Methods of treating pain using senolytics

Abstract

Provided are methods of treating pain in a subject in need thereof. The methods comprise administering to the subject a senolytic in an amount effective to treat the pain. According to some embodiments, the senolytic is administered as the only active agent to treat the pain. In other embodiments, the senolytic is administered in combination with a second active agent (e.g., a second senolytic or non-senolytic active agent) to treat the pain. Non-limiting examples of pain treatable by the methods include chronic inflammatory pain, surgery-induced pain, complex regional pain syndrome (CRPS) type 1 or type 2, neuropathic pain, diabetic peripheral neuropathy, and age-related pain. Also provided are kits that find use in practicing the methods of the present disclosure.

Description

[0001] METHODS OF TREATING PAIN USING SENOLYTICS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 613,596, filed December 21 , 2023, which application is incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with Government support under contracts AG075622 and AG08805201 awarded by the National Institute of Aging. The Government has certain rights in the invention.

[0006] INTRODUCTION

[0007] Aging impacts the peripheral nervous system (PNS) as evidenced by molecular, physiological, and functional changes in primary sensory neurons which ultimately alters sensory processing such as touch, proprioception, and pain. (1 -5) In part, these changes arise from the increased pro-inflammatory environment of the aged body, collectively termed “inflammaging”.(4, 6-10) In the context of aging, insults such as injury trigger more severe morphological and physiological changes in sensory neurons when compared to younger counterparts. (3, 1 1 , 12) Further mechanistic studies using aged animals are needed to fundamentally understand the interaction between aging and injury and to determine to what extent this may increase susceptibility to sensory neuron dysfunction. (7, 13)

[0008] One cellular mechanism common to both aging and injury (among other cellular stressors), is cellular senescence. (14, 15) Cellular senescence is a complex cellular state in which cells irreversibly cease cell division, resist apoptosis and cell death, and undergo long-term transcriptional changes that can manifest as expression of a pro-inflammatory senescence- associated secretory phenotype (SASP). Senescent cells gradually accumulate with age in essentially all tissues in the body, including the brain and spinal cord. They contribute to inflammaging through their secretion of SASP factors, which include cytokines and chemokines, making SASP a key deleterious feature of these cells. (14, 16-18) Senescent cells are found to further accumulate in the context of disease and injury, leading to so-called premature senescence, in either young or aged animals. (15, 19, 20) Identification of senescence in vivo relies on sets of markers including upregulation of p2lWAF1 / CIP1(p21 ) and p16INK4A(p16) , which function to drive early and late-stage senescence programs, senescence associated-B- galactosidase activity (SA-p-gal), and expression of associated SASP factors such as IL6. (21 , 22)

[0009] In mice, genetic or pharmacological elimination of senescent cells in disease and injury contexts can improve molecular and functional outcomes, demonstrating that senescent cells contribute to pathology. (20, 23-33) Importantly, senescent cells are heterogeneous with a phenotype (i.e. expression of senescent markers / SASP) that varies depending on the cell type, context of induction, and timing after induction(34). Early senescence can have beneficial functions while chronic / late-stage senescence can have deleterious functions resulting from longterm expression of proinflammatory SASP. (35-37) The heterogeneity of senescence demands a more detailed analysis of senescent phenotype to appropriately evaluate and target only deleterious cells.

[0010] Senescence has been extensively studied in mitotic cells, given its originally described feature of cell cycle-arrest, in the context of cancer where it is being pursued as a therapeutic target. However, there is now some evidence that post-mitotic cells can acquire senescent signatures with associated functional SASP. (38-40) These findings have recently sparked interest in evaluating the potential for senescence of post-mitotic neurons within the nervous system(27, 29, 41 , 42). There are limited reports thus far describing senescent phenotypes in neurons. In one study, mouse cortical, Purkinje, and myenteric ganglia neurons displayed p21 - driven senescence accompanied by pro-inflammatory and pro-oxidant phenotypes which became more severe with age. (18) Intriguingly, human pyramidal, cortical, and myenteric neurons can express p16 (Cdkn2a) suggesting human neurons may also senesce.(28, 43, 44)

[0011] It remains unknown whether primary sensory neuron senescence occurs with age or after injury, and if so, to what extent this impacts sensory processing. Sensory neurons are susceptible to damage of their peripheral axons in a variety of contexts including limb trauma or surgery. Damage to sensory axons results in persistent sensitization of neurons in the spinal dorsal horn (i.e. central sensitization); thus peripheral nervous system sensitization serves as a major driver of chronic pain. (45, 46) Further, chronic inflammation localized at the dorsal root ganglia (DRG) can contribute to long-lasting neuropathic pain triggered by nociceptors following injury.(47) For example, cytokine and chemokine signaling at nociceptors upregulates Trpvl ion-channel receptor expression and can modulate channel conductance, further exacerbating nociceptor hyperexcitability.(48-51 ) Inflammatory molecules that mediate nociceptor hyperexcitability are common SASP factors released by senescent cells. (52)

