macrophages after spinal cord injury. Am J Pathol 2017, 187:639-653.\u003c/li\u003e\n\u003cli\u003eCigognini D, Satta A, Colleoni B, Silva D, Donega M, Antonini S, et al. Evaluation of early and late effects into the acute spinal cord injury of an injectable functionalized self-assembling scaffold. PLoS One 2011, 6: e19782.\u003c/li\u003e\n\u003cli\u003eCigognini D, Silva D, Paloppi S, Gelain F. Evaluation of mechanical properties and therapeutic effect of injectable self-assembling hydrogels for spinal cord injury. J Biomed Nanotechnol 2014, 10:309-323.\u003c/li\u003e\n\u003cli\u003eRuzicka J, Romanyuk N, Hejcl A, Vetrik M, Hruby M, Cocks G, et al. Treating spinal cord injury in rats with a combination of human fetal neural stem cells and hydrogels modified with serotonin. Acta Neurobiol Exp (Wars) 2013, 73:102-115.\u003c/li\u003e\n\u003cli\u003eWang X, Xu XM. Long-term survival, axonal growth-promotion, and myelination of schwann cells grafted into contused spinal cord in adult rats. Exp Neurol 2014, 261:308-319.\u003c/li\u003e\n\u003cli\u003eSoleimani A, Oraee Yazdani S, Pedram M, Saadinam F, Rasaee MJ, Soleimani M. Intrathecal injection of human placental mesenchymal stem cells derived exosomes significantly improves functional recovery in spinal cord injured rats. Mol Biol Rep 2024, 51:193.\u003c/li\u003e\n\u003cli\u003eHong L, Kim YM, Park HH, Hwang DH, Cui Y, Lee EM, et al. An injectable hydrogel enhances tissue repair after spinal cord injury by promoting extracellular matrix remodeling. Nat Commun 2017, 8:533.\u003c/li\u003e\n\u003cli\u003eMarquardt LM, Doulames VM, Wang AT, Dubbin K, Suhar RA, Kratochvil MJ, Medress ZA, Plant GW, Heilshorn SC. Designer, injectable gels to prevent transplanted schwann cell loss during spinal cord injury therapy. Sci Adv 2020, 6: eaaz1039.\u003c/li\u003e\n\u003cli\u003eCorreia C, Peixoto D, Soares Da Costa D, Reis RL, Pashkuleva I, Alves NM. Development and in vitro assessment of injectable, adhesive, and self-healing chitosan-based hydrogels for treatment of spinal cord injury. Biomater Adv 2025, 167:214090.\u003c/li\u003e\n\u003cli\u003eSoderblom C, Luo X, Blumenthal E, Bray E, Lyapichev K, Ramos J, et al. Perivascular fibroblasts form the fibrotic scar after contusive spinal cord injury. J Neurosci 2013, 33:13882-13887.\u003c/li\u003e\n\u003cli\u003eHoll D, Hau WF, Julien A, Banitalebi S, Kalkitsas J, Savant S, et al. Distinct origin and region-dependent contribution of stromal fibroblasts to fibrosis following traumatic injury in mice. Nat Neurosci 2024, 27:1285-1298.\u003c/li\u003e\n\u003cli\u003eXue X, Wu X, Fan Y, Han S, Zhang H, Sun Y, et al. Heterogeneous fibroblasts contribute to fibrotic scar formation after spinal cord injury in mice and monkeys. Nat Commun 2024, 15:6321.\u003c/li\u003e\n\u003cli\u003eAyazi M, Zivkovic S, Hammel G, Stefanovic B, Ren Y. Fibrotic scar in CNS injuries: from the cellular origins of fibroblasts to the molecular processes of fibrotic scar formation. Cells 2022, 11.\u003c/li\u003e\n\u003cli\u003eShearer MC, Fawcett JW. The astrocyte/meningeal cell interface--a barrier to successful nerve regeneration? Cell Tissue Res 2001, 305:267-273.\u003c/li\u003e\n\u003cli\u003eByrnes KR, Fricke ST, Faden AI. Neuropathological differences between rats and mice after spinal cord injury. J Magn Reson Imaging 2010, 32:836-846.\u003c/li\u003e\n\u003cli\u003eZhou H, Wang X, Li Y, Wang D, Zhou X, Xiao N, et al. Dynamic development of microglia and macrophages after spinal cord injury. Neural Regen Res 2025, 20:3606-3619.\u003c/li\u003e\n\u003cli\u003eChen Y, Gao J, Yan H, Zhou S, Fang Y, Lyu X, et al. Identifying microglia and peripheral infiltrating macrophages in the injured spinal cords using flow cytometry. J Vis Exp 2025.\u003c/li\u003e\n\u003cli\u003eSun W, Dion E, Laredo F, Okonak A, Sepeda JA, Haykal E, et al. In vivo programming of adult pericytes aids axon regeneration by providing cellular bridges for SCI repair. Mol Ther 2025, 33:3968-3993.