DOI: 10.5061/dryad.fbg79cp5v
title: Aging and injury drive neuronal senescence in the dorsal root ganglia

description: 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.
description: <strong>Animals</strong> 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 <em>ad libitum</em> 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.
 <strong>Human samples</strong> 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. <strong>METHOD
 DETAILS</strong> <strong>Spared Nerve
 Injury</strong> 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<sup>TM</sup> 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.
 <strong>Senolytic Administration</strong>
 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.
 <strong>Quantitative Real Time RT-PCR
 (qPCR)</strong> 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<sup>TM</sup> VILO<sup>TM</sup> 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: <em>p21</em>
 F:5’-GTGAGGAGGAGCATGAATGGA-3’, R:5’-GCACCTTTTATTCTGCTGGCAA-3’;
 <em>p16</em> F:5’-GTGTGCATGACGTGCGG-3’, R:
 5’-CACCTGAATCGGGGTACGAC-3’; <em>p53(Trp53)</em>
 F:5’-TCATCCCTCCCCTTTTCTGTC-3’, R:5’-ATGGCGGGAAGTAGACTGGC-3’;
 <em>Il6</em> F:5’-GCTACCAAACTGGATATAATCAGGA-3’,
 R:5’-CCAGGTAGCTATGGTACTCCAGAA-3’; <em>Ccl2</em>
 F:5’-AGCACCAGCCAACTCTCACT-3’, R:5’-CGTTAACTGCATCTGGCTGA-3’;
 ms-<em>Tnfα</em> (Qiagen, PPM03113G);
 <em>ms-Il1b</em> (Qiagen, PPM03109F); housekeeping:
 <em>Tuba1a</em> F:5’-GTGCATCTCCATCCATGTTG-3’,
 R:5’-GTGGGTTCCAGGTCTACGAA-3’. Relative quantification of gene expression
 was performed via 2^-DDC(T)^ method.
 <strong>Tissue preparation</strong>
 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.
 <strong>Fluorescent in situ
 hybridization</strong> Fluorescent <em>in
 situ</em> 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 (<em>Cdkn1a</em>),
 p16* (Cdkn2a*), <em>IL6</em>, <em>Tprv1</em>).
 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.
 <strong>Immunohistochemistry</strong>
 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. <strong>SA-β-galactosidase activity
 assay</strong> 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.
 <strong>Neuron diameter analysis</strong>
 Fluorescent TIFF images taken from RNAscope experiments that
 labeled <em>p21</em>, <em>p16</em>,
 <em>IL6</em> 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 <em>p21</em>,
 <em>p16</em>, with <em>IL6</em>. 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. <strong>ELISA
 assay</strong> 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). <strong>DRG neuron dissociation and culture</strong> 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. <strong>Electrophysiology</strong> For <em>ex vivo</em> 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 <em>in vitro</em> (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. <strong>Mechanical nociception assays</strong> 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. <strong>Unweighting</strong> 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: <em>left</em>) hindlimb. <strong>Imaging/Image analysis</strong> 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). <strong>Quantification and statistical analysis</strong> Measurements of cohort sizes were determined based on historical data from our laboratory using a power analysis to provide &gt;80% power to discover 25% differences with p&lt;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 &gt;5 puncta; p16-positive &gt;10puncta; p21-positive &gt;20puncta; Trpv1-positive &gt;20puncta. Human DRG neuron cut offs for positive counted cells were as follows: IL6 &gt;10puncta, p16 &gt;15puncta, p21 &gt;20puncta, Trpv1 &gt;20puncta. Experimenter was blinded to age/sex/timepoint per experiment during RNAscope quantification. All counts were conducted assessing &gt;800 total DRG neurons per experiment per biological replicate. No experiment presented in this study failed to replicate. <strong>Transcriptomic Analysis of existing datasets</strong> 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. <strong>Data Availability</strong><br> 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. <strong>Code Availability</strong> 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.