[0012] SUMMARY

[0013] Provided are methods of treating pain in a subject in need thereof. The methods comprise administering to the subject a senolytic in an amount effective to treat the pain. According to some embodiments, the senolytic is administered as the only active agent to treat the pain. In other embodiments, the senolytic is administered in combination with a second active agent (e.g., a second senolytic or non-senolytic active agent) to treat the pain. Non-limiting examples of pain treatable by the methods include chronic inflammatory pain, surgery-induced pain, complex regional pain syndrome (CRPS) type 1 or type 2, neuropathic pain, diabetic peripheral neuropathy, age-related pain, chronic back pain, pain from osteoarthritis, pain associated with fibromyalgia or benign hypermobility, a widespread pain condition, musculoskeletal pain syndrome, pain associated with endometriosis or vulvodynia, chronic pelvic pain, neck pain, and pain associated with sacroiliac joint dysfunction. Also provided are kits that find use in practicing the methods of the present disclosure.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1A-1 F: Senescent neurons accumulate with age in the mouse DRG. A. Representative images of SA-p-gal activity staining (blue) in the lumbar DRG of young and aged mice. Percent SA-p-gal positive pixels per DRG area (right) (n= 5-6 mice per group; two-tailed unpaired t-test, *p<0.05 ). B. Representative RNAscope images for senescent markers p21 and p16 with SASP factor IL6 in whole DRG section. C. Quantification of neuronal expression of each marker or in combination (D) expressed as a percent of total DRG neurons (n=4-5 mice per group, two-tailed unpaired t-test, *p<0.05, **p<0.01 , ***p<0.001 ). E. Analysis of / L6-expressing DRG neuron population to show co-expression with senescence markers p21 and / or p16 in young and aged mice (n=4-5 mice per group). F. Quantification of IL6 protein levels by ELISA assay in young or aged plasma (n=5-6 mice per group, two-tailed unpaired t-test, *p<0.05). Data are expressed as the mean ± SEM.

[0016] FIG. 2A-2K: DRG neurons express senescent markers and SASP factors following peripheral nerve injury. A. Schematic of spared nerve injury (SNI) model in mice. Timeline which DRG tissues were analyzed following SNI. B. qPCR of a panel of senescent markers and SASP factor gene expression from whole lumbar DRG in controls or following SNI (3-weeks post) in young mice (n=4 control; n=4 SNI young male mice). C. Representative RNAscope image of whole DRG section from uninjured or 3-weeks post-SNI in young mice. D. Quantification of the number of L3 / 4 DRG neurons expressing either p21 (upper graph) or p16 (lower graph) in male and female young mice. E. Representative RNAscope image of whole DRG section from uninjured or 3-weeks post-SNI in aged mice. F. Quantification of the number of L3 / 4 DRG neurons expressing either p21 (upper graph) or p16 (lower graph) in male and female aged mice. G. Quantification of p21 +p16+ co-expressing DRG neurons as a percent of total DRG neurons in uninjured and 3-weeks post-injury in young and aged mice. H. Representative RNAscope images showing co-expressing senescent markers (p21 and p16) with SASP factor (IL6). I. Quantification of p21 +IL6+ co-expressing DRG neurons as a percent of total DRG neurons in uninjured and 3-weeks post-injury in young and aged mice. J. Quantification of p21 +p16+IL6+ triple positive DRG neurons as a percent of total DRG neurons in uninjured and 3-weeks postinjury in young and aged mice. K. Analysis of IL6-expressing DRG neuron population which coexpress senescent markers p21 and / or p16 at 3-weeks post-injury in young and aged mice.

[0017] FIG. 3A-3F: ATF3+ injured and neighboring non-injured DRG neurons express senescent markers after nerve injury. A. Quantification of number of ATF3+ neurons as a percent of total L3 / 4 DRG neurons in uninjured and multiple post-SNI time points in young and aged mice (n=3-5 mice per group / time-point, one-way ANOVA, *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 ). B. Representative images of dual immunohistochemistry / RNAscope labeling ATF3+ injured neurons (nuclear localized protein) and RNA puncta of p21 and p16 at 3-weeks post-SNI. Co-expression of ATF3 with p21 and or p16 (arrows). Asterisks represent ATF3- negative cells that express p21 and / or p16 senescent markers. C & D. Quantification of ATF3- positive neuron population which co-express p21 and / or p16 at multiple time points post-injury in young and aged DRG. E. Quantification of ATF3-negative population co-expressing senescent marker p21 at multiple time points post-injury in young and aged DRG (n=3 mice per group per time-point, one-way ANOVA, *p<0.05). F. Quantification of ATF3-negative population coexpressing senescent marker p16 at multiple time points post-injury in young and aged DRG (n=3-5 mice per group per time-point, one-way ANOVA, *p<0.05). Data are expressed as the mean ± SEM.