\u003c/li\u003e\n\u003cli\u003eLi Y, He X, Kawaguchi R, Zhang Y, Wang Q, Monavarfeshani A, et al. Microglia-organized scar-free spinal cord repair in neonatal mice. Nature 2020, 587:613-618.\u003c/li\u003e\n\u003cli\u003eTate CC, Garcia AJ, LaPlaca MC. Plasma fibronectin is neuroprotective following traumatic brain injury. Exp Neurol 2007, 207:13-22.\u003c/li\u003e\n\u003cli\u003eSakai T, Johnson KJ, Murozono M, Sakai K, Magnuson MA, Wieloch T, et al. Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis. Nat Med 2001, 7:324-330.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spinal cord injury, Cystic cavity, Fibroblast recruitment, PDGF-B, Fibronectin","lastPublishedDoi":"10.21203/rs.3.rs-8587175/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8587175/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eSpinal cord injury (SCI) induces substantial cell loss at the injury site, leading to the formation of cystic cavities, a major obstacle to neural repair in both humans and rats. The mechanisms driving cavity development remain elusive. Given the well-established role of platelet-derived growth factor-B (PDGF-B) in wound healing via activation of its receptor PDGFR-β, as demonstrated in mice, we propose that impaired PDGFR-β signaling may be a key contributor to cavity formation post-SCI in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e First, the spatiotemporal expression patterns of PDGF-B, PDGF-D, PDGFR-β, and key extracellular matrix (ECM) components (Fibronectin, Laminin, Collagen I) were assessed in rat spinal cord following crush injury using immunofluorescence, and the process of cavity formation was observed. Subsequently, the therapeutic potential of immediate and delayed in situ delivery of PDGF-B, PDGF-D, or Fibronectin on post-traumatic cavitation were evaluated. Additionally, the necessity of fibroblast activation and recruitment was assessed by intraperitoneal administration of the PDGFR-β inhibitor SU16f.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Expression of PDGFR-β, Fibronectin, laminin, and collagen I peaked at 7 days post-injury (dpi) and subsequently regressed within GFAP-negative regions, coinciding with progressive cystic cavity formation by 28 and 56 dpi in the injured rat spinal cord. Endogenous PDGF-D peaked at 3 dpi, while PDGF-B peaked at 14 dpi, both exhibiting lower levels thereafter. Immediate or delayed in situ injection of exogenous PDGF-B, but not PDGF-D, specifically prevented and reversed cavitation, upon PDGFRβ-mediated fibroblast recruitment and ECM deposition, especially Fibronectin upregulation, thereby yielding marked neurological and motor function recovery following SCI in rats. Fibronectin injection recapitulated the benefits of PDGF-B, whereas systemic SU16f abolished the therapeutic effects of both PDGF-B and Fibronectin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our findings demonstrate that PDGF-B specifically prevents SCI-induced cavitation in rats by orchestrating fibroblast recruitment and ECM remodeling, providing a translational framework for growth factor therapy after SCI.\u003c/p\u003e","manuscriptTitle":"PDGF-B, but not PDGF-D, prevents spinal cord injury-induced cavitation in rats via fibroblast recruitment and extracellular matrix remodeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 15:27:09","doi":"10.21203/rs.3.rs-8587175/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c048c177-f4c9-486e-93ef-0122608da9fa","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:17:13+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 15:27:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8587175","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8587175","identity":"rs-8587175","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}