Biology subjects

Clayton, S. W.

Publications and source records attributed to Clayton, S. W..

8 recordsLinked to original sources

bioRxiv2026

A PDMS-based microfluidic platform enabling dual biochemical and electric-field stimulation for modeling sensory neuron-intervertebral disc crosstalk

Inflammation-driven increases in nociception are prominent in pain pathologies associated with intervertebral disc (IVD) degeneration yet are difficult to model in vitro. Since neurons are exposed to multi-modal stimuli in vivo, it is critical for these exposures to be conserved in an in vitro test system. We developed a polydimethylsiloxane (PDMS)-based microfluidic platform to interrogate peripheral sensory neurons (SNs) in the presence of conditioned media from nucleus pulposus cells from the degenerated IVD, to model a potential impact of IVD cells secretome on pain sensing. Our platform enables controlled perfusion of cell-derived biochemical cues alongside a defined homogeneous electric field (EF) and supports real-time optical analysis. Computational modeling, fluid perfusion experiments, and conductivity measurements confirmed stable fluid transport and tunable homogeneous EF generation within the device. As proof of concept for neuronal stimulation, neuroblastoma (N2a) cells loaded with a fluorescent Ca2+ indicator exhibited a 56% increase in Ca2+ transient activity when exposed to media from degenerated IVDs, concomitant with increased IVD-derived IL-1{beta} production. Importantly, EF-stimulated Ca2+ transients increased in SNs derived from human induced pluripotent stem cells when exposed to conditioned media from primary human IVD cells, demonstrating the translation of this model system to human cells. Together, these results establish a versatile platform that enables controlled and simultaneous exposure to biochemical and electrical stimuli to quantify inflammation-driven peripheral neuronal hyperexcitability in tissue-neuron crosstalk.

bioengineering↗

bioRxiv2025

Ablation of VEGFA following a lumbar intervertebral disc injury attenuates intradiscal neurovascular features and prevents chronic low back pain symptoms

Despite its enormous burden on patients and society, chronic low back pain (LBP) has no effective therapeutic options. Innervation of the degenerating intervertebral disc (IVD) is suspected to cause discogenic LBP, but the mechanisms that orchestrate the IVDs neo-innervation and subsequent symptoms of LBP remain unknown. We hypothesize that Vascular Endothelial Growth Factor-A (VEGFA) critically mediates the neurite invasion in the IVD and contributes to chronic LBP. Initiating IVD degeneration through a mechanical injury, we evaluated the progression of neurovascular features into the IVD, as well as ensuring LBP symptoms and locomotive impairments at acute (3-weeks) and chronic (12-weeks) timepoints following the IVD injury. To determine the role of VEGFA, we utilized a mouse model with ubiquitously inducible recombination of the floxed Vegfa allele (UBC-CreERT2; Vegfafl/fl). The ablation of VEGFA after an IVD injury attenuated de novo neurite and vessel infiltration and impeded the expression of TRPA1, a nociceptive ion channel, in the dorsal root ganglion. The VEGFA-null animals, despite IVD degeneration, exhibited alleviated mechanical allodynia and improved locomotive performance. To determine the effects of IVD-derived VEGFA on endothelial cells and neurons, we co-cultured HMEC-1 endothelial cells and SH-SY5Y neurons with VEGFA-silenced human primary IVD cells. The endothelial cells co-cultured with VEGFA-silenced IVD cells exhibited reduced vessel growth and shifted their transcriptome and secretome from angiogenic to lymphangiogenic. The neurons co-cultured VEGFA-silenced IVD cells showed slowed growth and attenuated transcriptional programs for growth and elongation. These results show that VEGFA directs the growth of intradiscal vessels and neurites that cause low back pain and impaired function, and the inhibition of IVD-derived VEGFA during degeneration may be sufficient to prevent chronic pain behavior and motor impairment associated with discogenic low back pain. One Sentence SummaryVEGFA is a key mediator of neurovascular infiltration in the degenerating intervertebral disc and an essential driver of chronic low back pain, whose ablation prevents pain-related behaviors.

neuroscience↗

bioRxiv2024

Single cell RNA sequencing reveals a shift in cell function and maturation of endogenous and infiltrating cell types in response to acute intervertebral disc injury

