In vivo programming of adult pericytes aids axon regeneration by providing cellular bridges for SCI repair

Wenjing Sun; Elliot Dion; Fabio Laredo; Allyson Okonak; Jesse A Sepeda; Esraa Haykal; Min Zhou; Heithem M El-Hodiri; Andy J Fischer; Jerry Silver; Juan Peng; Andrew Sas; Andrea Tedeschi

doi:10.1016/j.ymthe.2025.04.020

. 2025 Apr 18;33(8):3968–3993. doi: 10.1016/j.ymthe.2025.04.020

In vivo programming of adult pericytes aids axon regeneration by providing cellular bridges for SCI repair

Wenjing Sun ¹, Elliot Dion ¹, Fabio Laredo ^1,², Allyson Okonak ¹, Jesse A Sepeda ³, Esraa Haykal ¹, Min Zhou ¹, Heithem M El-Hodiri ¹, Andy J Fischer ¹, Jerry Silver ⁴, Juan Peng ⁵, Andrew Sas ^3,⁶, Andrea Tedeschi ^1,^6,^∗

PMCID: PMC12461639 PMID: 40253585

Abstract

Pericytes are contractile cells of the microcirculation that participate in wound healing after spinal cord injury (SCI). Thus far, the extent to which pericytes cause or contribute to axon growth and regeneration failure after SCI remains controversial. Here, we found that SCI leads to profound changes in vasculature architecture and pericyte coverage. We demonstrated that pericytes constrain sensory axons on their surface, causing detrimental structural and functional changes in adult dorsal root ganglion neurons that contribute to axon regeneration failure after SCI. Perhaps more excitingly, we discovered that in vivo programming of adult pericytes via local administration of platelet-derived growth factor BB (PDGF-BB) effectively promotes axon regeneration and recovery of hindlimb function by contributing to the formation of cellular bridges that span the lesion. Ultrastructural analysis showed that PDGF-BB induced fibronectin fibril alignment and extension, effectively converting adult pericytes into a permissive substrate for axon growth. In addition, PDGF-BB localized delivery positively affects the physical and chemical nature of the lesion environment, thereby creating more favorable conditions for SCI repair. Thus, therapeutic manipulation rather than wholesale ablation of pericytes can be exploited to prime axon regeneration and SCI repair.

Keywords: spinal cord injury, pericytes, DRG neurons, PDGF-BB, vasculature remodeling, axon regeneration, extracellular matrix, functional recovery

Graphical abstract

Sun and colleagues discover that therapeutic manipulation rather than wholesale ablation of pericytes can be exploited to prime axon regeneration after SCI. Specifically, PDGF-BB-mediated pericyte conversion at the lesion site enables axon regeneration and positively affects the physical and chemical nature of the lesion environment, thereby promoting SCI repair.

Introduction

In mammals, injury to the adult spinal cord yields devastating neurological deficits and long-term disability due to axon regeneration failure.¹ Not only do central nervous system (CNS) neurons lose intrinsic axon growth ability especially during later stages of development and into adulthood,²^,³ there is also a hostile cellular environment at the lesion site that actively blocks regeneration after CNS injury.⁴ The regenerative capacity of neurons profoundly differs between animal lineages.⁵ In zebrafish and amphibians, a more permissive lesion environment supports axon regeneration,⁶^,⁷^,⁸ allowing complete restoration of function after spinal cord injury (SCI). Although described as a near absolute barrier to axonal regeneration after SCI in mammals, the fibrotic scar has mainly been understudied. In mammals, current strategies that allow the conversion of the growth-inhibitory lesion environment into a growth-supportive state primarily focus on the elimination of myelin-derived inhibitors,⁹ digestion of chondroitin sulfate proteoglycans (CSPGs),¹⁰^,¹¹^,¹² and, more recently, ablation of specific cell populations that participate to fibrotic scarring.¹³^,¹⁴^,¹⁵

Of the cellular sources contributing to fibrotic scarring, pericytes have been the subjects of genetic ablation studies aimed at promoting axon regeneration and functional recovery following SCI in adult mice.¹⁴^,¹⁵^,¹⁶ Pericytes are multifunctional mural cells of the microcirculation where they control blood flow, vascular permeability, and homeostasis.¹⁷^,¹⁸ While revascularization and vascular normalization ensure the delivery of oxygen and nutrients needed to sustain the high metabolic demands of neural networks, aberrant reorganization of microvasculature networks and perivascular cell function blunts axon regeneration after SCI in adult mice.¹⁶ Structured reorganization of vasculature networks at the lesion site is also instrumental for axon regeneration in the peripheral nervous system.¹⁹ Since pericytes are highly plastic and motile during vascular development,²⁰ manipulation of pericyte-neuron interaction may be exploited to prime regeneration after SCI by providing vascular bridges for axon elongation across the lesion site.

Here, we show that profound changes in vasculature architecture and pericyte coverage fail to fully restore vasculature function after SCI in adult mice. Pericytes also cause detrimental structural and functional changes in adult dorsal root ganglion (DRG) neurons associated with poor axon outgrowth. Importantly, PDGF-BB stimulation effectively converts adult pericytes into a permissive substrate for axon growth and regeneration. Mechanistically, reconfiguration of fibronectin matrix and integrin signaling are necessary to sponsor axon elongation on PDGF-BB-stimulated pericyte substrates. Finally, PDGF-BB-mediated pericyte conversion at the lesion site enables axon regeneration and positively affects the physical and chemical nature of the lesion environment, thereby promoting neurological recovery after SCI. Thus, our study underscores the strong potential for programming pericyte behavior to create more favorable conditions for SCI repair.

Results

SCI leads to profound changes in vasculature architecture and pericyte coverage

Aberrant reorganization of microvascular structures and perivascular cell function interferes with physiological recovery following CNS injury and disease. To determine the extent to which pericytes participate in the reorganization of microvasculature structure and vascular normalization at the lesion site, we assessed changes in vasculature architecture and pericyte coverage after SCI. To this end, we subjected adult FVB mice expressing membrane-anchored enhanced GFP (eGFP) under the control of the chondroitin sulfate proteoglycan 4 (Cspg4)—also known as neuron-glial antigen 2 (NG2)—promoter on a BAC transgene,²¹ hereafter called NG2-eGFP mice, to a thoracic (T)12 SCI that completely severed dorsal column axons on one side (Figure 1A). Of note, both pericytes and oligodendrocyte precursor cells (OPC) in the spinal cord expressed eGFP in this transgenic line (Figures S1A and S1B). Although it is possible to distinguish the two cell types based on their unique morphology without any specific staining procedure, we confirmed pericyte identity by immunostaining using canonical pericyte markers such as desmin, platelet-derived growth factor receptor β (PDGFrβ) and CD13 (Figure S1A).²² Consistent with previous reports,²³^,²⁴ eGFP-positive OPC express PDGFrα and display a highly branched morphology (Figure S1B). Under normal physiological conditions, we found that eGFP-positive pericytes were closely associated with microvasculature structures in the adult mouse spinal cord (Figure S1C). At each time point after SCI, mice were transcardially perfused and the entire vasculature was labeled by filling the blood vessel lumen with a fluorescent gel perfusate (Figure S1C). This procedure improves image contrast, allowing vasculature reconstruction at the capillary level.²⁵^,²⁶ After dissecting and clearing the spinal cords, we imaged the unsectioned tissues to visualize three-dimensional changes in vasculature architecture and pericyte coverage at the lesion site over days and months after SCI (Figures 1B and 1C). Newly formed eGFP filamentous structures displayed complex and tortuous trajectories (Figure 1B). Our data indicate that pericytes already migrated into the lesion site 3 days post-injury (DPI) during capillary sprouting as shown by the colocalization with the endothelial cell (EC) marker CD31 (Figure 1D).²⁷ However, pericyte decorated filamentous structures outnumbered the extent of microvasculature filling at the lesion epicenter at acute and subacute time points (e.g., 3, 7, and 14 DPI) (Figure 1B). Even at chronic time points (e.g., 28 and 90 DPI), microvasculature structures filled by the gel perfusate showed incomplete coverage of the lesion site (Figure 1B). Yet, pericyte-decorated structures continued to fill the injury site (Figures 1B and 1C). Given that pericytes are embedded in the vascular basement membrane,²⁸ the distance between eGFP-positive pericyte and the closest vasculature structure filled with albumin-tetramethylrhodamine (TRITC) was close to zero in the uninjured spinal cord (Figures 1D and 1E). In contrast, the distance increased at 3 DPI, suggesting that pericytes dissociated from mature microvasculature structures during capillary sprouting after SCI. Previous studies have shown that pericyte deficiency and detachment from the vasculature lead to increased vascular permeability.²⁹^,³⁰^,³¹ In line with this concept, we found that claudin 5, a major cell adhesion molecule of tight junctions in ECs maintaining barrier function,³² was only expressed along 30%–36% of the total vasculature length within the lesion site at 3 and 7 DPI, underscoring a compromised barrier function early after SCI (Figures 1F and 1G). Although claudin 5 expression continued to increase within the lesion core at subacute time points, ∼30% of the total vasculature showed no claudin 5 expression 1 month after SCI (Figure 1G).

Together, our data indicate that profound changes in vasculature architecture and pericyte coverage fail to fully restore vasculature function and homeostasis in the injured spinal cord, likely contributing to a lack of functional recovery in adulthood.

Pericytes cause detrimental structural and functional changes in adult DRG neurons

Next, we assessed the contribution of pericytes on axon growth and regeneration failure. Accordingly, we purified and cultured adult pericytes from the spinal cord of adult FVB wild-type mice. Isolated pericytes expressed classical pericyte markers including NG2, PDGFrβ, and desmin²² (Figures S2A and S2B). We confirmed the purity of the pericyte culture by staining cells with astrocyte (e.g., GFAP and Aldh1l1), EC (e.g., CD31), oligodendrocyte (e.g., CC1), macrophage/microglia (e.g., F4/80 and Iba1), and fibroblast (e.g., S100A4) markers. We found no additional contaminating cells in our pericyte culture except for a small percentage of macrophage/microglia (∼4% of all cells cultured) (Figure S2C). We further validated our pericyte isolation protocol using adult NG2-eGFP mice (FVB background) and confirmed that cultured pericytes express eGFP (Figure S2D). Not only do pericytes express NG2, a potent growth-inhibitory proteoglycan,³³ but they also express permissive substrates for axon growth including laminin and fibronectin (Figure S2E).³⁴^,³⁵ Thus, we questioned whether pericyte-neuron interaction inhibits or promotes axon growth. We created a confluent pericyte monolayer and, 24 h later, plated adult DRG neurons on its upper surface. Laminin-coated dishes served as a control. DRG neurons grown on a pericyte monolayer showed a drastic reduction in axon length and increased branching compared with the control condition (Figures 2A and 2B). Of note, axon growth inhibition was comparable with that seen in DRG neurons plated on growth-inhibitory CSPGs (Figure 2B). Interestingly, adult DRG neurons grown on the pericyte monolayer had disorganized microtubules around the cell body (Figure 2C) and collapsed growth cones resembling punctate adhesive contacts (Figures S2F and S2G). After SCI, a disorganized microtubule network is also present in dystrophic end bulbs,³⁶ a hallmark of regeneration failure in the adult mammalian CNS. Next, we performed patch-clamp recording and time-lapse calcium imaging to determine the extent to which pericytes impair the physiological properties of adult DRG neurons. Passive membrane properties including input resistance, capacitance, and resting membrane potential were comparable between DRG neurons cultured on laminin-coated dishes and pericyte monolayers (Figures 2D and 2E). In contrast, we found reduced voltage-gated sodium current in DRG neurons growing on pericytes (Figures 2D and 2F), indicating reduced neuronal excitability. We then performed calcium imaging and found that >40% of control neurons displayed spontaneous calcium events 24 h after plating (Figures 2G and 2H). In contrast, <20% of neurons cultured on pericyte monolayer showed spontaneous calcium activity (Figures 2G and 2H). Of note, neuron survival was not affected (Figure S2H).