[0018] FIG. 4A-4D: Trpvl + nociceptors express senescent marker p21 and SASP factor IL6 following nerve injury. A. Analysis of cell diameter (pm) of p21 +IL6+, p16+IL6+, or p21 +p16+IL6+ co-positive neurons in the DRG at 3-weeks post-nerve injury in young and aged mice (Young: n=215 p21+IL6+ neurons; n=51 p16+IL6+ neurons; n=102 p21 +p16+IL6+ neurons; Aged: n=155 p21 +IL6+ neurons; n=21 p16+IL6+ neurons; n=46 p21 +p16+IL6+ neurons). B. Representative RNAscope images of young or aged DRG co-labeled for the ion-channel Trpvl , senescent marker p21 , and SASP factor / cytokine IL6. Merged images also have DAPI overlay (grey). For I L6-signal, intense puncta signal with white center are positive for IL6, while fainter / dull blue is background. Scale bars 100pm and 20pm (insets). Quantification of Trpvl neuron population and its co-expression with p21 and / or IL6 in young (C) and aged (D) L3 / 4 DRG of uninjured (controls) and 3-weeks post-SNI (n=3 uninjured, n=3 SNI young mice; n=4 uninjured, n=2 SNI aged mice).

[0019] FIG. 5A-5J: Electrophysiological profiles are distinct in senescent neurons and in vivo elimination of senescent neurons using senolytics alleviates pain behaviors after nerve injury. A. Experimental design includes whole-cell electrophysiological recordings from ex vivo DRG in aged mice, followed by single-cell qPCR to identify neuronal markers of senescence, nerve injury, and cytokine expression. B. Representative traces from current injection protocols recorded in a neuron found to express p16 (senescence), ATF3 (injury), and Tubb3 (neuronal). C. Representative traces from current injection protocols from a p21 +, ATF3+, IL6+ (SASP / cytokine), and Tubb3-expressing neuron. D. Schematic of treatment paradigm. Young and aged mice were treated with senolytic (ABT263, 10Omg / kg, p.o. daily) or vehicle for 10 days, starting at 3-weeks post-spared nerve injury (SNI). Mechanical allodynia and weight bearing were assessed during and after treatment. E. Senolytics induce apoptosis in the DRG. Representative image of CC3-immunohistochemistry to capture apoptotic cells after 5-day treatment with ABT263 (white arrows = CC3-positive neurons, other red fluorescent signal is background lipofuscin). Quantification of cleaved-caspase-3 (CC3) positive neurons in the DRG following treatment with vehicle or ABT263 for 5 consecutive days (n=3 male, n=3 female aged mice per treatment group, unpaired t-test **p<0.01 ). F. Aged mice were treated with ABT263 or vehicle (light blue indicates treatment window) and their mechanical allodynia thresholds were assessed (n=13 female, n=9 male vehicle-treated mice; n=14 female, n=13 male ABT263-treated mice, mixed-effects analysis, Sidak's multiple comparisons test, *p<0.05, **p<0.01 , ****p<0.0001 ). G. Aged mice treated with ABT263 displayed improved weight bearing on injured limb compared to vehicle-treated mice at both Day 16 (n=10 female, n= 8 male vehicle-treated mice; n=9 female, n=9 male ABT263-treated mice, unpaired t-test, ***p<0.001 ) and Day 39 post-treatment start (n=9 female, n= 7 male vehicle-treated mice; n=9 female, n=9 male ABT263-treated mice, unpaired t-test, ****p<0.0001 ). H. Young mice treated with ABT263 or vehicle (light blue indicates treatment window) and their mechanical allodynia thresholds were assessed (n=12 male vehicle- treated mice, n=12 male ABT263-treated mice). I. Young mice treated with ABT263 displayed improved weight bearing on injured limb compared to vehicle-treated mice at both Day 16 (n=1 1 vehicle-treated, n=13 ABT263-treated male mice, unpaired t-test, ****p<0.0001 ) and Day 29 posttreatment start (n=5 vehicle-treated, n=5 ABT263-treated male mice, unpaired t-test **p<0.01 ). J. The fraction of CC3-positive neurons that co-express both senescence markers p16 and p21 was increased in ABT263-treated mice, indicating senescent neurons were targeted by ABT263.

[0020] FIG. 6A-6K: Human DRG neurons express senescent markers and SASP factor IL6 with age. A & B. Representative RNAscope images from young (33yr) or aged (65yr) human L4 DRG showing expression of p21 and p16 senescent markers (enlarged left images, scale bar 100pm). Overlayed non-puncta signal is lipofuscin fluorescence and not RNAscope signal. C. Quantification of p21 +, p16+, and co-positive p21 +p16+ neurons in the young and aged human DRG as a percent of total DRG neurons. D. Quantification of IL6-expressing neurons as a percent of total DRG neurons. E. Analysis of IL6-positive neuron population and quantification of the coexpression of senescent markers p21 and / or p16. F. Quantification of neurons co-expressing senescent markers p21 and / or p16 with IL6 as a percent of total DRG neurons. G. Percent of DRG neurons which are ATF3-positive in young and aged human DRG. H. Example image depicting a single human neuron positive for ATF3 (nuclear-localized, immunohistochemistry) and p21 (RNAscope). Scale bars 20pm. Analysis of ATF3-positive neuron population and quantification of the co-expression with p21 in young and aged human DRG (right, donuts). I. Total percentage of Trpv1 + neurons as a percent of total DRG neurons in young and aged human DRG. Boxed right, Quantification of the subsets of TRPV1 + neurons which co-express either p21 or p16 by RNAscope. J. Single human neurons showing co-expression of Trpvl with p21 and / or p16. DAPI in grey. Scale bars 20pm. K. Venn diagram of human DRG neurons which express TRPV1 , p16, and p21. Aged DRG display a greater overlapping fraction of TRPV1 + neurons expressing either or both senescent markers p21 and p16 compared to young neurons.

