Aging negatively impacts central nervous system function; however, there is limited information about the cellular impact of aging on peripheral nervous system function. Importantly, injury to vulnerable peripheral axons of dorsal root ganglion (DRG) neurons results in somatosensory dysfunction, such as pain, at higher rates in aged individuals. Cellular senescence is common to both aging and injury and contributes to the aged pro-inflammatory environment. We discovered DRG neuron senescence in the context of aging and pain-inducing peripheral nerve injury in young (~3mo) and aged (~24mo) male and female mice. Senescent neurons were dynamic and heterogeneous in their expression of multiple senescence markers, including pro-inflammatory factor, IL6. Senescence marker-expressing neurons had nociceptor-like profiles, included high-firing phenotypes, and displayed increased excitability following IL6 application. Furthermore, elimination of senescent cells resulted in improvement of nociceptive behaviors in nerve-injured mice. Finally, male and female-derived human DRG contained senescent neurons, which increased with age (~32 vs 65 years old). Overall, we describe a susceptibility of the peripheral nervous system to neuronal senescence - a potential targetable mechanism to treat sensory dysfunction, such as chronic pain, particularly in aged populations.

Animals

All animal procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care and Institutional Animal Care and Use Committee (IACUC; 34760) in accordance with American Veterinary Medical Association guidelines and the International Association for the Study of Pain. All mice were housed 2-5 per cage, maintained on a 12-hour light/dark cycle in a temperature-controlled environment (Temp:68-74°F; Humidity:30-70%) with ad libitum access to food and water. Young male and female mice used were 11-16 weeks old, wild-type C57BL/6J mice (Jax stock #00664). Aged male and female mice used were 20-24 months old, wild-type C57BL/6JN mice (NIA aged rodent colony). We did not note any sex differences in the expression of senescence markers and therefore combined male and female DRG throughout all analyses. Aged mice were pre-screened for abnormal masses and cataracts and were included in the study only if they appeared healthy.

Human samples

Use of human post-mortem DRG received Stanford University Institutional Review Board for human subjects’ exemption. Human lumbar L4 DRG tissues were obtained from one female donor (age 33) who died from head trauma, one female donor (age 32) who died from head trauma, one male donor who died from head trauma (age 65), and one female donor (age 65), who died from stroke. Human post-mortem DRG were obtained in collaboration with Donor Network West.

METHOD DETAILS

Spared Nerve Injury

To perform SNI surgery(60), mice were anesthetized with isoflurane and a small incision is made over the left thigh. Blunt dissection was performed through the biceps femoris muscle in order to expose the sciatic nerve and its three branches (common peroneal, tibial, and sural nerves). The common peroneal and tibial nerves were then ligated using an 5-0 nylon suture (ETHILON^TM ref#1668G) and these nerves were then axotomized using small-sized spring scissors. The sural nerve is left intact (the “spared nerve”). The incision was closed with surgical staples. Following surgery, mice were monitored for the study period, which varies from 1 day to 16 weeks depending on the time point of interest. Controls used for qPCR experiments, were sham surgery in which an incision was made followed by opening of muscle to reveal the nerve, without touching the nerve, followed by closure.

Senolytic Administration

ABT263 (Navitoclax) (Med Chem Express, Cat. No.: HY-10087) was dissolved in 60% Phosol40PG, 30% PEG400, 10% Ethanol at a concentration of 12.5 mg/mL using brief water bath sonication and vortexing. Young and aged mice were briefly anesthetized with isoflurane before they were dosed by oral gavage (p.o.) at 100 mg/kg daily for 5 days, with a 2-day rest period, followed by a second 5-day daily dosing.

Quantitative Real Time RT-PCR (qPCR)