Prolonged exposure to CSPG can cause over-adhesion with no axon outgrowth.³⁷ In addition, protein tyrosine phosphatase σ (PTPσ), a CSPG receptor,³⁸ actively contributes to the conversion of neuronal growth cones into dystrophic bulbs via stabilization with CSPG-rich substrates.³⁷ While searching for strategies to overcome pericyte-mediated inhibition on axon outgrowth, we found increased PTPσ expression within the dystrophic growth cones of neurons plated on the pericyte monolayer (Figures S2I and S2J). In turn, we asked whether incubation with a membrane-permeable peptide that binds to PTPσ and blocks CSPG-mediated inhibition of axon growth³⁷ would be sufficient to rescue axon growth defects in adult DRG neurons plated on a pericyte monolayer. Incubation with a PTPσ peptide rescued, at least in part, axon outgrowth in our co-culture system as it does on CSPG-coated dishes (Figures S2K and S2L). Taken together, these data support the conclusion that pericytes cause detrimental structural and functional changes in adult DRG neurons, likely due to over-adhesion to CSPG-rich substrates.

PDGF-BB converts adult pericytes into a permissive substrate for axon growth

PDGF play a crucial role during the development of the vascular system. PDGF-BB binds to PDGFrβ expressed on pericytes to stimulate their proliferation and migration as well as their recruitment to growing blood vessels and into the wound area.³⁹ Since pericytes are highly plastic and can respond to PDGF-BB exposure by secreting pro-regenerative molecules including numerous neurotrophic factors,⁴⁰ we next addressed whether PDGF-BB would be sufficient to convert adult pericytes into a permissive substrate for axon outgrowth. Strikingly, adult DRG neurons cultured for 24 h on a PDGF-BB-stimulated pericyte monolayer extended long and sparsely branched axons (Figures 3A and 3B). Of note, the extent of axon growth was comparable with that found in DRG neurons grown for 24 h on permissive laminin-coated dishes (Figures 2A and 2B). As PDGF-BB alone did not further improve axon growth of adult DRG neurons plated on laminin-coated dishes (Figure S3A), we tested whether PDGF-BB pericyte-conditioned medium promotes axon growth in adult DRG neurons. We plated adult mouse DRG neurons on laminin-coated dishes and, 2 h after plating, we replaced the medium with either pericyte control or PDGF-BB pericyte-conditioned media. Surprisingly, exposure to PDGF-BB pericyte-conditioned medium failed to enhance axon growth in adult DRG neurons (Figure S3B), indicating additional variables may be at play in neurons plated on a PDGF-BB-stimulated pericyte monolayer.

Adult pericytes exposed to PDGF-BB acquired an elongated phenotype (Figure S3C and S3D), as reported by others.⁴⁰ Fibronectin is a multifunctional adhesive glycoprotein and ubiquitous extracellular matrix (ECM) component that plays a critical role in tissue repair, cell attachment, and motility.⁴¹^,⁴² Since fiber structure alignment supports axon elongation and regeneration,⁴³^,⁴⁴ we questioned whether PDGF-BB stimulation also causes structural changes in fibronectin matrix assembly in cultured pericytes. Our immunocytochemistry and cryo-EM analysis confirmed that pericytes stimulated for 24 h with PDGF-BB display fibronectin elongation and fibril alignment (Figures 3C–3G). The overall expression of fibronectin and laminin, a basal lamina component, decreased in cultured pericytes exposed to PDGF-BB (Figures 3H, 3I, and S3E). In the adult mouse spinal cord, OPC and pericytes also express collagen I (Figures S3F–S3H), as shown by others.⁴⁵^,⁴⁶^,⁴⁷ Our data indicate that pericyte expression of NG2 and pro-α1 chain of type I collagen decreased upon exposure to PDGF-BB (Figures S3I and S3J). This may not be surprising as accumulating evidence indicates that fibronectin accelerates collagen nucleation and that the presence of collagen fibrils induces the formation of highly colocalized fibronectin fibrils and subsequent matrix assembly.⁴⁸^,⁴⁹

Neurons adhere to ECM components including fibronectin substrates through transmembrane receptor proteins of the integrin family.⁵⁰^,⁵¹ To test the role of fibronectin matrix assembly and integrin signaling during axon outgrowth on PDGF-BB-stimulated pericyte monolayers, we turned to well-established tools that enable acute fibronectin silencing and pharmacological disruption of integrin signaling. Indeed, silencing fibronectin expression in pericyte monolayers abrogated PDGF-BB-dependent axon outgrowth of adult DRG neurons (Figures 4A–4D). Similarly, pharmacological blockade of integrin signaling using the RGD peptide (a broad-spectrum inhibitor of integrin receptor function), cilengitide (inhibitor of α_vβ₃ and α_vβ₅ integrins), and K34c (α₅β₁ integrin inhibitor) strongly inhibited axon outgrowth of DRG neurons plated on PDGF-BB-stimulated pericyte monolayers (Figures S4E and S4F).

*In vivo* conversion of adult pericytes via PDGF-BB promotes axon regeneration after SCI

(A) Schematic of T12 SCI and experimental timeline. (B) Representative three-dimensional imaging of the unsectioned spinal cords of adult NG2-eGFP mice generated by automated tile scanning at 4 weeks after SCI. Dorsal column sensory axons were labeled by injecting AAV-tdTomato into the left sciatic nerve. The asterisk indicates the lesion epicenter (R, rostral; C, caudal). Scale bar, 200 μm. (C) Quantification of regenerating axons in (B). Scatterplot and mean (linear mixed model controlled on baseline measurements ∗p < 0.05; LS, lesion site; vehicle n = 6, PDGF-BB n = 7 biological replicates). (D) Representative three-dimensional imaging of the lesion site of adult NG2-eGFP mice at 4 weeks after SCI. Asterisk indicates the lesion epicenter (R, rostral; C, caudal). Scale bar, 200 μm. (E) Quantification of NG2-eGFP filament distribution at the lesion epicenter in (D). Mean and SEM (unpaired two-tailed Student’s t test ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant, vehicle n = 6 and PDGF-BB n = 7 biological replicates). (F) Higher-magnification image of the unsectioned spinal cord shown in (A). The asterisk indicates the lesion epicenter (R, rostral; C, caudal). Arrowheads indicate regenerating axons growing on pericyte-decorated cellular bridges. Scale bar, 200 μm. (G) Quantification of (F). Mean and SEM (unpaired two-tailed Student’s t test ∗∗∗p < 0.001, vehicle n = 5 and PDGF-BB n = 6 biological replicates). (H) Representative three-dimensional imaging of the unsectioned spinal cords of adult FVB mice generated by automated tile scanning at 4 weeks after SCI. Dorsal column sensory axons were labeled by injecting AAV-tdTomato into the left sciatic nerve. The asterisk indicates the lesion epicenter (R, rostral; C, caudal). Scale bar, 200 μm. (I) Quantification of regenerating axons in (H). Scatterplot and mean (linear mixed model controlled on baseline measurements ∗∗∗p < 0.001; LS, lesion site; PDGF-BB/vehicle n = 8, PDGF-BB/SU16F n = 7, PDGF-BB/CP673451 n = 7 biological replicates). (J) Representative three-dimensional imaging of the traced vasculature at the lesion site at 4 weeks after SCI from (H). Asterisk indicates the lesion epicenter (R, rostral; C, caudal). Scale bar, 100 μm.

To test the clinical relevance of PDGF-BB-mediated pericyte conversion, we plated adult DRG neurons on a PDGF-BB-stimulated human pericyte monolayer. Once again, adult mouse DRG neurons cultured for 24 h on human pericytes exposed to PDGF-BB extended longer and sparsely branched axons compared with the control (Figures 3J and 3K).

Together, these results indicate that PDGF-BB effectively converts adult pericytes from an inhibitory to a growth-promoting substrate for axon outgrowth and that reconfiguration of fibronectin matrix and integrin signaling are necessary to sponsor axon outgrowth on PDGF-BB-stimulated pericyte monolayers.

In vivo conversion of adult pericytes via PDGF-BB aids axon regeneration by providing cellular bridges that span the lesion

Next, we examined whether in vivo PDGF-BB administration promoted axon regeneration after SCI. We subjected adult (8–10 weeks old) NG2-eGFP mice (FVB background) to a T12 SCI that completely transected dorsal column sensory axons on one side (Figure 4A). Seven days later, at a clinically relevant therapeutic window, we administered either vehicle (ddH₂O) or PDGF-BB at the lesion site. To visualize dorsal column axons in the spinal cord, we injected adeno-associated viral (AAV1) particles expressing tdTomato into the left sciatic nerve to transduce dorsal column axons originating from L3-5 DRG. We dissected and cleared the spinal cords 28 days after SCI. Three-dimensional imaging of the unsectioned spinal cord showed that localized delivery of PDGF-BB at the lesion epicenter promoted robust regeneration of dorsal column axons into and beyond the lesion site (Figures 4B and 4C). As they crossed the lesion epicenter, these regenerating axons exhibited tortuous trajectories, a common feature often seen in experimental models associated with regenerative growth.³^,⁵² Imaging segmentation using AI-based convolutional reconstruction confirmed that one single administration of PDGF-BB 7 days after SCI led to the formation of pericyte-decorated cellular bridges at the lesion site (Figures 4D and 4E). Strikingly, we observed numerous if not all regenerating axons growing on these cellular bridges that had formed throughout the lesion site in response to PDGF-BB stimulation (Figures 4F and 4G).