[0021] FIG. 7: Confirmation of p16INK4A-specific RNA expression in the DRG. RNAscope using RNA probes spanning exons encoding both p16INK4Aand p19ARFprotein (Cdkn2a-tv1). Cdkn2a-tv2 RNA probe spans exons specific to p16INK4Aprotein. Complete cellular co-localization of the Cdkn2a variants in mouse lumbar DRG sections confirming p16-specific expression in the mouse lumbar DRG. Scale bar of upper panels are 25pm. Scale bars of inset are 15pm.

[0022] FIG. 8A-8B: Increased percentage of human DRG neurons filled with lipofuscin with age. A. Representative neurons in aged (65yr) human DRG with accumulated lipofuscin, a marker of senescence. Example of neuron either mostly filled (left arrow) or completely filled (right arrow). Scale bar is 10pm. B. Quantification of DRG neurons whose cell bodies were greater than 75% occluded by lipofuscin as a percentage of all DRG neurons in young and aged human DRG.

[0023] FIG. 9A-9B: SASP-expressing senescent neuron diameters in young (A) and aged (B) human DRG. Cell diameters (pm) of human DRG neurons co-expressing either p21 +IL6+, p16+IL6+, or p21 +p16+IL6+, as a percent of total neurons counted in each population.

[0024] FIG.10A-10E: Confirmation of human DRG senescence with age and in painful conditions using existing RNA-sequencing datasets. A-C. Re-analysis of Yu et al. (66) singlesoma human DRG RNAseq dataset. Bar graphs represent the percent positive p16 (CDKN2A cells (A) and p21 (CDKN1 A) cells (B) of all DRG cells, which are also negative for LMNB1 , MKI67, TOP2A to filter out any proliferating and otherwise non-senescent cells. C. Percent of ATF3+ neurons which are co-positive for wither p21 and / or p16 senescence markers. D & E. Re-analysis of North et al. (67) human DRG bulk RNAseq dataset for expression of p16 (CDKN2A) (D) or SenMayo gene set € across multiple ages using DRG samples taken from patients either with associated pain (filled box) or without pain (open box). Significant correlation found for CDKN2A expression with age (Spearman correlation, coefficient= 0.612, p=0.00321 ). Significant association of SenMayo gene expression in pain DRG samples.

[0025] FIG.11A-11C: FOXO4-DRI treatment. A. Mechanical allodynia in young and aged mice after SNI was significantly improved after treatment with the senolytic, FOXO4-DRI (***p<0.001 vs. age-matched vehicle by ANOVA). B. Senolytic treatment did not result in any change in latency to a 50°C hot plate or (C) 4°C cold plate, suggesting preservation of sensory function after senolytic treatment.

[0026] DETAILED DESCRIPTION

[0027] Before the methods and kits of the present disclosure are described in greater detail, it is to be understood that the methods and kits are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and kits will be limited only by the appended claims.

[0028] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and kits. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and kits, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and kits.

[0029] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and kits belong. Although any methods and kits similar or equivalent to those described herein can also be used in the practice or testing of the methods and kits, representative illustrative methods and kits are now described.

[0031] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and kits are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0032] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0033] It is appreciated that certain features of the methods and kits, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and kits, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and kits and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0034] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0035] METHODS OF TREATING PAIN

[0036] Aspects of the present disclosure include methods of treating pain. In certain embodiments, provided are methods of treating pain in a subject in need thereof, the methods comprising administering to the subject a senotherapeutic in an amount effective to treat the pain. The senotherapeutic may be a senolytic or a senomorphic / senostatic. In certain embodiments, provided are methods of treating pain in a subject in need thereof, the methods comprising administering to the subject a senolytic in an amount effective to treat the pain. In certain embodiments, provided are methods of treating pain in a subject in need thereof, the methods comprising administering to the subject a senostatic in an amount effective to treat the pain. The methods are based in part on the inventors’ discovery that primary sensory neurons (e.g., those of the dorsal root ganglia (DRG)) senesce with age and after peripheral injury, and that in vivo targeting of these senescent primary sensory neurons improves pain-related outcomes.

[0037] As used herein, a “senolytic” or “senolytic agent” is an agent that promotes apoptosis in senescent cells. In some instances, such an agent targets one or more proteins in a senescent cell anti-apoptotic pathway (SCAP). According to some embodiments, the methods of the present disclosure employ a senolytic that specifically targets senescent cells or a subtype thereof, e.g., senescent primary sensory neurons. For example, the methods may employ a senolytic that induces targeted apoptosis of senescent cells (TASC).