Whole DRG were collected, homogenized using a 1mL glass homogenizer, (PYREX® No. 7724-1) and placed in TRIzol Reagent (Invitrogen, Cat#15596018). RNA was isolated using miRNeasy® Mini kit (Qiagen, Cat #217004). The concentration and purity of RNA samples were determined using NanoDrop 2000 (Thermo Fisher Scientific). RNA was reverse transcribed using SuperScript^TM VILO^TM cDNA Synthesis Kit (Cat#11754-050). qPCR analysis was performed with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Cat#A25741) and run on an Applied Biosystems 7900HT or on an Applied Biosystems StepOnePlus. Appropriate no reverse-transcriptase and no template controls were used for each 384-well PCR reaction. The cycle conditions were as follows: 50°C for 2 min, 95°C for 2 min, then 40 cycles of 15 s at 95°C, 1min at 60°C. Dissociation analysis was performed at the end of each run to ensure specificity. qPCR primers used: p21 F:5’-GTGAGGAGGAGCATGAATGGA-3’, R:5’-GCACCTTTTATTCTGCTGGCAA-3’; p16 F:5’-GTGTGCATGACGTGCGG-3’, R: 5’-CACCTGAATCGGGGTACGAC-3’; p53(Trp53) F:5’-TCATCCCTCCCCTTTTCTGTC-3’, R:5’-ATGGCGGGAAGTAGACTGGC-3’; Il6 F:5’-GCTACCAAACTGGATATAATCAGGA-3’, R:5’-CCAGGTAGCTATGGTACTCCAGAA-3’; Ccl2 F:5’-AGCACCAGCCAACTCTCACT-3’, R:5’-CGTTAACTGCATCTGGCTGA-3’; ms-Tnfα (Qiagen, PPM03113G); ms-Il1b (Qiagen, PPM03109F); housekeeping: Tuba1a F:5’-GTGCATCTCCATCCATGTTG-3’, R:5’-GTGGGTTCCAGGTCTACGAA-3’. Relative quantification of gene expression was performed via 2^-DDC(T)^ method.

Tissue preparation

Mice were anesthetized using Pentobarbital (Vortech Pharmaceuticals, NDC 0298-9373-68, 150mg/kg in 0.9% saline) and transcardially perfused with 5mL 1XPBS followed by 30mL 10% formalin solution (ThermoFisher). Lumbar DRG tissues were dissected and placed temporarily in RNAlater solution at RT (ThermoFisher), then frozen in O.C.T. Compound (Sakura Finetek, Inc., Cat#4583) on dry ice, and stored at -80°C. Mouse DRG was sectioned at 14µm and mounted onto SuperFrost Plus glass slides, dried for 1hr, and stored at −80°C. Human lumbar DRGs were obtained from organ donors who were de-identified prior to collection. Extracted tissues were flash frozen immediately on dry ice and stored in screw cap 15mL conical tubes and stored at −80°C. DRGs were slowly embedded in OCT on dry ice and sectioned at 20µm onto SuperFrost Plus glass slides and stored at −80°C.

Fluorescent in situ hybridization

Fluorescent in situ hybridization using the RNAscope® Multiplex V2 Kit (ACD, Cat#323100) was performed to detect the RNA of senescence markers, cytokine, and DRG neuronal markers (p21 (Cdkn1a), p16* (Cdkn2a*), IL6, Tprv1). Briefly, DRG tissues were isolated and processed as described above. Dorsal root ganglion (DRG) sections (14µm) were mounted on glass slides and dried 1hr at room temperature and stored at −80°C. On Day 1 of RNAscope, slides were submerged in 10% formalin and incubated for 20 min at 4°C. Slides were washed (1XPBS) and dehydrated (EtOH) as described in the ACD RNAscope user manual (UM 323100). Sections were incubated for 10min in RNAscope® Hydrogen Peroxide solution, washed in Millipore water, and incubated in RNAscope® ProteaseIV for 1min (human) or 5min (mouse) at room temperature. Slides were incubated with appropriate RNAscope probes (Mouse probes: Mm-IL6-C1, Cat#315891, Mm-Cdkn1a-C2, Cat#408551-C2, Mm-Cdkn2a-C3, Cat#411011-C3, Mm-Cdkn2a-tv2-C2, Cat#447491, Mm-Trpv1, Cat#313331-C1 and -C3; Human probes: Hs-TRPV1-C1, Cat#415381, Hs-CDKN2A-C2, Cat#310181-C2, Hs-CDKN1A-C3, Cat#311401) at 40°C in a Hybez^TM II ^oven (ACD) for 2hrs and stored overnight in 5X SSC buffer at room temperature. On Day 2, slides were incubated in Amp1, Amp2, and/or Amp3 solutions followed by HRP-C1, HRP-C2, and/or HRP-C3 as appropriate. In each round, TSA VividTM (1:1000, ACD Bio., TSA Vivid 520 Cat#323271, TSA Vivid 570 Cat#323272, TSA Vivid 650 Cat#323273) dye reagents and HRP Blocker. Negative control probes (ACD, #321838) were used to assess background levels of RNAscope signal.