Intervertebral disc (IVD) degeneration contributes to disabling back pain. Degeneration can be initiated by injury, and progressively leads to irreversible cell loss and loss of IVD function. Attempts to restore IVD function through cell replacement therapies have had limited success due to knowledge gaps in the critical cell populations and molecular crosstalk after injury. Here, we used single cell RNA sequencing to identify the transcriptional changes of endogenous cells of the IVD and infiltrating cell populations following IVD injury. Control and Injured coccygeal IVDs were extracted from 12 week old female C57BL/6J mice 7 days post injury and subjected to single-cell resolution transcriptomic sequencing. Clustering, gene ontology, and pseudotime trajectory analyses determined transcriptomic divergences in the cells of the Injured IVD, flow cytometry identified they types of infiltrating immune cells, and immunofluorescence was utilized to define mesenchymal stem cell (MSC) localization. Clustering analysis revealed 11 distinct cell populations that included IVD, immune, vascular cells, and MSCs. Differential gene expression analysis determined that Outer Annulus Fibrosus, Neutrophils, Saa2-High MSCs, Macrophages, and Krt18+ Nucleus Pulposus (NP) cells were the major drivers of transcriptomic differences between Control and Injured cells. Gene ontology revealed that the most upregulated biological pathways were angiogenesis and T cell-related while wound healing and ECM regulation categories were downregulated. Pseudotime trajectory analyses revealed that IVD injury directed cells towards increased differentiation in all clusters, except for Krt18+ NP cells which remained in a less mature cell state. Saa2-High and Grem1-High MSCs populations drifted towards more differentiated IVD cells profiles with injury and localized distinctly within the IVD. This study revealed novel MSC populations in a heterogeneous landscape of IVD cell populations during injury, and these cells may be leveraged for future IVD repair studies. Lay SummaryThe intervertebral disc (IVD) is a spinal joint that accumulates damage with age but has limited tissue repair capabilities. IVD injuries progress into degeneration, and IVD degeneration is a leading cause of lower back pain. Understanding the cell populations that change and respond to injury will uncover targets to restore IVD function. Mesenchymal stem cells (MSCs) are cells within the IVD that can potentially replenish the cells lost after IVD damage. To better understand how IVD cell populations are affected by tissue damage, we performed single cell RNA sequencing of IVD tissue 7 days post injury and analyzed the differences in gene regulation. We identified diverse cells populations such as IVD, immune cells, vascular cells, and MSCs. We discovered the presence of Saa2 and Grem1 expressing MSCs that become less stem cell-like and express higher levels of IVD gene markers after injury. We also determined that Saa2 and Grem1 have varying gene expression patterns in IVD tissues that becomes attenuated after injury. These MSCs could be targeted for future stem cell therapies to prevent IVD degeneration.

cell biology↗

bioRxiv2024

Secreted chemokines reveal diverse inflammatory and degenerative processes in the intervertebral disc of the STZ-HFD mouse model of Type 2 diabetes

The chronic inflammation observed during type 2 diabetes (T2D) is associated with spinal pathologies, including intervertebral disc (IVD) degeneration and chronic spine pain. Despite the presence of confounding factors, such as obesity, studies have shown that after adjusting for age, body mass index, and genetics (e.g. twins), patients with T2D suffer from disproportionately more IVD degeneration and/or back pain. We hypothesize that chronic T2D fosters a proinflammatory microenvironment within the IVD that promotes degeneration and disrupts IVD homeostasis. To test this hypothesis, we evaluated two commonly used mouse models of T2D - the leptin-receptor deficient mouse (db/db) and the chronic high-fat diet in mice with impaired beta-cell function (STZ-HFD). Compared to their genetic controls--C57BL/6 wild-type mice for STZ-HFD and heterozygous littermates for db/db--STZ-HFD IVDs exhibited more severe degeneration and elevated chemokine expression profiles. RNA-seq further revealed extensive transcriptional dysregulation in STZ-HFD IVDs that was not observed in the db/db model. The STZ-HFD IVDs also expressed enzymes that enhanced production of glycolytic AGE precursors, impaired non-AGE DAMP pathways, and reduced suppressors of RAGE turnover. These results suggest that, under controlled genetic and environmental conditions, the STZ-HFD model more accurately reflect the multifactorial inflammatory milieu characteristic of T2D-induced IVD degeneration.

physiology↗

bioRxiv2024

Analysis of Infiltrating Immune Cells Following Intervertebral Disc Injury Reveals Recruitment of Gamma-Delta (????) T cells in Female Mice