[0038] A wide variety of senolytics may be employed when practicing the methods of the present disclosure. Suitable senolytics include the various senolytics currently known and available, as well as senolytics yet to be identified or currently in development. According to some embodiments, the senolytic targets the phosphatidylinositol 3-kinase (PI3K) / AKT pathway, a B- cell lymphoma-2 (BCL-2) protein family pathway, a BCL family member, MDM2 / p53 pathway, HSP90 pathway, Wnt / p-catenin pathway, MAPK pathway, histone deacetylase (HDAC) pathway, glycolysis pathway, caspase pathway, ATM pathway, ZNF91 pathway, Protein kinase C (PKC) pathway, poly ADP-ribose polymerase (PARP) and sirtuin pathway, interaction between P53 and FOXO4, the Bax / Bcl-2 pathway, the Nrf2 pathway, or any combination thereof. Strategies and senolytics for in vivo targeting of senescent cells are described, e.g., in Zhu et al. (2020) Ther Adv Chronic Dis. 11 :2040622320964125; Chaib et al. (2022) Nature Medicine 28(8):1556-1568; Gasek et al. (2021 ) Nature Aging 1 :870-879; Zhang et al. (2022) The FEBS Journal 290(5): 1362- 1383; and Barr et al. (2017) Cell 169(1 ):132-147.e16; the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0039] Examples of a senolytic targeting BCL-2 protein family pathway include Navitoclax (ABT263), A1331852, A1155463, ABT737, ABT199 (Venetoclax), and BI-97C1 (Sabutoclax). FOXO4-DRI is a senolytic that targets interaction between p53 and FOXO4. UBX0101 is a senolytic that targets the MDM2 / p53 pathway. Senolytics, 17-DMAG, Alvespimycin, Ansamycin, 17-AAG, and Geldanamycin target the HSP90 pathway. Examples of a senolytic targeting PI3K / AKT pathway include Dasatinib, Quercetin, Fisetin, Curcumin, Catechin, Procyanidin C1 , Apigenin, Epigallo-Catechin-Gallate (EGCG), and Enzastaurin. The senolytic Apigenin targets the Wnt / p-catenin pathway. The MAPK pathway is targeted by senolytics such as Apigenin, Curcumin, Epigallo-Catechin-Gallate (EGCG), and Luteolin. Examples of a senolytic targeting the HDAC pathway include Panobinostat, Vorinostat, Valproic acid, Trichostatin A (TSA), and D- P-hydroxybutyrate (DBHB). The senolytic 2-DG targets the glycolysis pathway. Caspase pathway- targeting senolytics include piperlongumine and OVD-OPh. KU-60019 is a senolytic that targets the ATM pathway. Resorcinol is a senolytic that target the AT ZNF91 M pathway. Senolytics Luteolin and Epigallo-Catechin-Gallate (EGCG) target the Nrf2 pathway. Enzastaurin is a senolytic that targets the PKC Pathway. Examples of a senolytic targeting the PARP & Sirtuin pathway include Nicotinic acid, Nicotinic acid (pyridine-3-carboxylic acid), and Nicotinamide (niacinamide or pyridine-3-carboxamide). Senolytics such as combination of Dasatinib and Quercetin, Quercetin, Catechin, Curcumin, Apigenin, Epigallo-Catechin-Gallate (EGCG), luteolin, and Enzastaurin may be used to target two or more of the above-listed pathways.

[0040] As used herein, a “senomorphic” or “senostatic” is an agent that suppress senescence- associated secretory phenotypes (SASP) of senescent cells. Examples include rapamycin and RAD001 which target the mammalian target of rapamycin (mTOR) pathway and metformin which targets the ataxia-telangiectasia mutated (ATM) pathway.

[0041] According to some embodiments, the senolytic administered to the subject is ABT263, FOXO4-DRI, dasatinib, quercetin, fisetin, A1331852, A1155463, procyanidin C1 , curcumin, catechin, ABT737, panobinostat, apigenin, epigallo-catechin-gallate (EGCG), piperlongumine, 17-DMAG, ansamycin, resorcinol, luteolin, enzastaurin, MK1903, D-P-hydroxybutyrate (DBHB), nicotinic acid, rapamycin, nicotinamide, or any combination thereof.

[0042] In certain embodiments, a single senolytic is administered to the subject as the only active agent to treat the pain. In some instances, a single senolytic is administered to the subject in combination with a second non-senolytic active agent to treat the pain. According to further embodiments, two or more senolytics are administered to the subject to treat the pain. For example, the methods may employ two or more senolytics independently selected from ABT263, FOXO4-DRI, dasatinib, quercetin, fisetin, A1331852, A1 155463, procyanidin C1 , curcumin, catechin, ABT737, panobinostat, apigenin, epigallo-catechin-gallate (EGCG), piperlongumine, 17-DMAG, ansamycin, resorcinol, luteolin, enzastaurin, MK1903, D-|3-hydroxybutyrate (DBHB), nicotinic acid, rapamycin, and nicotinamide. Non-limiting examples of such combinations of senolytics include a combination comprising dasatinib and quercetin.

[0043] According to some embodiments, the senolytic or the senostatic administered to the subject is a peripherally restricted senolytic or the senostatic. By “peripherally restricted” is meant the senolytic or the senostatic is excluded from the central nervous system (CNS) by the bloodbrain barrier. By virtue of being excluded from the CNS, the peripherally restricted senolytic or the senostatic may act on the rest of the body (e.g., on senescent primary sensory neurons) without producing side-effects related to its effect on the brain or spinal cord.