description: # Aging and injury drive neuronal senescence in the dorsal root ganglia
 Dataset DOI:
 [10.5061/dryad.fbg79cp5v](https://doi.org/10.5061/dryad.fbg79cp5v) ##
 Description of the data and file structure Data includes individual data
 points generated from multiple independent experiments published at: 
 PMID: 40369367   PMCID:
 [PMC12081305](https://pmc.ncbi.nlm.nih.gov/articles/PMC12081305/)DOI:
 [10.1038/s41593-025-01954-x](https://doi.org/10.1038/s41593-025-01954-x)
 ### Files and variables **File: Donovan_SourceData_ED_Fig1-6.xlsx**
 **Description:** File contains raw data generated in Figures 1 through 6
 of the extended data file (DOI:
 [10.1038/s41593-025-01954-x]).(https://doi.org/10.1038/s41593-025-01954-x)
 Data are separated into Excel file tabs. **Variables** Tab titled
 "Figure 1"  a)      Histological analysis- SA-β-galactosidase
 activity assay using young and aged mouse DRG tissue. Displayed as
 %positive pixels/area (μm). b)     Generated fluorescence images
 representative of quantified data in c-e below. c)      Histological
 analysis using RNAscope methods to detect RNA expression in young and aged
 mouse DRG tissue. Data is displayed as %p21-positive DRG neurons/ total
 DRG neurons. d)     Histological analysis using RNAscope methods to detect
 RNA expression in young and aged mouse DRG tissue. Data is displayed as
 %p21, IL6 co-positive DRG neurons/ total DRG neurons. e)      Histological
 analysis using RNAscope methods to detect RNA expression in young and aged
 mouse DRG tissue. Data is displayed as %IL6 co-positive with either p21 or
 p16+ DRG neurons/ total IL6+ DRG neurons. f)      Protein quantification
 of IL6 from young and aged plasma measured in pg/mL. Tab titled
 "Figure 2"  b)     Quantitative PCR (qPCR) detecting RNA of p21,
 p16, p53, IL6, IL1B, CCL2, and TNFα from young mice with either sham
 (surgery) or spared nerve injury (SNI) surgery. c)      Histological
 analysis using RNAscope methods to detect RNA expression in young mouse
 DRG tissue. Data is displayed as %p21-positive DRG neurons/ total DRG
 neurons across uninjured conditions and post-SNI injury time points, 7-day
 (7d SNI), 3-week (3wk-SNI), 7-week (7wk-SNI), shown in left-hand columns.
 Same data format for p16%-positive DRG neurons shown on the right. d)    
 Re-analysis of Renthal et al. RNA sequencing data displaying the
 percentage of p16 (*Cdkn2a*+)-expressing senescent cells relative to all
 DRG cells after ScNT. Cells are negative for *Lmnb1* and *Top2a* to filter
 out any non-senescent cells. Glia, satellite glia and Schwann cells;
 Immune & other cells, neutrophils, macrophages, B cells, fibroblasts,
 endothelial cells and pericytes; Neuron, all DRG neurons. e)      Dot plot
 generated of senescence marker gene expression by DRG neurons after ScNT.
 Source data generated by Renthal et al. f)      Histological analysis
 using RNAscope methods to detect RNA expression in aged mouse DRG tissue.
 Data is displayed as %p21-positive DRG neurons/ total DRG neurons across
 uninjured conditions and post-SNI injury time points, 7-day (7d SNI),
 3-week (3wk-SNI), 7-week (7wk-SNI), shown in left-hand columns. Same data
 format for p16%-positive DRG neurons shown on the right. g)    
 Histological analysis using RNAscope methods to detect RNA expression in
 young and aged mouse DRG tissue. Data is displayed as %p21+p16+
 co-positive DRG neurons/ total DRG neurons across uninjured conditions and
 post-SNI injury time point of 3 weeks (3wk-SNI). h)     Representative
 fluorescent images of RNAscope methods to detect p21, p16, and IL6 RNA
 expression in mouse DRG tissue. i)       Histological analysis using
 RNAscope methods to detect RNA expression in young and aged mouse DRG
 tissue. *IL6*+ DRG neurons coexpressing *p21* and/or *p16* (*n* = 5 young
 uninjured mice, *n* = 4 aged uninjured mice, *n* = 4 young 3-week post-SNI
 mice, *n* = 3 aged 3-week post-SNI mice. j)       Histological analysis