As genetic variations impact axon regeneration and CNS repair,⁵³^,⁵⁴ we sought to replicate the regenerative phenotype associated with PDGF-BB-mediated pericyte conversion in a common mouse background strain. Accordingly, we subjected adult NG2-CreERT2/Ai9 (RCL-tdTomato) mice (C57Bl6/J background) to T12 SCI and, 7 days later, injected vehicle (ddH₂O) or PDGF-BB at the site of injury (Figure S5A). To visualize pericytes, we administered tamoxifen for four consecutive days beginning on the same day we injected vehicle and PDGF-BB. Two weeks after SCI, we injected AAV1 particles expressing GFP into the left sciatic nerve to transduce dorsal column axons originating from L3-5 DRG. We then dissected and imaged in three-dimensional the spinal cords 28 days after SCI. Our results confirmed that localized delivery of PDGF-BB at the lesion epicenter also promoted robust regeneration of dorsal column axons into and beyond the lesion site in a common mouse background strain (e.g., C57BL/6J) (Figures S5B and S5C). Analysis of tdTomato-expressing pericytes confirmed that one single PDGF-BB administration increased pericyte density at the lesion site (Figures S5D and S5E), as reported by others.⁵⁵

Given that PDGF-BB can bind to PDGFrβ (expressed by pericytes and fibroblasts) and PDGFrα (expressed by fibroblasts and OPC), we tested the extent to which PDGF-BB/PDGFrβ interaction is necessary to sponsor axon regeneration after SCI. We subjected adult FVB mice to a T12 SCI that completely transected dorsal column sensory axons on one side (Figure 4H). Seven days later, we administered PDGF-BB at the lesion site and, beginning 1 h later, injected the PDGFrβ inhibitors SU16F (10 mg/kg, intraperitoneally [i.p.], 1 time/day) and CP673451 (10 mg/kg, i.p., 1 time/day) for 2 weeks. At 2 weeks after SCI, we injected AAV1 particles expressing tdTomato into the left sciatic nerve to transduce dorsal column axons originating from L3-5 DRG. We then traced the vasculature at 28 days after SCI by filling the blood vessel lumen with a fluorescent gel perfusate. Three-dimensional imaging of the cleared spinal cord showed that PDGFrβ inhibition blunted regeneration in SCI mice-administered PDGF-BB (Figures 4H and 4I), thereby confirming that PDGF-BB/PDGFrβ interaction is necessary to sponsor axon regeneration after SCI. Three-dimensional imaging of the vasculature at the lesion site showed disorganized vessel structures in SCI mice-administered PDGF-BB/PDGFrβ inhibitors (Figure 4J).

Taken together, these results indicate that adult pericytes can be programmed in vivo to sponsor axon regeneration after injury to the adult mammalian spinal cord.

In vivo conversion of adult pericytes via PDGF-BB creates favorable conditions for SCI repair

As a result of SCI, astrocytes surrounding the lesion site become reactive and undergo hypertrophy.⁵⁶^,⁵⁷ Reactive astrogliosis is part of the tissue repair process,⁵⁸ but can lead to the formation of a dense astrocyte border that produces growth inhibitory molecules such as CSPG, which curtail axon regeneration beyond the lesion site following SCI.⁵⁹^,⁶⁰ Our results indicate that would-be scar-forming astrocytes in mice receiving PDGF-BB were loosely entwined and had reduced CSPG deposition compared with control cells (Figures 5A and 5B), thus facilitating axon regeneration into and beyond the lesion site. As microglia coordinate the cellular response at the lesion site in either a pathological or protective fashion,⁶¹^,⁶² we tested whether localized delivery of PDGF-BB also impacts the behavior of myeloid cells. To test this, we subjected NG2-eGFP mice to a T12 SCI. On day 7, we injected vehicle and PDGF-BB at the lesion site and, 3 days later, dissected the lesion site and subjected it to cell sorting (Figures 5C and S6). The total number of CD45⁺ myeloid cells present at the lesion site was comparable in all experimental conditions (Figure 5D). Whereas the percentage of neutrophils and microglia was comparable, we found an increase in the percentage of macrophages in mice-administered PDGF-BB (Figure 5D). This may not be surprising as inflammatory signals and macrophages are essential for pericyte-mediated angiogenesis⁶³ and blood vessel formation to potentially support nerve regeneration,⁶⁴ respectively. A more detailed characterization of the different macrophage populations did not identify a specific enrichment other than a non-significant downtrend in the percentage of CD14⁺ macrophages (Figure 5E). In contrast, we found a decrease in the percentage of CD14⁺ microglia at the lesion site of mice-administered PDGF-BB (Figure 5E). Given that CD14 acts as a key organizer in shaping microglia response after tissue damage,⁶⁵ a decrease in CD14 expression in microglia could be supportive of decreased proinflammatory microglia leading to diminished production of inflammatory chemokines and cytokines. In turn, we measured the expression of classical chemokines and cytokines associated with SCI pathology 3 days after vehicle and PDGF-BB administration. Our multiplex data confirmed that mice-administered PDGF-BB had decreased expression of TNF-α, IL-1β, CXCL2, and IL-6 at the lesion site (Figure 5F).

PDGF-BB localized delivery at the lesion site positively affects the physical and chemical nature of the lesion environment

(A) Representative fluorescence images of astrocyte reactivity 4 weeks after SCI. The asterisk indicates the lesion epicenter (R, rostral; C, caudal). Scale bar, 200 μm. (B) Quantification of (A). Mean and SEM (unpaired two-tailed Student’s t test ∗p < 0.05, ∗∗p < 0.01, vehicle n = 4 and PDGF-BB n = 4 biological replicates). (C) Schematic of T12 SCI and experimental timeline for flow cytometry. (D) Flow cytometry shows no change in the total number of myeloid cells and the percentage of neutrophils and microglia 3 days after vehicle and PDGF-BB administration at the lesion site. In contrast, the number of macrophages increased in mice-administered PDGF-BB. All samples were collected 10 days after SCI. Mean and SEM (one-way ANOVA ∗∗p < 0.01; ns, not significant, SCI n = 5, SCI/vehicle n = 5 and SCI/PDGF-BB n = 5 biological replicates). (E) Flow cytometry shows detailed characterization of Arg-, MRC-, and CD14-positive macrophage and microglia populations 3 days after vehicle and PDGF-BB administration at the lesion site. All samples were collected 10 days after SCI. Mean and SEM (one-way ANOVA ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant, SCI n = 5, SCI/vehicle n = 5 and SCI/PDGF-BB n = 5 biological replicates). (F) Multiplex analysis shows expression of inflammatory chemokines and cytokines 3 days after vehicle and PDGF-BB administration at the lesion site. All samples were collected 10 days after SCI. Mean and SEM (unpaired two-tailed Student’s t test ∗p < 0.05; ns, not significant, SCI/vehicle n = 3–4 and SCI/PDGF-BB n = 5 biological replicates).

Altogether, these results indicate that PDGF-BB localized delivery at the lesion site positively affects the physical and chemical nature of the lesion environment thereby creating more favorable conditions for SCI repair.

PDGF-BB administration leads to changes in pericyte gene transcription and cellular crosstalk at the injury site without impacting pericyte identity

To gain insight into the pericyte transcriptional signature, the pericyte trajectory and cell-cell interaction at the lesion site associated with PDGF-BB administration after SCI, naive and injured spinal cords from NG2-eGFP mice were subjected to single-cell RNA sequencing (scRNA-seq). RNA was barcoded from 37,627 single cells isolated from fixed spinal cords pooled from sham and SCI mice at 3, 7, and 14 days after vehicle or PDGF-BB administration (Figure 6A). Uniform Manifold Approximation and Projection (UMAP) for dimension reduction⁶⁶ clustering and annotation based on cellular markers identified 10 cell clusters including pericytes/ECs (3.68%), fibroblasts (7.8%), microglia/macrophages (17.68%), erythroid cells (0.39%), Schwann cells (1.06%), OPC (3.91%), astrocytes (9.1%), ependymal cells (0.77%), neurons (29.5%), and oligodendrocytes (26.08%) (Figures S7A–S7D). To determine the impact of SCI on pericyte gene signature and cell identity, we computationally selected the pericyte/EC and fibroblasts clusters and, after further clustering, identified pericytes based on Cspg4, Kcnj8, Rgs4, and eGFP expression (Figures 6B and 6C). The identity of fibroblasts, perivascular fibroblasts, ECs, tip cells, perivascular macrophages, and vascular-associated (V-) oligodendrocytes was confirmed based on annotated marker genes (Figure 6C). However, the identity of cluster 4 was not defined. Pcsk1n, Atp1b1, Spock2, and Syp were the top four genes expressed in cluster 4. Pericytes were present at all time points (Figure 6D). Interestingly, pericyte and perivascular fibroblast abundance showed opposing complementary profiles in vehicle/SCI but not in the PDGF-BB/SCI group (Figure 6E).

Injury- and PDGF-BB-dependent transcriptional changes in pericytes revealed by scRNA-seq

(A) Schematic of T12 SCI and experimental timeline. (B) UMAP plot showing eight cell types after reclustering the pericyte/EC and fibroblasts clusters from the original dataset (Figure S7A). Each dot represents a single cell. (C) Dot plot showing cluster identity based on annotated marker genes. Color scale encodes the average gene expression after scaling. Gray dots show clusters with expression below the mean expression across the dataset in (B). (D) Proportions of cell clusters in (B) at each time point and experimental condition. (E) Fold change in the abundance of pericytes and perivascular fibroblasts at each time point following vehicle and PDGF-BB administration. (F) Volcano plot showing upregulated genes (orange) and downregulated genes (blue) in pericytes after SCI. (G) GO analysis of DEGs in (F). (H) Volcano plot showing upregulated genes (orange) and downregulated genes (blue) in pericytes after PDGF-BB/SCI. (I) GO analysis of DEGs in (H). (J) Venn diagram showing overlap between DEGs in (F) and (H). (K) GO analysis of 26 of the downregulated genes in PDGF-BB/SCI that showed opposite regulation in vehicle/SCI. (L and M) Venn diagrams showing overlap between DEGs in PDGF-BB/SCI at all time points.