[0044] Antibody-Drug Conjugates (ADCs):

[0045] Senolytics or senostatics may be linked to an antibody that binds to a cell surface antigen expressed on senescent cells. Examples of such antigens include cell surface proteins / receptors such as Dipeptidyl peptidase-4 (DPP4), Decoy receptor 2 (DcR2), and EphA2.

[0046] Exemplary anti-DPP4 antibodies include anti-DPP4 antibody [EPR18215] (Abeam, ab187048), anti-DPP4 antibody [EPR21945] (Abeam, ab222716), anti-DPP4 antibody [EPR22233-135] (Abeam, ab229018), and BEGEDINA® (Begelomab) (a murine immunoglobulin G (IgG) 2b monoclonal antibody against CD26 (dipeptidyl peptidase-4; DPP4)).

[0047] Exemplary anti-DcR2 antibodies include anti-DcR2 antibody [EPR3588(2)] (Abeam, ab108421 ), anti-DcR2 antibody (Abeam, ab2019), anti-DcR2 antibody [EPR3588(2)] - BSA and Azide free (Abeam, ab247666).

[0048] Exemplary anti- EphA2 receptor antibodies include Anti-Eph receptor A2 antibody [EPR24711 -1 1] (Abeam, ab2731 18), Anti-Eph receptor A2 antibody [EPR17660-120] (Abeam, ab185156), Anti-EphA2 antibody [BLR136H] - BSA free (Abeam, ab314085), Anti-Eph receptor A2 antibody [RM-0051 -8F21 ] (Abeam, ab73254), and antibody DS-8895a against EphA2 that has been used in a patient clinical trial, NCT0225221 1 , with no dose-limiting toxicities or treatment-related adverse events reported (PMID: 35404015).

[0049] Bivalent Molecules:

[0050] Another example of a senolytic includes bivalent molecules linking CDK9, CDK12, or CDK13 inhibitors to BCL-2 family members ligands (i.e. BCL-2, BCL-6, BCL-XL, MCL-1 , BCL-W) to specifically kill BCL2 family-overexpressing cells, which include senescent cells. These molecules function by re-localizing transcriptional kinases (CDKs) to activate cell death gene expression ordinarily repressed by BCL2 family members to activate apoptosis in senescent neurons. (Sarott et al., Science 2024, Vol 386, Issue 6717). Proteolysis-targeting Chimeras (PROTACs):

[0051] PROTACs are bivalent small molecules containing a ligand that recognizes a target protein linked to another ligand that recruits a specific E3 ubiquitin ligase. In senescent cells, the anti-apoptotic proteins like BCL-2 and BCL-xL are often overexpressed, contributing to the resistance of these cells to apoptosis. A senolytic may be a PROTAC that targets these proteins for degradation by ubiquitin ligase and induces senescent cell death by inhibiting anti-apoptotic pathway expression. {Angew. Chem. Int. Ed. Engl. 55, 1966-1973 (2016); Lai, A. C. & Crews, C. M. Nat. Rev. Drug Discov. 16, 101-114 (2017)).

[0052] Small Molecule Prodrugs:

[0053] Prodrugs are chemically modified drugs that become active only after being metabolized by target cells. Examples of senescent cell specific senolytic prodrug include [3-galactosidase prodrugs, based on increased beta-galactosidase activity which occurs in senescent cells specifically. The design of the [3-galactosidase prodrug includes any combination of selected compound with cytotoxicity for senescent cells (such as gemcitabine), linked to an acetyl group to improve cell permeability and [3-gal-responsive moiety to improve senescent cell specificity.

[0054] An example includes SSK1 , which includes gemcitabine, a potent molecule which eliminates non-dividing senescent cells and is FDA-approved with a proven safety profile and short plasma circulation time. (Cai et al., Cell Research, 2020, volume 30, pages574-589)

[0055] SSK'1

[0056] An example of a senolytic may include pro-drug combination with proteolysis-targeting chimeras (PROTACs). For example, the senolytic agent may include galactose, the SA-[3- galactosidase substrate, and the PROTAC (e.g. PZ15227 (PZ)) He et al., Nature Communications, 2020; PMID: 32332723). Examples of this include Gal-ARV-771 and Gal- MS99, which is effective in eliminating senescent cancer cells in lung tissue (Chang et al., J Med Chem, 2024; PMID: 38635879). Targeted Peptide Conjugates:

[0057] Senolytic / senostatic disclosed herein may be conjugated to peptides that have high affinity for specific receptors on senescent cells (e.g., Dipeptidyl peptidase-4 (DPP4), Decoy receptor 2 (DcR2), EphA2).

[0058] DPP-4 inhibitors, also known as gliptins, are a class of oral medications used to treat type 2 diabetes in adults which may serve as ligands for this receptor. Some examples of FDA- approved DPP-4 inhibitors include: sitagliptin, saxagliptin, linagliptin, and alogliptin. DcR2 blocking peptides are commercially available from ProSci, CATALOG NUMBER: 2021 P. YSA and SWL peptides are used to bind EphA2.