Immunohistochemistry

For immunohistochemistry performed immediately following RNAscope protocol (dual labeling), slides were first washed in 1XPBS and blocked (10% Normal Donkey Serum, 0.3 % Triton-X 100, in PBS) for 1hr at room temperature. Slides were incubated with Rabbit anti-ATF3 (1:200, Novus Bio, Cat#NBP1-85816) in 1% blocking solution in 1XPBS at 4°C overnight. Slides were washed 3X in 1XPBS for 5 min each, incubated with AlexaFluor secondary antibodies (1:1000, Donkey anti-rabbit-A488, LifeTechnologies, Cat# A21206), and mounted with Fluoromount G with DAPI (ThermoFisher, Cat#00-4959-52).

For cleaved caspase-3 immunostaining, mice were transcardially perfused as described and DRG tissues were extracted and frozen in OCT. Slides were then blocked (5%Normal Donkey Serum, 0.3 % Triton-X 100, in PBS) for 1hr at room temperature. Rabbit Cleaved-caspase-3 primary antibody (1:200, Cell Signaling Technology, Cat#9661) was incubated overnight at 4°C. Slides were incubated with secondary antibody, (1:1000, Donkey anti-rabbit Alexa-555, LifeTechnologies, Cat#A31572) for 2hr in the dark, washed, and mounted with Fluoromount G with DAPI. Primary antibody controls (no-primary conditions) were used throughout to validate immuno-positive signal.

SA-β-galactosidase activity assay

Mice were perfused with cold 1XPBS. L3-L5 dorsal root ganglion (DRG) was extracted and mounted onto OCT. DRG were sectioned at 14µm onto glass slides. Slides were removed from the freezer, and 1X of fixative solution provided by Senescence β-Galactosidase Staining Kit (Cell Signaling Technology kit, Cat#9860S) was added to the slides for 15min. Slides were rinsed in PBS, and a wax barrier was drawn around the sections. Fresh β-Galactosidase Staining Solution at pH 6.1 was added to the slides and incubated at 37°C for 22hrs. β-Galactosidase Staining Solution was removed and slides were rinsed twice in PBS and twice in distilled water before mounting and imaging. FIJI v2.9.0 was used to outline the area of the DRG and the percentage of positive SA-β-Gal pixels in the area was acquired and normalized to area. Sections (5-10) were analyzed per mouse.

Neuron diameter analysis

Fluorescent TIFF images taken from RNAscope experiments that labeled p21, p16, IL6 RNA, and DAPI were used to measure diameters of neurons in both mouse and human DRG. Cells were categorized for the co-expression of markers p21, p16, with IL6. Using Fiji v2.9.0 software, the scale (µm) was appropriately set based on objective used in image. The longest end-to-end cell diameters, with line centered through each neuron (line segment tool). The line was measured using ‘Measure’ as an output in µm unit.

ELISA assay

1mL syringes were coated with heparin and used to withdraw ~0.8ml of blood from mice anesthetized mice. Blood was centrifuged (1mL tubes) at 2000xg at 4°C for 10 minutes. The supernatant was removed, aliquoted, and stored at -80°C. IL-6 plasma concentration levels were measured by IL6 ELISA kit (ThermoFisher, Cat#KMC0061). Samples were tested in duplicate and diluted 1:2. The final concentration was corrected for the dilution factor. A VERSAmax tunable microplate reader (Molecular Devices) was used to calculate optical density (OD) values at 450 nm. Data was analyzed with Boosterbio’s 5PL regression model and subtracting the blank well’s OD value from the sample’s OD values (https://www.bosterbio.com/biology-research-tools/elisa-data-analysis-online).