Inadequate repair of injured intervertebral discs (IVD) leads to degeneration and contributes to low back pain. Infiltrating immune cells into damaged musculoskeletal tissues are critical mediators of repair, yet little is known about their identities, roles, and temporal regulation following IVD injury. By analyzing longitudinal changes in gene expression, tissue morphology, and the dynamics of infiltrating immune cells following injury, we characterize sex-specific differences in immune cell populations and identify the involvement of previously unreported immune cell types, {gamma}{delta} and NKT cells. Cd3+Cd4-Cd8-T cells are the largest infiltrating lymphocyte population with injury, and we identified the presence of {gamma}{delta} T cells in this population in female mice specifically, and NKT cells in males. Injury-mediated IVD degeneration was prevalent in both sexes, but more severe in males. Sex-specific degeneration may be associated with the differential immune response since {gamma}{delta} T cells have potent anti-inflammatory roles and may mediate IVD repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/582950v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@90ba37org.highwire.dtl.DTLVardef@1b17f87org.highwire.dtl.DTLVardef@2baa2corg.highwire.dtl.DTLVardef@b155c5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Schematic of the workflow to obtain longitudinal analyses of the acute IVD injury response Injured caudal IVDs, CC5/6-CC9/10, were bilaterally punctured with a 30G needle to induce a traumatic injury. Injured IVDs and neighboring uninjured internal Control IVDs, CC12/13-CC16/17, were isolated during the acute injury response every 2-3 days until 21 days post injury (dpi) and a chronic injury time point at 42 dpi in female mice. Male mice samples were only collected at key injury response time points (red numbers): 3, 7, 12, 19, and 42 dpi. Longitudinal analyses of the temporal regulation of immune cell gene expression and cell infiltration were measured with qPCR, flow cytometry, immune fluorescence, and histology analyses to identify a sex-divergent immune response. C_FIG

cell biology↗

bioRxiv2022

Single Cell RNA-Sequence Analyses Reveal Uniquely Expressed Genes and Heterogeneous Immune Cell Involvement in the Rat Model of Intervertebral Disc Degeneration

Intervertebral disc (IVD) degeneration is characterized by a loss of cellularity, and changes in cell-mediated activity that drives anatomic changes to IVD structure. In this study, we use single cell RNA-sequencing analysis of cells extracted from the degenerating tissues of the rat IVD following lumbar disc puncture. Two control, uninjured IVDs (L2-3, L3-4) and two degenerated, injured IVDs (L4-5, L5-6) from each animal were examined either at two- and eight-week post-operative time points. The cells from these IVDs were extracted and transcriptionally profiled at a single-cell resolution. Unsupervised cluster analysis revealed the presence of 4 known cell types in both non-degenerative and degenerated IVDs based on previously established gene markers: IVD cells, endothelial cells, myeloid cells, and lymphoid cells. As a majority of cells were associated with the IVD cell cluster, sub-clustering was used to further identify the cell populations of the nucleus pulposus, inner and outer annulus fibrosus. The most notable difference between control and degenerated IVDs was the increase of myeloid and lymphoid cells in degenerated samples at 2- and 8- weeks post-surgery. Differential gene expression analysis revealed multiple distinct cell types from the myeloid and lymphoid lineages, most notably macrophages and B lymphocytes and demonstrated a high degree of immune specificity during degeneration. In addition to the heterogenous infiltrating immune cell populations in the degenerating IVD, the increased number of cells in the AF sub-cluster expressing Ngf and Ngfr, encoding for p75NTR, suggest that NGF signaling may be one of the key mediators of the IVD crosstalk between immune and neuronal cell populations. These findings provide the basis for future work to understand the involvement of select subsets of non-resident cells in IVD degeneration.

cell biology↗

bioRxiv2021

TGFβ signaling is required for sclerotome resegmentation during development of the spinal column in Gallus gallus.

We previously showed the importance of TGF{beta} signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGF{beta} signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGF{beta}R1 inhibitor, SB431542, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in SB431542 injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGF{beta} signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro- caudal markers within sclerotome was disrupted at E3.5 after treatment with SB431542 with rostral domains expressing both rostral and caudal markers. We propose that TGF{beta} signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

developmental biology↗

bioRxiv2020

Canonical and noncanonical TGF-β signaling regulate fibrous tissue differentiation in the axial skeleton

Previously, we showed that embryonic deletion of TGF-{beta} type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-{beta} regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-{beta} stimulated expression of Scleraxis mRNA by two hours and Fibromodulin and Adamtsl2 mRNAs by eight hours of treatment. Regulation of Scleraxis by TGF-{beta} did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-{beta}-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-{beta}-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-{beta} treatment. Next, the role of several noncanonical TGF-{beta} pathways were tested. We found that ERK1/2 was activated by TGF-{beta} and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-{beta} regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGF{beta}-mediated fibrous differentiation in sclerotome.

developmental biology↗