[0059] RNA-based Targeting:

[0060] A small interfering RNA (siRNA) or antisense oligonucleotides (ASOs) that bind to CDKN1 A or CDKN2A encoding RNA, and enable apoptosis of the cell may be used in the method for treating pain. siRNA or ASOs would be designed toward anti-apoptotic regulators such as BCL-2 family proteins (e.g., siRNA or ASOs that bind to mRNA encoding BCL-2, BCL-6, BCL-XL, MCL-1 , BCL- W) so as to disable these pathways, enabling the pro-apoptotic pathways known to be upregulated in senescent cells to induce apoptosis in senescent cells, thereby acting as a senolytic agent. These ASOs may be administered in combination with an antibody (e.g., anti- Dipeptidyl peptidase-4 (DPP4), Decoy receptor 2 (DcR2), or EphA2 antibody) conjugated to a senolytic / senostatic. The administration may be local injection to enhance specific targeting of senescent neurons in the DRG. Alternatively, ASOs may be packaged into nanoparticles or exosomes.

[0061] The senolytics and senostatics disclosed herein may be linked to cell penetrating peptides to enhance uptake of the senolytic or senostatic by cells. Any of the above listed senolytics and senostatics may be used in any combination to enhance the specificity, delivery, and efficacy of the senolytics and senostatics.

[0062] ROUTES OF ADMINISTRATION

[0063] The senolytic or the senostatic may be administered to the subject via any route of administration suitable for reaching the target senescent neurons, e.g., senescent primary sensory neurons (e.g., the DRG). In some instances, the senolytic or the senostatic is administered orally, e.g., as a tablet, capsule or the like. Other suitable routes of administration include parenteral routes of administration, non-limiting examples of which include transforaminal, intravenous, transdermal, intrathecal, intramuscular, or subcutaneous administration.

[0064] Focused ultrasound delivery may be used for targeted delivery of the senolytic or the senostatic. This technique may be used for enhancing the permeability of certain cell membranes in a localized manner, allowing for targeted delivery of the senolytic or the senostatic while minimizing systemic exposure. For DRG targeting, this approach may be used as an alternative to a local injection or in addition to local injection. A senolytic or senostatic may be packaged in microbubbles, liposomes, nanoparticles, micelles, nanoemulsions, or sonosensitive hydrogels, delivered to a location in the subject, e. g., spine, and uncaged by applying ultrasound at location.

[0065] PHARMACEUTICAL COMPOSITIONS

[0066] The methods may comprise administering a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises the senolytic or the senostatic (which may include a combination of senolytics or senostatics) and a pharmaceutically acceptable carrier. The pharmaceutical compositions generally include a therapeutically effective amount of the senolytic or the senostatic. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a pain parameter, as compared to a control. An effective amount can be administered in one or more administrations.

[0067] The pharmaceutical compositions may include liposomes, nanoparticles, e.g., polymeric or gold nanoparticles encapsulating the senolytic or senostatic or senolytic-linked or senostatic- linked prodrug / PROTACs. These liposomes and nanoparticles may display ligands or antibodies for targeting these liposomes and nanoparticles to senescent cells.

[0068] The pharmaceutical compositions may include exosomes, naturally occurring extracellular vesicles, engineered to enclose a senolytic or senostatic and to express ligands that direct them to specific senescent cells.

[0069] The pharmaceutical compositions may include microbubbles, liposomes, nanoparticles, micelles, nanoemulsions, or sonosensitive hydrogels, encapsulating the senolytic or senostatic.

[0070] Senolytics may be packaged into exosomes consisting of specialized coating or modifications with target ligands or antibodies to increase specificity of targeting to DRG senescent neurons.

[0071] The senolytic or the senostatic can be incorporated into a variety of formulations for therapeutic administration. More particularly, the senolytic or the senostatic can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

[0072] Formulations of the senolytic or the senostatic suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration. In pharmaceutical dosage forms, the senolytic or the senostatic can be administered in the form of its pharmaceutically acceptable salt, or it may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0073] For oral preparations, the senolytic or the senostatic can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0074] The senolytic or the senostatic can be formulated for parenteral (e.g., transforaminal, intravenous, transdermal, intrathecal, intra-arterial, intraosseous, intramuscular, subcutaneous, intraperitoneal, etc.) administration. In certain embodiments, the senolytic or the senostatic is formulated for injection by dissolving, suspending or emulsifying the senolytic in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0075] Pharmaceutical compositions that include the senolytic or the senostatic may be prepared by mixing the senolytic or the senostatic having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0076] The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.

[0077] An aqueous formulation of the senolytic or the senostatic may be prepared in a pH- buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0078] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0079] A surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w / v.

[0080] A lyoprotectant may also be added in order to protect the senolytic against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 nM.

[0081] In some embodiments, the pharmaceutical composition includes a senolytic or a senostatic and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v).

[0082] SUBJECTS

[0083] A variety of subjects are treatable according to the methods of the present disclosure. Generally such subjects are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the subject is a human. The human subject may be male or female. The human subject may be an older person, e.g., at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 years of age.

[0084] The methods of the present disclosure find use in treating a variety of pain types. In certain embodiments, the subject has chronic inflammatory pain, and the methods treat the chronic inflammatory pain. A non-limiting example of chronic inflammatory pain is injury-induced pain.

[0085] In some instances, the subject has surgery-induced pain, and the methods treat the surgery-induced pain. Non-limiting examples of surgery-induced pain include post-fracture pain, orthopedic injury, and complex orthotrauma.