DRG neuron dissociation and culture

Lumbar L3-6 dorsal root ganglia (DRG) were excised from aged mice (24mo) following transcardial perfusion with 3mL 1XPBS (Corning Ref #: 21-031-CV). DRG were placed into DMEM media supplemented with 10%FBS and Penicillin/streptomycin (10U per mL/10µg per mL; Thermo Fisher Scientific, Cat#: 15070063) on ice during collection. DRG were then washed with HBSS and placed in 3mL warmed DispaseII(2.5mg/mL)/CollagenaseA(1.25mg/mL) and incubated at 37°C shaking (200rpm) for 30min (Sigma-Aldrich, Cat#: 10103578001, Cat #: D4693-1G). DRG were then washed with 5mL HBSS and placed in 1mL neurobasal media (Thermo Fisher Scientific Cat #: 10888022) supplemented with 5%FBS (Sigma-Aldrich, Cat #: F4135-500ML), 1X GlutaMAX (Gibco, Cat#: 35050061), 1X B-27 supplement (Thermo Fisher Scientific, Cat#: 10889038), and Penicillin/streptomycin. DRG were then triturated ~4-6X with P1000 pipette, followed by a series of 4 fire-polished glass pipettes of decreasing bore size. Dissociated cells were filtered through a 40µm sterile mesh filter. Cells were spun at 300xg for 4min and resuspended in supplemented neurobasal media without FBS for subsequent culture. 24-well culture plates containing sterilized glass coverslips were pre-coated with 1/30th mixture of Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo, A1413202) with Neurobasal media, and incubated for 2hr at 37°C. Coverslips were rinsed with Neurobasal media and left to dry in the culture hood for 1hr. Cells were plated in 1mL of supplemented neurobasal media without FBS. Coverslips were collected for electrophysiology recordings 48hr after plating.

Electrophysiology

For ex vivo recording preparations, all extracellular solutions in contact with live tissue were bubbled with a 95% O2/5% CO2 gas. Animals were deeply anesthetized with a ketamine/xylazine bolus and transcardially perfused with a sucrose-based dissection solution (containing in mM: 250 sucrose, 2.5 KCl, 25 NaHCO3, 1 NaH2PO4, 6 MgCl2, 0.5 CaCl2, and 25 glucose). The vertebral column was removed and placed in dissection solution. The DRG were removed and stripped of epineurium. The tissue was transferred to collagenase (1 mg/ml in dissection solution) to incubate for 30 minutes at 35°C to digest the perineurium. Recordings were performed in a chamber (RC-26GLP; Warner Instruments) within an upright microscope (Nikon Eclipse FN1) and secured with a platinum wire-based anchor, and tissue was constantly perfused with artificial cerebrospinal fluid (aCSF; composition in mM: 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.0 NaH2PO4, 1.0 MgCl2, 2.0 CaCl2, and 25 glucose). For in vitro (culture) preparations, coverslips were preincubated with either IL6 (50 ng/mL with carrier, prepared from 100 mg/mL stock solution in 0.1% BSA; R&D systems, Cat. #406-ML) or control media (with 0.00005% BSA) for one hour prior to recording, and aCSF containing the same concentration of either IL6 or control BSA was applied (29-32°C). Coverslips were discarded one hour following transfer to the recording chamber. Patch pipettes were pulled (P-97; Sutter Instruments) from single-filament borosilicate glass capillaries (1.5 mm OD, 1.1 mm ID; Sutter Instruments) with resistances from 5–8 MΩ, and filled with internal patch solution as follows (in mM): 120 potassium gluconate, 20 KCl, 165 2 MgCl2, 2 Na2ATP, 0.5 NaGTP, 20 HEPES, 0.5 EGTA, pH adjusted to 7.2–7.3 with KOH. Signals were amplified (Multiclamp 700B; Molecular Devices), digitized (Digidata 1440A; Molecular Devices), filtered with a 4 kHz Bessel and sampled at 10 kHz (pClamp 10.6 software; Molecular Devices). Liquid junction potentials (-14 mV) were corrected for (JPCalc software, P. Barry, University of New South Wales, Sydney, Australia; modified for Molecular Devices). In current clamp, depolarizing current steps were applied from resting membrane potential to determine excitability parameters. Following recordings, images were taken of the neuron to estimate size (the average of two separate diameter measurements), and the cytoplasm was aspirated into the patch pipette for subsequent polymerase chain reaction (PCR). PCR was performed using primers for p16 (Mm.PT.58.42804808; IDT), p21 (Mm.PT.58.5884610; IDT), IL6 (Mm.PT.58.10005566; IDT), GFAP (To determine glia presence in sample; Mm01253033_m1; Thermo Fisher) and Tubb3 (To confirm neuronal tissue was sampled; Mm.PT.58.32393592; IDT) in combination with TaqMan™ Gene Expression Master Mix (Cat. #4369016; Thermo Fisher). Samples were then subjected to real-time PCR with the same primers and the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Cat. #12574018; Thermo Fisher). Uniform Manifold Approximation and Projection (UMAP; python implementation from https://github.com/lmcinnes/umap) was performed to integrate and connect the high-dimensional neuronal parameters (33) in low-dimensional 2D space. Training was performed using the train_test_split function from Scikit-Learn over 1000 epochs. UMAP hyperparameters were as follows: number of neighbors=5, minimum distance=0.82, local connectivity=2, random state=42. Clusters were then estimated via the hierarchical density-based clustering algorithm HDBSCAN (python implementation from https://github.com/scikit-learn-contrib/hdbscan/blob/master/docs/index.rst) with the following parameters: minimum cluster size=4, cluster selection epsilon=5, cluster selection method='eom' or Excess of Mass. Parameters were normalized for heatmap visualization using the following equation: (p - min(p)) / (max(p) - min(p)) where p is a vector containing all measurements of a given parameter.