 using RNAscope methods to detect RNA expression in young and aged mouse
 DRG tissue post SNI-injury. Data is displayed as %IL6 co-positive with
 either p21 or p16+ DRG neurons/ total IL6+ DRG neurons. Tab titled
 "Figure 2e_dotplot"  * columns A displaying index, column B
 displays gene name, column C gene group, column D cell group displays
 either sciatic nerve transection time points (ScNT) at various hours post
 injury, column E displays the log2 fold change (log2fc), column F is
 ranksum p-value, column G is rank sum false discovery rate (fdr) and
 column H is true or false for differential expression. Tab titled “Figure
 3” a)      Histological analysis-percentage ATF3-positive cells out of all
 DRG neurons from young and aged mouse DRG tissue at post-injury time
 points) in columns B-I. b)     Representative fluorescent images of
 RNAscope methods to detect p21, p16, RNA expression, and
 immunohistochemistry to detect ATF3 protein expression in young and aged
 mouse DRG tissue. c)      Histological analysis using RNAscope methods to
 detect RNA expression in young mouse DRG tissue. Data is displayed as
 %ATF3-positive DRG neurons co-expressing either p21 or p16 / total DRG
 neurons across uninjured conditions and post-SNI injury time points, 7-day
 (7d SNI), 3-week (3wk-SNI), 7-week (7wk-SNI) in young mouse tissue. d)    
 Same data analysis as above ‘c’ in aged mouse tissue. e &f)
 Histological analysis using RNAscope methods to detect RNA expression in
 young mouse DRG tissue. Data is displayed as %ATF3-negative DRG neurons
 co-expressing either p21 or p16 / total DRG neurons. Tab titled “Figure
 3g_dotplot” * Re-analysis of Renthal et al., columns A displaying index,
 column B displays gene name, column C gene group, column D cell group
 displays either sciatic nerve transection time points (ScNT) at various
 hours post injury, column E displays the log2 fold change (log2fc), column
 F is ranksum p-value, column G is rank sum false discovery rate (fdr) and
 column H is true or false for differential expression. Tab titled “Figure
 4” a)      Histological analysis showing cell diameters in μm of p21+IL6+,
 p16+IL6+, or p21+p16+IL6+ DRG neurons from young (columns A-D) and aged
 (columns F-I) mouse DRG tissue at the post-injury 3-week time point.
 b)     Representative fluorescent images of RNAscope methods to detect
 TRPV1, p21, and IL6 RNA expression in young and aged mouse DRG tissue.
 c)      Histological analysis using RNAscope methods to detect RNA
 expression in young mouse DRG tissue. Data is displayed as %TRPV1-positive
 DRG neurons co-expressing either p21 or IL6 / total DRG neurons across
 uninjured conditions and post-SNI injury time point 3-week (3wk-SNI) in
 young mouse tissue. d)     Same data analysis as above ‘c’ in aged mouse
 tissue. Tab titled “Figure 5B-F_UMAP” Electrophysiology whole-cell
 patch-clamp recording from DRG neurons. 33 electrophysiological parameters
 were recorded and clusters identified with the hierarchical density-based
 algorithm HDBSCAN after UMAP alignment of individual neurons constructed
 with diameter (range, 14–41 μm), firing properties, and intrinsic
 currents. Discrete clusters 1–5 are identified (*n* = 82 recorded DRG
 neurons from young (11–16 weeks) and aged (20–24 months) mice). * Column A
 displays cell cluster that each individual cell grouped into, column B
 displays cell diameter, column C is rmp, column D is action potential (AP)
 threshold value, column E displays rheobase, column F is action potential
 latency (ms), column G-J displays either a 1 or 0 indicating expression
 (1) or no expression (0) of each gene in respective column, columns K-N
 are hyperpolarization currents at various current intensities (pA),
 columns O-AK show action potential firing at various current intensities
 (pA), and column AL displays overall action potential firing values. Tab
 titled “Figure 5g” Electrophysiology whole-cell patch-clamp recording from
 DRG neurons in monolayer culture conditions. * Column A displays the date