The main effect of SCI was to decrease gene expression in pericytes as shown by the 2,195 genes (20.39% of the total) that were downregulated after SCI as opposed to the 501 genes (4.65% of the total) that were upregulated (Figure 6F). The biological processes associated with the SCI-dependent decrease in gene expression include small GTPase-mediated signal transduction, regulation of supramolecular fiber organization, and vesicle-mediated transport (Figure 6G). When looking at the PDGF-BB/SCI-dependent pericyte signature, 157 genes were downregulated (1.48% of the total) and 35 were upregulated (0.33% of the total) (Figure 6H). The largest effect on decreased gene transcription was found at 7 days with 200 downregulated genes (2.04% of the total). The biological processes associated with the PDGF-BB/SCI-dependent suppression of gene expression include response to metal ion, regulation of angiogenesis and vasculature development, response to hypoxia and reactive oxygen species (Figure 6I). Functional terms associated with increased gene expression in pericytes originating from SCI mice-administered PDGF-BB include ECM organization, extracellular structure organization, actin-mediated cell contraction and collagen-containing ECM (Figure 6I). Interestingly, 26 of the downregulated genes in PDGF-BB/SCI showed opposite regulation in vehicle/SCI (Figure 6J). These genes are involved in ameboid cell migration and regulate angiogenesis, vasculature development, and transport across plasma membrane (Figure 6K). To visualize consistent gene signatures between 3, 7, and 14 days after PDGF-BB administration, differentially expressed genes (DEGs) were displayed using Venn diagrams (Figures 6L and 6M). Among the downregulated genes, Hbb-bs, Slc7a5, and Hba-a2 were present at all time points. Hbb-bs and Hba-a2 encode for the hemoglobin subunits β and α, respectively. The Slc7a5 gene encodes for a sodium-independent transporter for essential amino acids across barrier structures. Given that pericytes play a vital role in maintaining barrier function and can regulate the clearance and phagocytosis of cellular debris including damaged red blood cells and hemoglobin breakdown products such as free iron may explain the association of hemoglobin genes with pericytes. Importantly, Fn1 expression in pericytes exposed to PDGF-BB decreased at 7 days with a −0.81 average log2 fold change (Table S1), confirming our in vitro results. Of the upregulated genes, none of the genes were consistently present at all time points. Cavin 1 was the only upregulated gene shared between 3 and 7 days (Figure 6M). Cavin1 encodes for the caveolae-associated protein 1, which plays a crucial role in the formation and maintenance of caveolae, small invaginations of the plasma membrane important for a variety of cellular processes including endocytosis, lipid metabolism, signal transduction, and barrier properties. The upregulated genes shared between 3 and 14 days include Col1a1, Col1a2, Acta2, Col3a1, Col8a1, Sema3g, and Cd74 (Figure 6M). CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor that plays a role in signaling pathways related to inflammation, tissue repair and angiogenesis, and therefore pericyte behavior after SCI. The induction of Col1a1, Col1a2, and Acta2 expression and decreased expression of the pericyte markers Atp13a5 may be associated with the conversion of pericytes to fibroblasts or myofibroblasts as suggested by others.⁴⁵^,⁶⁷^,⁶⁸ To study the pericyte trajectory following SCI and determine the extent to which PDGF-BB exposure impacts pericyte identity, we performed pseudotime analysis. After dimensional reduction, pericytes spatially segregated from fibroblasts and perivascular fibroblasts (Figure 7A). Given that two pericyte subclusters (e.g., 5 and 12) were identified, a starting point in naive was defined within the pericyte cluster with the highest Atp13a5, Cspg4, Kcnj8, and Pdgfrb expression (e.g., cluster 12) (Figure 7B). Pseudotime analysis identified only one pericyte trajectory following SCI and PDGF-BB administration that merged into pericyte subcluster no. 5 characterized by a rapid decrease in the expression of the classical pericyte markers Cspg4, Kcnj8, Pdgfrb, and Rgs4 and a gradual induction of an angiogenic signature with increased expression of Cldn5, Col1a2, Fn1, Slc2a1, and Vwa1 (Figures 7C and 7D). When comparing pericyte subclusters 5 and 12, an increase in gene expression was found in pericyte subcluster 5 as shown by the 2,744 genes (987 or 8.3% of the total with a log2 fold change >1) that were upregulated, as opposed to the 2,529 genes (287 or 2.4% of the total with a log2 fold change <−1) that were downregulated (Figure 7E; Table S2). The biological processes associated with the increase in gene expression include small GTPase-mediated signal transduction, regulation of cellular catabolic process, regulation of supramolecular fiber organization, and macroautophagy (Figure 7F; Table S2).

Pericytes retain cellular identity after SCI and PDGF-BB administration but acquire a proangiogenic signature

(A) UMAP plot shows selected cell types pooled from all experimental groups in Figure 6B after dimensional reduction in Monocle3. Pericytes segregate from other cell clusters. Within the pericyte cluster, subclusters 5 and 12 were identified and differentially colored to allow visualization. (B) Violin plots show expression of the pericyte markers *Cspg4*, *Pdgfrb*, *Kcnj8*, and *Atp13a5* in pericyte subclusters. (C) Pseudotime trajectory in pericytes using Monocle3. (D) Expression profiles of *Cspg4*, *Kcnj8*, *Pdgfrb*, *Rgs4*, *Cldn5*, *Col1a1*, *Col1a2*, *Fn1*, *Slc2a1*, and *Vwa1* along pseudotime. (E) Volcano plot showing upregulated genes (orange) and downregulated genes (blue) in pericyte subcluster 5 compared with subcluster 12. (F) GO analysis of upregulated genes in (E).

Our data showed that PDGF-BB administration positively impacted the lesion environment after SCI. Accordingly, we interrogated our scRNA-seq dataset to determine the extent to which PDGF-BB localized administration influences the crosstalk between pericytes and other cells known to play a role in SCI pathophysiology. We computationally selected the pericyte/EC, fibroblast, astrocyte, OPC, oligodendrocyte, and microglia/macrophage clusters from the original dataset (Figure S8A). CellChat⁶⁹ analysis indicated SCI negatively impacted cell-cell communication as shown by the decrease in the number and strength of ligand-receptor interactions (Figures S8B and S8C). However, cell interactions between pericytes, astrocytes, and fibroblasts increased at 3 and 7 days (e.g., 10 and 14 days after SCI) (Figure S8C). This may not be surprising as formation and consolidation of the glial scar spans the first 2 weeks after SCI in adult mice. Pericyte to astrocyte and fibroblast communication was suppressed at 3 and 7 days after PDGF-BB administration (Figure S8C). We then investigated the SCI- and PDGF-BB-dependent alterations in the major signaling pathways present within the pericyte/EC cluster. The pathways subjected to the most drastic decrease following SCI were those associated with ECM (e.g., laminin, collagen) and cell-cell adhesion (e.g., CNTN, NOTCH, NRXN, ADGRE, ADGRL) signaling (Figure S8D). Interestingly, the pericyte-astrocyte and fibroblast-astrocyte interactions based on fibronectin signaling increased after SCI but not in SCI mice-administered PDGF-BB (Figure S8E).

When looking at individual ligand-receptor pairs, we found that cell-cell interactions based on Fn1 expressed in pericytes/EC and fibroblasts and its receptors expressed in astrocytes (e.g., Sdc4, Cd44, Itgav+Itgb8, Itgav+Itgb1, Itga5+Itgb1, and Itga3+Itgb1) decreased 7 days after PDGF-BB administration (Figure S8F). While pericytes, ECs, and fibroblasts all express Fn1, EC do not express Pdgfra or Pdgfrb (Figure S8G), suggesting that PDGF-BB-dependent decrease in cell-cell interaction based on fibronectin signaling may be caused by changes in pericyte and fibroblast but not EC behavior.

These results demonstrate that PDGF-BB administration at the lesion site is associated with transcriptional changes in pericytes and alteration of cellular crosstalk that may positively impact functional repair after SCI. Our analysis of cell trajectory after SCI and PDGF-BB administration indicates that pericytes retain cellular identity but acquire a pro-angiogenic signature, in agreement with others.⁷⁰

Mice-injected PDGF-BB recover hindlimb function after thoracic SCI

Lastly, we examined the extent to which PDGF-BB administration at the lesion site promotes functional reorganization of neural circuits and recovery of hindlimb function after SCI. To this end, we subjected adult mice to a T12 SCI that severed ascending and descending pathways on one side (Figure 8A). On day 7, we injected vehicle and PDGF-BB at the lesion site. To probe functional connectivity in the injured spinal cord, we recorded the evoked local field potentials (LFPs) upon electrical stimulation of sensory axons of anesthetized mice 1 month after SCI. The multichannel recording electrodes were inserted in two locations: caudal and rostral to the site of injury (Figures S8A–S8C). In both groups, electrical stimulation evoked a large response caudal to the lesion, serving as a positive control for the stimulation parameters. On the rostral side, instead, larger LFPs were recorded in SCI mice-administered PDGF-BB (Figures 8B and 8C), indicating that PDGF-BB creates favorable conditions for functional connectivity within the injured spinal cord. Kinematic analysis showed that mice-administered PDGF-BB substantially recovered step height during ground locomotion 28 days after SCI (Figures 8D and 8E). This may be due to the different angular displacements of the hip and knee joints in SCI mice receiving PDGF-BB (Figure 8D), underscoring changes in the recruitment of muscle synergies. Of note, the ankle joint angles were comparable between the two conditions (Figure 8D). Whereas SCI mice-administered vehicle developed chronic pain, PDGF-BB ameliorated mechanical hypersensitivity (Figure 8F). Collectively, these data indicate that PDGF-BB administration at the lesion site promotes functional reorganization of neural circuits and recovery of hindlimb function after SCI.

PDGF-BB localized delivery at the lesion site promotes functional connectivity and recovery of hindlimb function after thoracic SCI

(A) Schematic of T12 SCI and experimental timeline. (B) Representative traces from *in vivo* recording of stimulus-evoked LFPs using a 32-channel electrode array. The color code represents LFPs recorded from four electrode shanks. (C) Heatmap (blue to red) of average LFP amplitude from SCI mice receiving vehicle and PDGF-BB to visualize connectivity changes of regenerating sensory axons rostral to the site of injury. Each box represents averaged data from a single channel, each column a single electrode shank (mixed model with random subject effect to account for within-subject correlation arising from repeated measurements ∗∗∗p < 0.001, vehicle n = 5 and PDGF-BB n = 6 biological replicates). (D) Kinematics of displacement during the step cycle 28 days after SCI. Left panels show a representative set of stick figures of left hindlimbs. Arrow indicates the direction of movement. Scale bar, 25 mm. Average angular displacements of joint angles of the left hindlimb were normalized based on step phase (right panels). Shaded contour indicates SEM (vehicle n = 6 and PDGF-BB n = 6 biological replicates). (E) Step height during swing phase of ground locomotion (one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ns, not significant; naive n = 8, vehicle/SCI n = 6 and PDGF-BB/SCI n = 6 biological replicates). (F) Von Frey hair threshold of the plantar surface of the left hind paw examined 28 days after SCI (one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ns, not significant; naive n = 8; vehicle/SCI n = 6 and PDGF-BB/SCI n = 6 biological replicates).

Discussion

Evidence suggests that pericytes play a role in the pathophysiology of CNS injury and disease.¹⁵^,⁷¹^,⁷²^,⁷³^,⁷⁴ Wound-associated pericytes also impair capillary blood flow and functional recovery at chronic stages of SCI. Although experimental data indicate that partial pericyte ablation enables some axon regeneration and restoration of function after SCI in adult mice,¹⁴ the extent to which pericyte manipulation alone may be sufficient to create more favorable conditions for SCI repair has never been explored. This study provides evidence that pericytes can be converted into a growth-permissive substrate at the lesion site, enabling robust regeneration after SCI. In addition, the same molecular strategy enabling pericyte conversion positively impacts the physical and chemical nature of the lesion environment. Given the simplicity of our delivery approach, our study identifies a treatment strategy that is readily translational across different species, CNS injury, and neurodegenerative conditions.

Lack of neurological recovery after SCI is associated with long-term impairment of sensation and locomotion as well as bladder and bowel function among many other complications, contributing to deterioration of quality of life in SCI individuals. Axon sprouting and regeneration can restore connectivity within the injured spinal cord. Moreover, revascularization and normalization of vascular function are necessary to diffuse nutrients and metabolites needed for neuronal growth and function and to eliminate waste material at acute and chronic stages after SCI. In mammals, however, adult neurons have limited capacity to sprout and even less to regenerate,⁷⁵ often failing to restore connectivity across the lesion. In addition, detrimental changes in vasculature structure and pericyte function contribute to a lack of physiological recovery after injury and disease.⁷¹^,⁷⁴^,⁷⁶ The development of genetic tools that specifically label pericytes with durable fluorescent markers²¹ together with vascular tracing methods,²⁵^,²⁶ tissue clearing, three-dimensional microscopy, and volumetric analysis⁷⁷^,⁷⁸^,⁷⁹ allowed us to study changes in vascular architecture and pericyte coverage over the course of the days, weeks, and months after SCI. Our data indicate that the injured spinal cord fails to completely restore normal vasculature function at acute and chronic time points, likely contributing to poor functional recovery in adulthood. In contrast, early perinatal injuries to the murine spinal cord enable full restoration of vasculature architecture and robust regeneration in the absence of fibrotic scarring.³⁴

After tissue injury in the adult, pericytes detach from the mature vasculature and migrate to the lesion epicenter during capillary sprouting,⁷⁰ contributing to the deposition of the growth inhibitory ECM.¹⁵^,⁴⁵ Our data show pericyte detachment and reduced claudin 5 expression along newly formed microvessels at the lesion epicenter, indicating compromised barrier function early after SCI. An increase in vascular permeability with consequent leakage of larger molecules and cells into the spinal cord results in edema, inflammation, and progression of acute SCI pathophysiology.⁸⁰ Pro- and antiangiogenic pathways are tightly regulated during embryonic vasculature development. An imbalance of such signaling after injury and disease is often associated with uncontrolled vessel growth that results in the formation of immature microvascular networks with profound structural and functional abnormalities.⁸¹ Whether normalization of pro- and antiangiogenic signaling may be required to promote the formation of structurally sound and functionally normal vasculature networks after SCI remains to be tested.