Mechanical nociception assays

To evaluate mechanical reflexive hypersensitivity, we used a logarithmically increasing set of 8 von Frey filaments (Stoelting), ranging in gram force from 0.007 to 6.0 g. These were applied perpendicular to the plantar hindpaw with sufficient force to cause a slight bending of the filament. A positive response was characterized as a rapid withdrawal of the paw away from the stimulus filament within 4 s. Using the up-down statistical method,(62) the 50% withdrawal mechanical threshold scores were calculated for each mouse and then averaged across the experimental groups.

Unweighting

An incapacitance device (IITC Life Science) was used to measure hindpaw unweighting. Mice were placed in the plexiglass apparatus with a ramp with the hindpaws resting on separate metal scale plates. Measurements were taken when the hindpaws were supporting the weight of the mouse with forepaws on the ramp. Each measurement was 4-6 s, and 6 consecutive measurements were taken at 60 s intervals. Six readings were averaged to calculate the bilateral hindpaw weight-bearing values. The calculation of weight bearing on the injured hindlimb was as follows: 2*(L)/(L + R)*100 to get percent weight bearing on injured (L: left) hindlimb.

Imaging/Image analysis

All imaging was performed using a Keyence BZ-X810 fluorescent microscope (2018 Keyence Corporation) using a 40X objective (mouse DRG) or a 20X objective (human DRG). 8-12 DRG sections were imaged per mouse and 3-5 DRG sections per human sample. Images were saved as stitched/full focus TIFF files using Keyence BZ-X800 Analyzer v1.1.1.8. Fiji 2.9.0 was used for subsequent image processing and analysis. All images were similarly adjusted for brightness and contrast (across the entire image) per experiment, with no additional alterations made to the image. Lipofuscin signal was defined by strong autofluorescence signal across all channels (488nm, 550nm, 647nm).

Quantification and statistical analysis

Measurements of cohort sizes were determined based on historical data from our laboratory using a power analysis to provide >80% power to discover 25% differences with p<0.05 between groups to require a minimum of 4 animals per group for all behavioral outcomes, and 2 animals per group for RNAscope analyses. All experiments were randomized by cage and performed by a blinded researcher. Researchers remained blinded throughout histological, biochemical, electrophysiological, and behavioral assessments. Groups were unblinded at the end of each experiment before statistical analysis. All data are expressed as the mean values ±SEM. Data were collected using Excel v2108. Statistical analysis was performed using GraphPad Prism v10.0.2, Python, or R, as described in Methods. Data were analyzed using two-tailed Student’s t test or Mann-Whitney tests, depending on the normality of the distribution, as indicated in the main text or figure captions, as appropriate. A combination of male and female mice young or aged were used throughout the study. No data were excluded from analyses. In some cases, the different sections of the same DRG from the same mouse was used to detect multiple senescence markers for RNAscope analyses. DAPI nuclear stain was used to determine glial-neuronal boundary to carefully associate RNAscope puncta with neuronal cell bodies. For mouse DRG, neurons expressing RNA were quantified per probe set as: IL6-positive >5 puncta; p16-positive >10puncta; p21-positive >20puncta; Trpv1-positive >20puncta. Human DRG neuron cut offs for positive counted cells were as follows: IL6 >10puncta, p16 >15puncta, p21 >20puncta, Trpv1 >20puncta. Experimenter was blinded to age/sex/timepoint per experiment during RNAscope quantification. All counts were conducted assessing >800 total DRG neurons per experiment per biological replicate. No experiment presented in this study failed to replicate.