 that each individual cell was patched, column B displays whether young or
 aged mouse source, column C is sex, column D is injury status, column E
 displays number of cell, column F is action potential half width (ms),
 column G-I displays either AMP (gene expression) or NO_AMP (indicating no
 gene expression) of IL6, p21, or p16. Tab titled “Figure 5h”
 Electrophysiology whole-cell patch-clamp recording from DRG neurons in
 monolayer culture conditions, displaying the number of action potentials
 fired with or without IL6 application. * Column A displays the date that
 each individual cell was patched, column B displays experimental condition
 (control or IL6-application), column C is sex, column D is cell number,
 columns E-J displays either a 1 or 0 indicating expression (1) or no
 expression (0) of each gene in respective column, and column K shows total
 action potentials fired. Tab titled “Figure 6” b) Histological
 analysis-percentage CC3-positive cells out of all DRG neurons from aged
 mouse DRG tissue at post-injury time point after treatment with vehicle
 (column A) or ABT263 (column B). c) Histological analysis-percentage
 CC3-positive cells co-expressing p21 or p16 out of all CC3+ DRG neurons
 from aged mouse DRG tissue at post-injury time point after treatment with
 vehicle (columns B-G) or ABT263 (columns H-M). d) Mouse behavior analyses
 representing von Frey filament thresholds calculated as a percentage of
 baseline responses from aged mice treated with either vehicle (columns
 B-W) or ABT263 (columns B-AC). e) Mouse behavior analysis of weight
 bearing with either vehicle or ABT263 treatment in aged mice at either DAY
 16 or DAY 29 post-treatment start. The calculation of weight bearing on
 the injured hindlimb was as follows: 2 × (L) / (L + R) × 100 to get the
 percent weight bearing on the injured (L, left) hindlimb. f) Mouse
 behavior analyses representing von Frey filament thresholds calculated as
 a percentage of baseline responses from young mice treated with either
 vehicle (columns B-M) or ABT263 (columns B-M). e) Mouse behavior analysis
 of weight bearing with either vehicle or ABT263 treatment in young mice at
 either DAY 16 or DAY 29 post-treatment start. The calculation of weight
 bearing on the injured hindlimb was as follows: 2 × (L) / (L + R) × 100 to
 get the percent weight bearing on the injured (L, left) hindlimb. Tab
 titled “Figure 7” c) Histological analysis of human DRG tissues.
 Percentage of p21+ or p16+ DRG neurons / all DRG neurons in young DRG
 (columns B-C) or aged DRG (columns D-E). d) Histological analysis of human
 DRG tissues. Percentage of IL6+ DRG neurons / all DRG neurons in young DRG
 (columns B-C) or aged DRG (columns D-E). e) Histological analysis of human
 DRG tissues. Percentage of p21+, p16+, or IL6+ co-positive DRG neurons /
 all IL6+ DRG neurons in young DRG (columns B-C) or aged DRG (columns D-E).
 f) Histological analysis of human DRG tissues. Percentage of p21+, p16+,
 IL6+ DRG neurons / all DRG neurons in young DRG (columns B-C) or aged DRG
 (columns D-E). g) Histological analysis-immunohistochemistry (IHC) of
 human DRG tissues. Percentage of ATF3+  DRG neurons / all DRG neurons in
 young DRG (columns B-C) or aged DRG (columns D-E). h) Histological
 analysis-immunohistochemistry (IHC) of human DRG tissues. Percentage of
 ATF3+  co-expressing p21 (RNA) DRG neurons / all ATF3+ DRG neurons in
 young DRG (columns B-C) or aged DRG (columns D-E). i) Histological
 analysis of human DRG tissues. Percentage of TRPV1+ DRG neurons / all DRG
 neurons in young DRG (columns B-C) or aged DRG (columns D-E). k)
 Histological analysis of the number of TRPV1, p21, p16 positive or
 co-positive neurons in young (column B) or aged (column E) human DRG
 tissues. **File: Donovan_SourceData_Fig1-7.xlsx** **Description:** File
 contains raw data generated in Figures 1 through 7 of the main figure data
 file (DOI: [10.1038/s41593-025-01954-x]).