Both OPC and pericytes express NG2, a potent axon growth inhibitory proteoglycan upregulated after SCI.⁸² Whereas OPC preferentially localize within the glial scar and lesion penumbra,¹⁶^,⁸³ pericytes migrate into the fibrotic core after SCI.¹⁵^,¹⁶ In addition to NG2, our data indicate that adult pericytes also express large quantities of laminin and fibronectin, also known as positive substrates for axon growth and regeneration. Under normal physiological conditions, however, pericytes isolated from the adult mouse spinal cord cause detrimental structural and functional changes in adult DRG neurons associated with diminished axon growth. Filous et al. reached a similar conclusion after testing axon outgrowth of DRG neurons plated on NG2-expressing cells.⁸³ Indeed, exposure to CSPG, laminin, and fibronectin can cause over adhesion leading to minimal neurite outgrowth.³⁷ Our data indicate that incubation with a membrane-permeable peptide that binds to a CSPG receptor rescues, at least in part, axon growth defects of adult DRG neurons growing on a pericyte monolayer.

PDGF-BB plays a key role during the development of the vasculature system. Upon binding to PDGFrβ expressed on pericytes, PDGF-BB stimulates the proliferation and migration of pericytes and their recruitment to growing vessels. Pericytes are highly plastic and can respond to PDGF-BB exposure by secreting trophic factors and regenerative molecules needed to repair and restore neuron structure and function after injury and disease.⁴⁰ Accordingly, we tested whether PDGF-BB administration would be sufficient to convert adult pericytes into a permissive substrate for axon growth and regeneration. To our surprise, PDGF-BB stimulation fully rescues axon growth defects of adult DRG neurons plated on a pericyte monolayer originating from the adult mouse spinal cord as well as the adult human brain. Similarly, our in vivo data prove that one single administration of PDGF-BB at the lesion site at a clinically relevant time point (e.g., 7 days after SCI) promotes robust regeneration of sensory ascending dorsal column axons in adult mice. Our three-dimensional imaging clearly indicates that axons regenerate into and beyond the lesion site by “riding” pericytes-decorated cellular structures that formed in response to PDGF-BB. Independent studies provide further evidence that vascular bridging across the lesion site is crucial for axon regeneration in the central and peripheral nervous systems.¹⁹^,⁸⁴

Our structural and functional characterization using gain- and loss-of-function experiments indicates that PDGF-BB supports the reorganization of fibronectin matrix and the depletion of NG2 and collagen I expression in adult pericytes and that integrin signaling is necessary for axonal elongation on their associated fibronectin matrix. Along this line, a large body of work underscores the importance of integrin signaling for axon growth and regeneration.⁸⁵ The fact that fibronectin acts as a permissive molecular substrate for axon growth is well established in the CNS injury field.⁸⁶ In the absence of collagen I deposition, injury to the spinal cord in neonatal mice leads to the formation of fibronectin bridges by microglia that promote the growth of long projecting axons across the lesion site.³⁴ Furthermore, another recent study showed that SCI in zebrafish leads to the recruitment of PDGFrβ myoseptal and perivascular cells to the site of injury, where they contribute to the deposition of a permissive ECM that promotes axon regeneration and functional recovery.⁸

PDGF-BB has been shown to stimulate the conversion of pericytes to fibroblasts in the tumor microenvironment.⁸⁷ Several studies suggest that perivascular cells, including fibroblasts and pericytes, give rise to stromal fibroblasts,⁸⁸^,⁸⁹ the main cellular source of fibrotic scarring. However, a comprehensive transcriptional analysis of virtually all cells of the brain vasculature indicates that perivascular fibroblasts and pericytes show a different gene signature.⁹⁰ As such, it is unlikely that the two cell types may be interchangeable in terms of contribution to fibrotic scarring.⁹¹ A recent study showed that, after stroke in mice, bona fide pericytes activate a transient angiogenic profile different from the fibrotic program coordinated by perivascular fibroblasts.⁷⁰ Controversy also exists about the number of pericyte subtypes in the adult CNS. Betsholtz and colleagues identified only one type of pericyte in the brain.⁹⁰ Our single-cell transcriptomic analysis of the injured spinal cord identified only one pericyte trajectory associated with SCI and PDGF-BB administration that converged into a pericyte subcluster characterized by a rapid decrease in the expression of classical pericyte markers and the induction of a pro-angiogenic signature. Understanding the molecular mechanisms controlling pericyte and fibroblast behaviors after CNS injury and disease will be an important direction for future investigation.

Intrinsic neuronal and extrinsic non-neuronal mechanisms must be targeted simultaneously to promote successful regeneration and SCI repair.⁹² Not only does one single administration of PDGF-BB promote axon regeneration across the lesion site, but it also dampens astrogliosis and microglia reactivity, leading to reduced expression of classical chemokines and cytokines associated with SCI pathology. Quantitative inference and analysis of cell-cell communication at the lesion site allowed us to identify fibronectin, collagen, and laminin as major signaling inputs that enable pericytes-coordinated cell behavior during PDGF-BB-dependent adaptive CNS repair.

Axon regeneration represents a critical step in rebuilding neuronal circuits after SCI. Regenerating sensory axons in SCI mice-administered PDGF-BB integrate into spinal circuits as shown by the large LFPs recorded rostral to the site of injury. Nonetheless, a comprehensive topographic mapping of neuronal connectivity within sensory and motor domains and the identification of postsynaptic targets will be required to dissect circuit rewiring and the functional implication of adaptive reorganization of spinal circuits in SCI mice-administered PDGF-BB. In this context, several studies demonstrated that specific classes of spinal interneurons are involved in neurological recovery after SCI.⁹³ We show that localized delivery of PDGF-BB at the lesion site promotes recovery of hindlimb function 4 weeks after thoracic SCI. Increased step height during ground locomotion and amelioration of mechanical hypersensitivity in SCI mice receiving PDGF-BB underscores adaptive rather than maladaptive reorganization of spinal circuits. In-depth characterization of flexor hindlimb muscle electromyographic activity (e.g., tibialis anterior) during stance and swing phases will be required to further our understanding of muscle synergies driving functional recovery after PDGF-BB administration. Whether our treatment strategy may synergize with other promising interventions targeting intrinsic neuronal mechanisms of axon growth and regeneration or neuromodulation remains to be tested. Moreover, the extent to which descending motor pathways effectively respond to our treatment strategy is awaiting further investigation.

These observations highlight the strong potential for pericyte programming as a promising treatment strategy for SCI repair and other neurodegenerative conditions.

Materials and methods

Mice

All animal experiments were performed following protocols approved by the Institutional Animal Care and Use Committee at The Ohio State University. Adult (7–9 weeks old) female and male FVB mice (stock no. 001800, RRID: IMSR_JAX:001800) were used for all experiments except those specifying NG2-eGFP,²¹ NG2-CreER,⁹⁴ and Ai9 (RCL-tdTomato).⁹⁵ NG2-eGFP (stock no. 022735, RRID: IMSR_JAX:022735), NG2-CreER (stock no. 008538, RRID: IMSR_JAX:008538), and Ai9 (RCL-tdTomato) (stock no. 007909; RRID: IMSR_JAX:007909) were purchased from The Jackson Laboratory. Mice were kept in an animal house in individually ventilated housing on a 12:12 h light-dark cycle with ad libitum access to food and water. Ambient temperature was maintained at 21°C–22°C and humidity at 50%. Mice were randomly assigned to experimental groups. Experimenters were blind to group assignment and experimental conditions.

Antibodies

The following antibodies were used: goat polyclonal anti-CD13 (AF2335, RRID: AB_22272 88, 1:200 immunohistochemistry [IHC], R&D Systems), rat anti-CD31 (550274, RRID: AB_39357 1, 1:200 IHC, BD Biosciences), rabbit polyclonal anti-claudin 5 (34-1600, RRID: AB_25331 57, 1:200 IHC, Invitrogen), rabbit polyclonal anti-COL1A1 (PA5-29569, RRID: AB_25470 45, 1:1,000 WB, Invitrogen), mouse monoclonal anti-CS-56 (C8035, RRID: AB_47687 9, 1:400 IHC, Sigma), goat polyclonal anti-desmin (AF3844-SP, RRID: AB_20924 19, 1:200 IHC, 1:1,000 WB, R&D Systems), rabbit polyclonal anti-fibronectin (F3648, RRID: AB_47697 6, 1:500 ICC, 1:1,000 WB, Sigma), rabbit monoclonal anti-GAPDH (2118, RRID: AB_561053, 1:1,000 WB, Cell Signaling Technology), mouse monoclonal anti-GFAP (G3893, RRID: AB_47701 0, 1:1,000 IHC, Sigma), rabbit polyclonal anti-GFAP (Z0334, RRID: AB_10013 382, 1:1,000 IHC, Dako), rabbit polyclonal anti-laminin (L9393, RRID: AB_47716 3, 1:1,000 ICC, Sigma), mouse monoclonal anti-NG2 (MAB5384, RRID: AB_17764 6, 1:500 IHC, 1:1,000 WB, Millipore), goat polyclonal anti-PDGFrα (AF1062, RRID: AB_22368 97, 1:500 IHC, R&D Systems), rabbit anti-PDGFrβ (SAB4502148, RRID: AB_10744 524, 1:500 ICC/IHC, 1:1,000 WB, Sigma), mouse monoclonal anti-R-PTPσ (MM-0020-P, RRID: AB_18083 57, 1:1,000 ICC, Medimabs), rabbit anti-β-III tubulin (Tuj1) (T2200, RRID: AB_26213 3, 1:1,000 ICC, Sigma), mouse anti-β-III tubulin (Tuj1) (801201, RRID: AB_23137 73, 1:1,000 ICC, BioLegend), and mouse monoclonal anti-vimentin (V2258, RRID: AB_26185 6, 1:500 ICC, Sigma).