Transcriptomic Analysis of existing datasets

Single nucleus and single cell RNA sequencing datasets were acquired from the sources described below. Transcriptomic data was processed in python 3.10 using scanpy-1.10.1, anndata-0.10.7, numpy-1.26.4, scipy-1.11.4, pandas-2.2.2, statsmodels-0.14.2 software packages. Differential gene expression significance was calculated using the Wilcoxon rank-sum test.  P-values were FDR corrected using the Benjamini/Hochberg method. A gene is considered differentially expressed if the Log2FC is greater than |0.6| and the adjusted p-value is less than 0.05. To detect senescent cell populations, discrete counts and percentages of senescent cells were calculated using several binary signatures as described in each figure. A cell was only considered positive for a signature if it contained a non-zero expression of all positive genes in the signature, and zero expression of all negative genes in the signature. For example, a cell would be considered positive for the signature CDKN2A+, LMNB1-, TOP2A- if it had non-zero expression of CDKN2A, and zero expression of LMNB1 and TOP2A. For SenMayo gene-set scoring, single-cell and single-nucleus data was log1p transformed, centered and scaled around the gene mean and variance respectively using Scanpy. To address differences in basal expression in Senmayo genes between cell types and highlight the changes in Senmayo scoring for each cell type, the same scaling was performed for each annotated cell type from Renthal, et. al, 2019 independently. Scoring performed on the North et. al, 2019(38) bulk-RNA sequencing data was also scaled in the same manner, but across the dataset. Gene-set scoring was performed using the ScanPy score genes method(63). Scores assessed for individual cells. Displayed heatmaps show the mean gene-set score per annotated subtype and model time-point.  

For analysis of Renthal et al.(27), we downloaded post-QC raw count matrices from GEO Accession GSE154659 (158,785 nuclei). In addition to their pre-processing, we removed a small number of probable doublets using the ‘scrublet’ tool from the scanpy library (294 nuclei), leaving us with 158,758 nuclei. We restricted our analysis to the nuclei from the C57 mouse SpNT model and Naive samples. This left us with 16,895 neuronal nuclei and 11,957 non-neuronal nuclei. For consistency we used the cell-type annotations provided by Renthal et. al. Raw count data was normalized to counts/10,000 for analysis.

For analysis of Wang et al.(28) we aligned raw count matrices from GEO Accession GSE155622 (114,243 cells). Our QC process removed 11,595 cells that had fewer than 500 UMI or fewer than 500 unique genes detected in a cell, had greater than 25% of UMI from mitochondrial genes, had greater than 10% of UMI from MALAT1, or were determined likely to be a doublet. After QC we had 102,648 cells left for analysis. For consistency we are using the cell-type annotations provided by Wang et. al. Raw count data was normalized to counts/10,000 for analysis.

For analysis of Yu et al. 2023(37) we aligned raw count matrices from GEO Accession GSE249746 (1,136 cells).  Consistent with the Smart-Seq2 protocol, somewhat higher mitochondrial gene count ratios were observed, but we chose not to remove any cells from the analysis. Raw count data was normalized to counts/10,000 for analysis.

For analysis of North et al.(67) (https://apps.utdallas.edu/bbs/painneurosciencelab/sensoryomics/hdrgclinical/) bulk RNA sequencing data in transcripts-per-million (TPM) and sample metadata were parsed from the supplemental files 1 and 2 in the associated article. No additional normalization or post-processing was performed.

Data Availability
Data that support the findings of this study will be available on Dryad at the following location https://doi.org/10.5061/dryad.fbg79cp5v within 6 months of publication. We re-analyzed data from the following existing sources: Renthal et al. GEO Accession GSE154659; Wang et al. GEO Accession GSE155622; Yu et al. GEO Accession GSE249746; North et al. (https://apps.utdallas.edu/bbs/painneurosciencelab/sensoryomics/hdrgclinical/). Source data used to make all figures are available as Source Data files for main figures and extended data figures.

Code Availability

Publicly available code was implemented in Python for the UMAP (https://github.com/lmcinnes/umap) and HDBSCAN (https://github.com/scikit-learn-contrib/hdbscan) analyses. Figures in these analyses were generated using Matplotlib (https://matplotlib.org/) and Seaborn (https://seaborn.pydata.org/). All code used to re-analyze publicly available datasets and generate associated figures is posted at: https://github.com/Tawfik-Lab/Donovan_Senescence_2024 and https://doi.org/10.5281/zenodo.14902120.