 (https://doi.org/10.1038/s41593-025-01954-x) **Variables** Tab titled
 “Extended_Data_2a_dotplot” Data used to generate a dot plot representing
 RNA-sequencing data (source data: Wang et al.) from mouse DRG tissues with
 spared nerve injury (SNI) at post-injury time points, 1-day (1d), 2-days
 (2d), 6 hours (6h), 7-day (7d), 14-day (14d), 28-day (28d). * Column A
 displays the index, column B displays gene name, column C is the category
 that gene falls under, column D is post-injury time point, columns E is
 the log2fc gene expression from baseline, column F shows ranksum p-value,
 column G shows ranksums-false discovery rate (fdr), and column H either
 notes TRUE or FALSE, describing significance or not. Tab titled
 “Extended_Data_Fig2b_heatmap” RNA sequencing (Renthal et al.) data used to
 generate a heatmap display showing DRG neuron subtype expression summary
 of gene expression that falls under the SenMayo score, normalized by cell
 type post-SNI. * Column A displays summary, column B displays DRG neuron
 subtype, column C is the hours post-spared nerve injury (SNI), column D is
 background naïve, column E is foreground mean, column H shows ranksum
 p-value, column I shows the t-test p-value, and column J either notes TRUE
 or FALSE, describing significance or not. Tab titled “Extended_Data_Fig3”
 c) Mouse behavioral data-The mechanical threshold of the contralateral
 (uninjured) hindlimb was measured as a percent of pre-injury baseline
 after application of senolytic ABT263 or vehicle control in aged mice. d)
 Mouse behavioral data-The mechanical threshold of the contralateral
 (uninjured) hindlimb was measured as a percent of pre-injury baseline
 after application of senolytic ABT263 or vehicle control in young mice. e)
 Mouse behavioral data- Open field showing distance travelled (meters, m)
 of mice post-nerve injury, treated with either vehicle or ABT263. Tab
 titled “Extended_Data_Fig4”  b) Histological analysis of human DRG
 tissues. Percentage of DRG neurons / all DRG neurons which show &gt;75%
 lipofuscin-filled neurons in young DRG (columns B-C) or aged DRG (columns
 D-E). Tab titled “Extended_Data_Fig5” a) Re-analysis of RNA sequencing
 datasets (Yu et al.) from human DRG showing percentage p16+ DRG neurons /
 all DRG neurons in three independent human DRG aged 23 years (column B),
 aged 56 years (column C), or 61 years (column D). b) Re-analysis of RNA
 sequencing datasets (Yu et al.) from human DRG showing percentage p21+ DRG
 neurons / all DRG neurons in three independent human DRG aged 23 years
 (column B), aged 56 years (column C), or 61 years (column D). c)
 Re-analysis of RNA-sequencing datasets (Yu et al.) from human DRG showing
 percentage ATF3+ co-positive with either p21 or p16 DRG neurons / all
 ATF3+ DRG neurons in three independent human DRG aged 23 years (column B),
 aged 56 years (column C), or 61 years (column D). d,e) Re-analysis of
 RNA-sequencing datasets (Renthal et al.) from human DRG from 21
 independent human DRG samples. Column A is the sample number, column B is
 the index describing the level of DRG excised, column C denotes whether a
 human DRG sample was taken from a patient with at-level pain, column D
 shows age of the human at time of excision, column E shows cdkn2a (p16)
 gene expression value, and column F shows the senmayo gene set correlation
 score. Tab titled “Extended_Data_Fig6” a)      Histological analysis
 showing cell diameters in μm of p21+IL6+, p16+IL6+, or p21+p16+IL6+ DRG
 neurons from young (columns A-G) human DRG tissue. b)     Histological
 analysis showing cell diameters in μm of p21+IL6+, p16+IL6+, or
 p21+p16+IL6+ DRG neurons from aged (columns A-G) human DRG tissue. ##
 Access information Re-analysis of RNA sequencing data was derived from the
 following sources: * North, R. Y. et al. Electrophysiological and
 transcriptomic correlates of neuropathic pain in human dorsal root
 ganglion neurons. Brain 142, 1215–1226 (2019). DOI:
 [10.1093/brain/awz063](https://doi.org/10.1093/brain/awz063) * Yu, H. et
 al. Leveraging deep single-soma RNA sequencing to explore the neural basis
 of human somatosensation. Nat Neurosci. 27, 2326–2340 (2024). DOI:
 [10.1038/s41593-024-01794-1](https://doi.org/10.1038/s41593-024-01794-1) *
 Renthal, W. et al. Transcriptional reprogramming of distinct peripheral
 sensory neuron subtypes after axonal injury. Neuron 108, 128–44 (2020).
 DOI:
 [10.1016/j.neuron.2020.07.026](https://doi.org/10.1016/j.neuron.2020.07.026) * Wang, K. et al. Single-cell transcriptomic analysis of somatosensory neurons uncovers temporal development of neuropathic pain. Cell Res. 31, 904–918 (2021). DOI: [10.1038/s41422-021-00479-9](https://doi.org/10.1038/s41422-021-00479-9) ## Human subjects data Data has been de-identified by only providing age, sex and cause of death. All samples are post-mortem and patients were registered organ donors consented prior to death.