The following antibodies were used for flow cytometry: arginase 1-APC (AlexF5, 17-3697-82, 1:25, Invitrogen), CD101-PE-Cy7 (Moushi101, 25-1011, 1:100, eBioscience), CD115-BUV395 (T38-320, 743642, RRID: AB_27416 52, 1:100, BD Biosciences), CD11b-BUV786 (M1/70, 740861, RRID: AB_27405 14, 1:500, BD Biosciences), CD11c-BUV737 (HL3, 612797, RRID: AB_28701 24, 1:100, BD Biosciences), CD124-BV421 (mIL4r-M1, 564086, RRID: AB_27385 84, 1:100, BD Biosciences), CD14-BV650 (rmC5-3, 740486, RRID: AB_27402 09, 1:100, BD Biosciences), CD16/32 (2.4G2, 14-0161, 1:200, eBioscience), CD206-AR700 (MR6F3, 56-2061-82, 1:50, Invitrogen), CD3e-PE-Cy5 (145-2C11, 553065, RRID: AB_39459 8, 1:100, BD Biosciences), CD45-BUV805 (30-F11, 752415, RRID: AB_29174 29, 1:100, BD Biosciences), CD45R/B220-PerCP-Cy5.5 (RA3-6B2, 552771, RRID: AB_39445 7, 1:100, BD Biosciences), CD84-BV711 (mCD84.7.rMAb, 749563, RRID: AB_28738 91, 1:100, BD Biosciences), F4/80-PE-CF594 (T45-2342, 565613, RRID: AB_27347 70, 1:100, BD Biosciences), I-A/I/E-BUV496 (M5/114.15.2, 750281, RRID_AB_28744 72, 1:100, BD Biosciences), goat CX3CR1-PE polyAb (FAB5825P, 1:100, R&D Systems), and Ly6G-BUV563 (1A8, 612921, RRID: AB_28702 06, 1:50, BD Biosciences).

Spinal cord injury

Adult NG2-eGFP mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight) and a T12 laminectomy was performed. The spinal cord was crushed with modified forceps (no. 5, 11254-20, FST). The forceps were positioned to completely sever ascending dorsal column sensory axons on the left side. For the control surgery, a laminectomy was performed without any direct manipulation of the spinal cord. In another cohort, NG2-eGFP mice were allowed to recover for 7 days before reopening the wound and injecting either vehicle (ddH₂O) or PDGF-BB (10 ng/μL, SRP3229, Sigma) into the lesion site (volume, 2 μL; depth, 500 μm; speed, 200 nL/min). The same experimental procedure was repeated in NG2-CreER/Ai9 (RCL-tdTomato) mice (C57Bl6/J background) to confirm results in a common genetic background and to determine pericyte density following PDGF-BB stimulation. NG2-CreER/Ai9 (RCL-tdTomato) mice were subjected to T12 SCI. At 7 days after SCI, vehicle and PDGF-BB were injected directly at the lesion site as described above. Tamoxifen (100 mg/kg of body weight, T5648, Sigma) or the corresponding volume of corn oil (C0136, Spectrum Chemical) was administered (i.p. injections, 2 times/day for 4 days) beginning 1 h after vehicle and PDGF-BB administration. To determine the extent to which PDGF-BB/PDGFrβ interaction is necessary to sponsor axon regeneration after SCI, an independent cohort of adult FVB mice was subjected to a T12 SCI as described above. At 7 days after SCI, PDGF-BB was administered at the lesion site and, beginning 1 h later mice were injected the PDGFrβ inhibitors SU16F (10 mg/kg, i.p., 1 time/day, Tocris) and CP673451 (10 mg/kg, i.p., 1 time/day, Tocris) for 2 weeks. DMSO served as vehicle.

To trace sensory dorsal column axons, 1.5 μL of AVV1 particles expressing either tdTomato (>1 × 10¹² vg/mL, 105554-AAV1, Addgene) or eGFP (8 × 10¹² vg/mL, 105530-AAV1, Addgene) were injected into the left sciatic nerve 2 weeks after SCI. At 28 days after SCI, mice were transcardially perfused and tissue dissected for further histological analysis or cleared for three-dimensional imaging. If necessary, the vasculature was traced as described below.

Vasculature tracing, in toto staining, three-dimensional imaging, and reconstruction

The vasculature was traced using the method we described earlier.²⁶^,⁷⁹ Briefly, adult NG2-eGFP mice were transcardially perfused with 4% paraformaldehyde (PFA) in PBS (pH 7.4). Mice were then perfused with 5 mL of 0.025% albumin-TRITC isothiocyanate bovine (A2289, Sigma) in 2% gelatin from porcine skin (G1890, Sigma). At the time of injection, the temperature of the gel solution was maintained at 45°C. After clamping the heart, mice were placed on ice to lower their body temperature and allow for gel formation. In another cohort of adult NG2-eGFP mice, the spinal cord was fixed, dissected, and subjected to CD31 in toto staining 3 days after SCI. Specifically, a 3 mm segment of the spinal cord containing the lesion site was blocked for 12 h at room temperature (RT) with 2.5% bovine serum albumin (A3059, Sigma-Aldrich) in PBS with 0.5% Triton X-100 (T8787, Sigma) and incubated for 48 h at RT with a rat monoclonal anti-CD31 (1:100). After washing 3–4 times (3 h/each) with PBS, tissues were incubated for 24 h at RT with Alexa Fluor-conjugated secondary antibodies (1:200, Life Technologies) and finally washed 3–4 times (3 h/each) with PBS.

The spinal cords were cleared using the advanced CUBIC protocol⁷⁸ and imaged in three-dimensional using a confocal microscope (C2 plus, Nikon). Vasculature networks were reconstructed and analyzed using the visualization and analysis software Imaris (version 10.0.0, Oxford Instruments). To exclude any confounding variable originating from OPC in NG2-eGFP mice, the deposition of eGFP-positive fibrous structure from pericytes located within the lesion site was calculated using the ImageJ plugin “SNT.” We then used the Imaris software to determine the density and position of eGFP- (for NG2-eGFP mice) or tdTomato-positive (for NG2-CreER/Ai9 (RCL-tdTomato) mice) pericytes at the lesion site in relation to vasculature structures filled with albumin-TRITC or stained with an antibody directed against CD31.

Immunohistochemistry

At the end of the study, mice were transcardially perfused with 4% PFA in PBS (pH 7.4). The spinal cords were dissected, post-fixed at 4°C in 4% PFA for 1 h, and dehydrated in 30% sucrose. Tissues were then embedded in optimum cutting temperature (OCT) compound (Tissue-Tek), frozen, sectioned (20 μm thick, HM525 NX, Thermo Fisher Scientific), and mounted on slides. Slides were warmed at 37°C for 30 min and OCT was washed away with PBS. Sections were then blocked at RT with 2.5% bovine serum albumin (A3059, Sigma-Aldrich) in PBS with 0.1% Triton X-100 for 1 h and incubated overnight at 4°C with the primary antibody. After washing 3 times with PBS, sections were incubated with Alexa Fluor-conjugated secondary antibodies (1:400, Life Technologies). When necessary, sections were counterstained with DAPI (1:10,000, D9542, Sigma-Aldrich). Images were taken using a confocal (C2 plus, Nikon) or epifluorescence microscope (Axio Observer Z1, Zeiss) and linear fluorescence intensity was calculated using Fiji (version 2.0.0-rc-69/1.52p) after background subtraction. The number of biological replicates analyzed for each condition is indicated in the corresponding figure legend.

Primary pericyte culture

The spinal cords from two adult (6–7 weeks) FVB mice were rapidly dissected and placed in cold Hank’s balanced salt solution (HBSS) medium (14175103, Gibco). After removing the meninges, the spinal cords were cut into 3–5 mm segments, washed twice with sterile HBSS and dissociated in 0.125% trypsin (25200056, Gibco) for 30 min at 37°C. After incubation, fetal bovine serum (FBS) (16000044, Gibco) was added to stop digestion and the spinal cords were dissociated in 30% Percoll (17-0891-01, GE Healthcare) in MEM without Ca²⁺ and Mg²⁺ (11380037, Gibco) using a 10 mL sterile pipette. The cell suspension was then layered into 70% Percoll. After centrifugation at 2,000 rpm for 30 min at 4°C, the 30%–70% interface was removed and collected into a sterile tube, washed, and spun down at 1,000 rpm for 5 min. The cell pellet was re-suspended in DMEM/F12 (21041025, Gibco) supplemented with 10% FBS and plated at the desired density on poly-D-lysine (P6407, Sigma)-coated dishes. The medium was changed 2 h after plating to remove any dead cells. Half of the medium was replaced with fresh medium 2 times/week. In a separate set of experiments, the same protocol was used to purify pericytes from NG2-eGFP mice (stock no. 022735). In another set of experiments, vehicle (e.g., ddH₂O) and PDGF-BB (20 ng/mL) were added to the medium immediately after plating pericytes. Human brain pericytes (HMP104, Neuromics) were cultured in pericyte growth medium (PGB001, Neuromics). The culture was maintained in a humidified atmosphere containing 5% CO₂ in air at 36.5°C.

RNA isolation and RT-PCR

Total RNA was extracted from mouse cultured pericytes using the RNeasy kit (QIAGEN) and cDNA was synthesized from 0.1–2 μg of RNA using random hexamers from the SuperScript VILO cDNA synthesis kit (11754050, Thermo Fisher Scientific). In separate experiments, the same RNA extraction and cDNA synthesis protocol was applied to sorted OPC and pericytes originating from the spinal cord (thoracic/lumbar region) of adult NG2-eGFP mice. The subsequent cDNA was used in PCR reactions using Choice Taq Master Mix (cb4070-7, Denville Scientific). PCR products were run on 1.5% agarose gel. The sequences of the primers used were as follows:

Mouse Ng2 forward 5′-CAGGCCGGTCGGGTGACCTA-3′

Mouse Ng2 reverse 5′-GGGCCACGTGGAAGACACGG-3′

Mouse Pdgfrb forward 5′-ACCTGCAGAGACCTCAAAAGTAGGT-3′

Mouse Pdgfrb reverse 5′-ACCACGGTGACCTCCTGCGA-3′

Mouse Desmin forward 5′-AGCCAGCGCGTGTCCTCCTA-3′

Mouse Desmin reverse 5′-AGCGTCGGCCAGGGAGAAGT-3′

Mouse Col1a1 forward 5′-CGATGGATTCCCGTTCGAGT-3′

Mouse Col1a1 reverse 5′-CGATCTCGTTGGATCCCTGG-3′

Mouse Col1a2 forward 5′-CTGGTCCTGTTGGAAGTCGT-3′

Mouse Col1a2 reverse 5′-CAGATGCACCTGTTTCTCCA-3′

DRG neuron culture

Murine thoracic and lumbar DRG were dissected and collected in ice-cold HBSS (Gibco). The ganglia were transferred into a sterile tube, washed twice with HBSS, and incubated in Neurobasal-A medium (Gibco) containing collagenase type I (3,000 U/mL, LS004194, Worthington) at 36.5°C for 15 min, followed by 30 min with trypsin (0.25%, 25200056, Gibco). Serum was then added to stop trypsin digestion. Ganglia were dissociated by gently pipetting up and down. The cell suspension was filtered using a nylon cell strainer (70 μm, 22-363-548, Fisher Scientific) and centrifuged at 900 rpm for 5 min. Dissociated neurons were re-suspended in DMEM/F12 supplemented with 10% FBS and plated at low density on laminin (5 μg/mL, 11243217001, Roche), CSPG (10 μg/mL, CC117, Millipore), or pericyte-coated coverslips.

When needed, RGDS peptide (0.02 mg/mL, 3498, Tocris), cilengitide (2 μM, 5870, Tocris), and K34c (20 μM, 5114, Tocris) were added to the culture medium while plating the neurons on pericyte-coated dishes. In a separate set of experiments, dissociated neurons were re-suspended in human pericyte growth medium (Neuromics) and plated on human pericyte-coated coverslips. The culture was maintained in a humidified atmosphere containing 5% CO₂ in air at 36.5°C. For the siRNA gene-silencing experiment, dissociated mouse DRG neurons were plated on mouse pericyte-coated dishes 24 h after pericytes were transfected with pre-designed siRNA oligos (Silencer select pre-designed siRNA (Fn1) 4390771 ID:s66182, Silencer select pre-designed siRNA (Fn1) 4390771 ID:s66183, and Silencer select negative control siRNA 4390843, Ambion) using the polyfect transfection reagent (301105, QIAGEN). Neurons were then fixed 24 h after plating.

For co-culture experiments, mouse and human pericytes were plated at 80% confluency on poly-D-lysine-coated 13 mm glass coverslips and dissociated mouse DRG neurons were added 24 h after plating pericytes.

Immunocytochemistry

Cells (e.g., pericytes, DRG neurons, and pericyte-DRG neuron co-cultures) were fixed with 4% PFA (158127, Sigma) in PBS. Coverslips were then blocked at RT for 1 h with 2.5% BSA and 0.1% Triton X-100 in PBS and incubated at 4°C overnight with the appropriate primary antibodies. After three rinses in PBS, the coverslips were incubated with Alexa Fluor-conjugated secondary antibodies (1:400, Life Technologies) and washed in PBS before mounting them onto microscope slides. Fluorescence images were randomly taken with a confocal microscope (C2 plus, Nikon) or epifluorescence microscope (Axio Observer Z1, Zeiss). This process was repeated for at least three independent experiments. The number of cells quantified for each condition is indicated in the corresponding figure legend.

Morphometric analysis

DRG neurons were fixed with 4% PFA in PBS and stained for Tuj1 (Covance). Images were randomly taken with an epifluorescence microscope (Zeiss) and analyzed using Fiji software (NIH). This was carried out for at least three independent experiments. The number of neurons quantified for each condition is indicated in the corresponding figure legend.

Immunoblotting

Cultured pericytes were lysed 24 h after plating on ice in RIPA buffer (0.5 M Tris-HCl [pH 7.4], 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) containing phosphatase and protease (04693132001, Sigma) and phosphatase (4906845001, Sigma) inhibitors. The homogenates were then centrifuged, and the supernatant was collected. Using Bradford reagent (Bio-Rad), the protein concentration of the lysate was determined and a portion of the lysate (10 μg total protein) was then fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a 0.2 μm nitrocellulose membrane (1620122, Bio-Rad), which was stained to confirm equal loading and transfer of the samples with Ponceau S (P7170, Millipore Sigma). After blocking at RT with 5% non-fat milk (1706404XTU, Bio-Rad) in Tris-buffered saline with 0.1% Tween 20 detergent for 1 h, the membrane was probed with mouse monoclonal anti-NG2 (Millipore), rabbit polyclonal anti-PDGFrβ (Sigma), goat polyclonal anti-desmin (R&D Systems), and rabbit polyclonal anti-COL1A1 (Invitrogen). For the siRNA experiment, transfected pericytes were lysed 48 h after plating and the membrane was probed with rabbit polyclonal anti-fibronectin (Sigma). Rabbit monoclonal anti-GAPDH (Cell Signaling Technology) antibody was used as the loading control. Densitometry analysis was performed using ImageJ (NIH). After background subtraction, the area of fibronectin bands was measured and normalized to the loading control (e.g., GAPDH). Three biological replicates for each experimental condition were analyzed.

Patch-clamp recording

Whole-cell patch-clamp recordings were performed on cultured mouse DRG neurons plated on either laminin or pericyte monolayers. One coverslip was transferred at a time to a submerged recording chamber mounted on a Nikon upright FN1 microscope, and the neurons were visualized through a CMOS camera (ORCA-Flash 4.0LT, Hamamatsu). Neurons were recorded in voltage-clamp mode (Axopatch 200A, Molecular Devices) and held at −70 mV while being continuously perfused with artificial cerebrospinal fluid (ACSF) at RT. The ACSF contained: 124 mM NaCl, 3 mM KCL, 1.25 mM NaH₂PO₄, 2 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mM glucose (gassed with 95% O₂/5% CO₂). The perfusion rate was set at 1.5 mL/min. To record the minimum current to trigger an action potential, the recording was switched to the current-clamp mode and we maintained the resting potential at −70 mV by adjusting the baseline current injection. Patch pipettes were pulled using a vertical pipette puller (Model PC-100, Narishige) with a resistance of 3.0–4.0 MΩ. Pipette solution contained: 125 mM K-gluconate, 4 mM Na₂-ATP, 2 mM MgCl₂, 10 mM HEPES, 20 mM KCl, 3 mM NaCl, 0.5 mM EGTA [pH 7.3], 280–290 mOsm). We corrected the liquid junction potential by adjusting the zero-current position to −10 mV before the sealing procedure. The recordings were acquired using WinWCP software (Strathclyde Electrophysiology Software, University of Strathclyde, Glasgow). The sampling rate was set at 20 kHz (NI USB-6229, National Instruments) and all recordings were low-pass filtered at 5 kHz. Data analysis was performed using Igor Pro software (WaveMetrics). For voltage-clamp recording in Figure 2D, an offline leak subtraction procedure was performed using a P/-4 protocol. A series of voltage steps from −90 to +40 mV with 10 mV increments were applied. The traces show both inward sodium and outward potassium currents. To generate the I-V curve in Figure 2F, the peak inward current amplitude was used.

To calculate the passive properties of recorded neurons, a negative voltage step pulse (−5 mV, 400 ms duration) was applied 5 times every 5 s. We then calculated the series resistance (Rs), cell input resistance (Rm), and cell capacitance (Cm) as previously described⁹⁶ using the average response. The average Rs from all recorded neurons was 7.02 ± 0.4 MΩ (n = 23 across both experimental groups).

Calcium imaging

The membrane-permeable calcium indicator Fluo-4 AM (F14201, Invitrogen) was added to the cell culture medium (final concentration: 5 μM) and kept at 36.5°C in the cell culture incubator for 45–60 min. The coverslip was then transferred to a submerged imaging chamber mounted on a Nikon upright confocal microscope. Plated neurons were then washed and bathed with gassed (95% O₂/5% CO₂) ACSF at RT. Spontaneous calcium signals were acquired (interval, 3 s; duration, 5 min) in frame-scan mode using a 60× water-dipping objective (NA 1.0, Nikon) mounted on a laser-scanning confocal system (C2 plus, Nikon) with a 525/40 bandpass filter. The data analysis was performed with Fiji (NIH) and Igor Pro software (Wavemetrics). Calcium profiles for each individual neuron analyzed were then normalized to baseline level and displayed as ΔF/F.

Cryo-EM imaging

Pericytes were grown on EM grids (01882G-F, Ted Pella) coated with PDL (0.1 mg/mL, P6407, Sigma). At 24 h after plating, the grids were flash-frozen using a manual cryo-plunger. The frozen grids were screened using Thermo Scientific Glacios Cryo-TEM. Grids with suitable ice thickness were transferred to a Thermo Scientific Titan Krios for cryo-ET data collection. The Titan Krios was equipped with an AMETEK Gatan K3 direct detector and AMETEK Gatan energy filter. Cryo-ET data were collected at 300 kV, with a pixel size of 0.39 nm/pixel at a magnification of 19,500×. The tilting range was from −60° to 60°. Data were collected at 3° or 4° intervals. A dose-symmetric tilt scheme was applied. The total dose for one dataset was 80–100 electrons per square Ǻngstrom. The angle of deviation (fibril structures) was calculated using Fiji.

Quantification of dorsal column regeneration after SCI

For each image of the unsectioned injured spinal cord, the number of regenerating axons at different distances from the lesion epicenter was normalized to the number of labeled axons caudal (200–400 μm) to the lesion. The lesion epicenter was identified based on axon morphology and the presence of infiltrating macrophages containing autofluorescent phagocytic material.⁹⁷ Samples with poor tracing for each specific experiment or altered by clear experimental flaws (e.g., incomplete lesion) were excluded from the analysis. Blinding strategies were adopted.

Flow cytometry

Three days (e.g., 10 days after SCI) after vehicle and PDGF-BB administration, mice were perfused with 1× PBS to remove the blood. The spinal segment containing the lesion site (1–1.5 mm of tissue) was carefully dissected and collected in DMEM containing 10% FBS. For each sample, a single-cell suspension was generated and cells were then labeled with fixable viability dye (eFluor506 or eFluor780; eBioscience), blocked with anti-CD16/32 (clone 2.4G2), and stained with fluorochrome-conjugated antibodies specific for CD3e (145-2C11), B220/CD45R (RA-36B2), CD11b (clone M1/70), Ly6G (1A8), CD45 (30-F11), CD84 (mCD84.7), CD124 (mIL4R-M1), CD14 (rmC5-3), CD115 (T38-320), F4/80 (T45-2342), I-A/I-E (M5/114.15.2), and CD11c (HL3), which were all purchased from BD Biosciences, CD101 (Moushi101) from eBioscience, and CX3CR1 (Polyclonal) from R&D Systems. For intracellular staining, the cells were fixed and permeabilized with BD cytofix and cytoperm solutions and then stained with fluorescent antibodies specific for arginase-1 (A1exF5) and CD206 (MR6F3) from Invitrogen. Flow cytometry was performed with a FACS Symphony A3 cell analyzer (BD Biosciences). The cells were gated on forward and side scatter after double exclusion and analyzed on FlowJo version 10 software.

In another set of experiments, the spinal cords (thoracic/lumbar region) of adult NG2-eGFP mice were homogenized with an 18-gauge needle then centrifuged at 800 × g for 5 min at 4°C. The tissue pellet was re-suspended in HBSS with 1 mg/mL collagenase A (11088793001, Roche) and 1 mg/mL DNase I (BP28184, Millipore Sigma) and incubated at 37°C for 20 min. Cells were separated from myelin via centrifugation at 800 × g for 5 min at 4°C in a 27% Percoll solution (89428-524, GE Healthcare). The cells were labeled with fixable viability dye (eFluor780, 65-0865-14, eBioscience), blocked with anti-CD16/32 (clone 2.4G2), and stained with fluorochrome-conjugated antibodies specific for CD45 (30-F11). Flow cytometry sorting (FACS) was performed with a BD FACSMelody cell sorter (BD Biosciences). Sorted cells were gated on viable, CD45-negative cells expressing eGFP.

Multiplex chemokine and cytokine assay

At 3 days after vehicle and PDGF-BB administration (e.g., 10 days after SCI) at the lesion site, spinal cord segments (∼1–1.5 mm of tissue) containing the lesion site were homogenated. Chemokine and cytokine levels in spinal cord homogenates were measured by Luminex multiplex analysis (LXSAMSM, Biotechne) using the Bio-Plex 200 system (Bio-Rad) according to the manufacturer’s protocol.

scRNA-seq and bioinformatic analysis

Single-cell suspensions were generated as described for the Chromium Fixed RNA Kit (1000496, 10X Genomics) and Chromium Next GEM Chip Q Single Cell Kit (1000422, 10X Genomics) with the following modifications. Briefly, spinal cords were obtained from adult (12–14 weeks old) NG2-eGFP mice at 3, 7, and 14 days (e.g., 10, 14, and 21 days after SCI) after vehicle and PDGF-BB administration. Three (for naive, 3 and 7 days) and two (for 14 days) cords (∼2 mm centered at the lesion site) were pulled to obtain ∼20–25 mg of tissue mass for each time point/experimental condition. Spinal cords from naive mice served as control. Tissues were chopped on a glass Petri dish maintained on ice and transferred to a 2 mL sterile tube with 1 mL of fixation buffer and stored at 4°C for 20 h. Samples were then processed for long-term storage at −80°C according to 10X Genomics instructions. After completing sample collection, the samples were thawed at RT and incubated at 37°C in a dissociation solution (2 mL/sample containing 0.4 mg/mL liberase) for 1 h in agitation. Tissues were then dissociated using a glass pipette (∼30 strokes/sample). Dissociated tissues were passed through a 30 μm cell strainer to remove debris. Samples were further dissociated (∼10 strokes/sample) by adding 1 mL of PBS to the remaining tissue pellet. After passing all cell suspension on the 30 μm cell strainer, cells were centrifuged at 850 rcf for 5 min at RT. After removing the supernatant, cells were re-suspended in 1 mL chilled quenching buffer.

Cell suspensions were mixed with propidium iodide dye and counted (Countess 3 FL; Invitrogen). Single-cell libraries were prepared using Chromium next GEM Fixed RNA Profiling Kits for Multiplexed Samples (10X Genomics). Barcoded probe hybridization was carried out with up to 2,000,000 cells per sample. Customized probes for eGFP spike-in were included during the hybridization step based on the technical note provided by 10X Genomics (technical note no. CG000621). Each single cell cDNA library was prepared for a target of 10,000 post-probe hybridization cells per sample. Cells and 10X Genomics Chromium 3′ Flex Next-GEM reagents were loaded onto chips to capture cells using a 10X Genomics Chromium iX Controller. Library preparation was according to manufacturers’ protocols. Sequencing was conducted using an Ilumina NovaSeq X-Plus flowcell (Novogene) with the following parameters: Read1 – i7 Index – i5 Index – Read2: 28 cycles – 10 cycles – 10 cycles – 90 cycles. Fastq sequence files were de-multiplexed, aligned, and annotated to the mm10 genome using 10X Genomics Cell Ranger version 9.0 software using 10X Cloud (URL: https://www.10xgenomics.com/products/cloud-analysis).

Using Seurat toolkits (version 5),⁹⁸ UMAP for dimensional reduction plots were generated from aggregates of multiple scRNA-seq libraries. Seurat FindMarkers function was used to construct gene lists for DEGs using a Poisson generalized linear model with the following cutoffs: min.pct = 0.01, logfc.threshold = 0.1, p_val_adj = 0.05. Top gene lists for each cluster were generated using FindAllMarkers (Figure S7C) or FindConservedMarkers functions (Figure S7D). Heatmaps and dot plots were generated using Seurat package. Volcano plots were generated using the EnhancedVolcano package (https://github.com/kevinblighe/EnhancedVolcano). GO term analysis was performed using the clusterProfiler package.⁹⁹ Upregulated (p_Val_adj < 0.05, avg_log₂FC > 0.1) or downregulated (p_Val_adj < 0.05, avg_log₂FC < −0.1) genes were used for enrichGO function to detect enrichment in biological processes (OrgDb = org.Mm,eg.db, ont = “BP”). Monocle3 package was used to construct pseudotime trajectories and gene signatures for selected cell clusters across pseudotime.¹⁰⁰ The root node to start the pseudotime was computationally selected within the naive group from pericyte cluster no. 12 using the get_earliest_principal_node function. Monocle3 function plot_genes_in_pseudotime was used to plot genes of interests along the projected pseudotime. CellChat package was used to assess potential ligand-receptor interactions between cells and aggregated cell-cell interactions between different cell types within single or multiple scRNA-seq datasets.¹⁰¹ The outgoing and incoming interaction strengths of individual cell types were plotted using the netAnalysis_signalingRole_scatter function (Figure S8B). Function netVisual_heatmap was used to generate heatmaps showing numbers of outgoing and incoming interactions between cell types (Figure S8C). To compare two different datasets, we merged individual CellChat objects and used the netAnalysis_signalingChanges_scatter function to demonstrate signaling pathways that were altered in pericytes/ECs when comparing two different conditions (naive vs. SCI, or vehicle vs. PDGF-BB treatment, Figure S8D). A circle plot of cell-cell interaction in FN1 signaling was plotted using the netVisual_aggregate function (Figure S8E) and the interactions of individual ligand-receptor pairs within the FN1 signaling pathway were plotted using the netVisual_bubble function (Figure S8F). The CellChat StackedVlnPlot function was used to generate violin plot to show gene expression among different cell clusters.

In vivo multichannel recording of LFP and data analysis

Four weeks after SCI, the mice were anesthetized and a laminectomy was performed to expose the spinal cord between T10 and T13. The vertebral columns were stabilized by clamps attached to either side of the laminectomy site. A 32-channel silicon electrode (A4X8-5mm-50-200-177-A32, NeuroNexus Technologies) connected to a stereotaxic frame was inserted at a depth of 600 μm into the spinal cord at the following locations: 1,000 μm rostral/ipsilateral and 500 μm caudal/ipsilateral to the lesion site. The left sciatic nerve was exposed at the mid-thigh level and electrically stimulated (amplitude, 0.3 mA; pulse duration, 1 ms biphasic) using an isolated pulse stimulator (A-M Systems Model 2100). For each animal, LFPs were recorded at a 25 kHz sampling rate and low-pass filtered at 50 Hz offline. LFPs obtained from the caudal site were used as positive control for the stimulation parameters. To record LFPs from the rostral site, 3–5 stimuli were applied with a ∼5 s interval. For each animal, stimulus-evoked LFPs were then averaged and the peak amplitude (either positive or negative peak) within a 100 ms window after the stimulus was calculated. The absolute values were used to generate the heatmap representing LFP amplitude. Of note, one animal was excluded from the analysis as it failed to consistently elicit a response upon stimulation (e.g., 1 response out of 4 trials).

Behavioral testing

Ground locomotion and kinematics analysis: before injury, mice were trained to walk across a transparent corridor to an enriched cage in one direction. One month after injury, mice were placed at one end of the corridor and video recorded while moving to the other end where the enriched cage was located. The day before, mice were briefly anesthetized with isoflurane, shaved and hindlimb reference points (e.g., iliac crest, hip joint, knee joint, ankle joint, and metatarsophalangeal) were marked with a black permanent marker on the shaved skin. Naive mice served as control. The recordings were then analyzed frame by frame using ImageJ and joint tracking was plotted using Igor pro.

Von Frey test: the mice were placed in a testing chamber, and the plantar threshold was measured using retractable monofilaments (Ugo Basile) and the “up-down” method. A quick withdrawal of the left hind paw was considered as a positive response. A pause of 20–30 s was given to allow sensory receptors to reach baseline levels between each monofilament application. The response threshold was calculated as the lowest force (in grams) that produced a retraction at least 50% of the time.

For all behavioral tests described above, experimenters collecting and analyzing data were blinded to the treatment.

Statistical analysis

Statistical analysis was performed using Prism (version 9.3.1, GraphPad) and SAS (SAS 9.4, SAS Institute). For all analyses performed, significance was defined as p < 0.05. The exact values of n, the definition of measures, and the type of statistical analysis are shown in the corresponding figure legends. Randomization and blinding strategies were adopted to eliminate any potential bias in the interpretation of the results.

Data availability

All data are included in the manuscript and supplemental information. The sequencing data included in this study were deposited to Gene Expression Omnibus (GSE293953). All raw and processed data will be made available upon request.

Acknowledgments

We want to thank Dr. Yingjie Shen for discussion on PTPσ antibodies, Dr. Binbin Deng (Center for Electron Microscopy and Analysis, CEMAS, The Ohio State University) for cryo-EM imaging and all members of the laboratory for discussion. This work was supported by the National Institute of Neurological Disorders and Stroke (R01NS110681), Chronic Brain Injury Program (The Ohio State University) and Marina Romoli Association.

Author contributions

A.T. conceived the project and designed the research. W.S., E.D., F.L., A.O., J.A.S., E.H., M.Z., H.M.E., A.J.F., J.P., A.S., and A.T. performed the research and analyzed the data. J.S. shared resources and expert knowledge. A.T. supervised the research and wrote the paper. All authors provided feedback and contributed to editing the manuscript.

Declaration of interests

The authors declare no competing interests.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2025.04.020.

Supplemental information

Document S1. Figures S1–S9

mmc1.pdf^{(8.4MB, pdf)}

Table S1. DEG list and GO analysis, related to Figure 6

mmc2.xlsx^{(883.8KB, xlsx)}

Table S2. DEG list and GO analysis, related to Figure 7

mmc3.xlsx^{(828.5KB, xlsx)}

Document S2. Article plus supplemental information

mmc4.pdf^{(26.6MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S9

mmc1.pdf^{(8.4MB, pdf)}

Table S1. DEG list and GO analysis, related to Figure 6

mmc2.xlsx^{(883.8KB, xlsx)}

Table S2. DEG list and GO analysis, related to Figure 7

mmc3.xlsx^{(828.5KB, xlsx)}

Document S2. Article plus supplemental information

mmc4.pdf^{(26.6MB, pdf)}

Data Availability Statement

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PERMALINK

In vivo programming of adult pericytes aids axon regeneration by providing cellular bridges for SCI repair

Wenjing Sun

Elliot Dion

Fabio Laredo

Allyson Okonak

Jesse A Sepeda

Esraa Haykal

Min Zhou

Heithem M El-Hodiri

Andy J Fischer

Jerry Silver

Juan Peng

Andrew Sas

Andrea Tedeschi

Abstract

Graphical abstract

Introduction

Results

SCI leads to profound changes in vasculature architecture and pericyte coverage

Figure 1.

Pericytes cause detrimental structural and functional changes in adult DRG neurons

Figure 2.

PDGF-BB converts adult pericytes into a permissive substrate for axon growth

Figure 3.

Figure 4.

In vivo conversion of adult pericytes via PDGF-BB aids axon regeneration by providing cellular bridges that span the lesion

In vivo conversion of adult pericytes via PDGF-BB creates favorable conditions for SCI repair

Figure 5.

PDGF-BB administration leads to changes in pericyte gene transcription and cellular crosstalk at the injury site without impacting pericyte identity

Figure 6.

Figure 7.

Mice-injected PDGF-BB recover hindlimb function after thoracic SCI

Figure 8.

Discussion

Materials and methods

Mice

Antibodies

Spinal cord injury

Vasculature tracing, in toto staining, three-dimensional imaging, and reconstruction

Immunohistochemistry

Primary pericyte culture

RNA isolation and RT-PCR

DRG neuron culture

Immunocytochemistry

Morphometric analysis

Immunoblotting

Patch-clamp recording

Calcium imaging

Cryo-EM imaging

Quantification of dorsal column regeneration after SCI

Flow cytometry

Multiplex chemokine and cytokine assay

scRNA-seq and bioinformatic analysis

In vivo multichannel recording of LFP and data analysis

Behavioral testing

Statistical analysis

Data availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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