[0086] According to some embodiments, the subject has complex regional pain syndrome (CRPS) type 1 or type 2 and the methods treat the CRPS type 1 or CRPS type 2. When the pain is CRPS type 1 or CRPS type 2, in some instances, the pain is reflex sympathetic dystrophy or causalgia.

[0087] In certain embodiments, the subject has neuropathic pain, and the methods treat the neuropathic pain. Examples of neuropathic pain include, but are not limited to, mononeuropathy, nerve injury- associated pain, post-herpetic neuralgia, small fiber neuropathy, polyneuropathy, nerve entrapment such as carpal tunnel syndrome, neuroma, chemotherapy-induced peripheral neuropathy, phantom limb pain, or thoracic outlet syndrome.

[0088] In some instances, the subject has diabetic peripheral neuropathy, and the methods treat the diabetic peripheral neuropathy. According to some embodiments, the subject has age-related pain and the methods treat the age-related pain. The subject may be a human subject at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 years of age.

[0089] In some instances, the subject has chronic back pain, and the methods treat the chronic back pain. The chronic back pain may be lumbar radiculopathy, discogenic back pain, or spinal stenosis.

[0090] In some instances, the subject has osteoarthritis, and the methods treat the pain caused by osteoarthritis. In some instances, the subject has pain associated with fibromyalgia or benign hypermobility, has a widespread pain condition, or has musculoskeletal pain syndrome and the methods treat the pain associated with fibromyalgia or benign hypermobility, the widespread pain condition, or the musculoskeletal pain syndrome.

[0091] According to some embodiments, the subject has been diagnosed as having the pain prior to administering the senolytic or the senostatic. In certain embodiments, the methods comprise diagnosing the subject as having the pain, and administering the senolytic or the senostatic to the subject subsequent to the diagnosis to treat the pain. In some instances, the subject to whom the senolytic or the senostatic is administered does not have cancer.

[0092] By “treat” or “treatment” is meant at least an amelioration of one or more symptoms associated with the subject’s pain, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the pain. As such, treatment also includes situations where the pain, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the pain, or at least the symptoms that characterize the pain. A variety of approaches are available to clinically assess pain and such approaches may vary depending upon the type of pain experienced by the subject. In certain embodiments, assessment of pain comprises using a pain scale. Non-limiting examples of pain scales include the Visual Analog Scale (VAS), Numerical Rating Scale (NRS), Defense and Veterans Pain Rating Scale (DVPRS), Verbal Rating Scale (VRS), Adult Non-Verbal Pain Scale (NVPS), Pain Assessment in Advanced Dementia Scale (PAINAD), Behavioral Pain Scale (BPS), the Faces Pain Scale-Revised (FPS-R), and the Critical-Care Observation Tool (CPOT).

[0093] KITS

[0094] Aspects of the present disclosure further include kits. In certain embodiments, the kits comprise a senolytic or a senostatic and instructions for administering the senolytic or the senostatic to treat pain in a subject in need thereof. The kits find use in practicing the methods of the present disclosure, e.g., methods of treating pain in a subject in need thereof, the methods comprising administering to the subject a senolytic in an amount effective to treat the pain.

[0095] A kit of the present disclosure may include one or any combination of senolytics or senostatics, including one or any combination of the senolytics described in the Methods of Treating Pain section above and the Experimental section below, which are not reiterated herein for purposes of brevity. In some instances, a kit of the present disclosure includes one or any combination of senolytics selected from ABT263, FOXO4-DRI, dasatinib, quercetin, fisetin, A1331852, A1155463, procyanidin C1 , curcumin, catechin, ABT737, panobinostat, apigenin, epigallo-catechin-gallate (EGCG), piperlongumine, 17-DMAG, ansamycin, resorcinol, luteolin, enzastaurin, MK1903, D-p-hydroxybutyrate (DBHB), nicotinic acid, rapamycin, and nicotinamide.

[0096] The kits of the present disclosure may include a quantity of the senolytic or the senostatic, present in unit dosages, e.g., tablets, capsules, ampoules, or a multi-dosage format. As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages of the senolytic or the senostatic. The term “unit dosage”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the senolytic calculated in an amount sufficient to produce the desired effect, e.g., reduction of pain. The amount of the unit dosage depends on various factors, such as the particular senolytic employed, the effect to be achieved, and the pharmacodynamics associated with the senolytic or the senostatic, in the subject. In yet other embodiments, the kits may include a single multi dosage amount of the senolytic or the senostatic.

[0097] A kit may include the senolytic or the senostatic present in a pharmaceutical composition comprising the senolytic (which may include a combination of senolytics) and a pharmaceutically acceptable carrier. Non-limiting examples of pharmaceutical compositions include those described above in the Methods of Treating Pain section above.

[0098] The instructions (e.g., instructions for use (I FU)) included in the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0099] The following examples are offered by way of illustration and not by way of limitation.

[0100] EXPERIMENTAL

[0101] Example 1 - Senescent sensory neurons increase in number with age in the mouse DRG

[0102] Assessed in this example was whether cellular senescence occurs within the lumbar DRG of aged mice. A classic marker of senescent cells, SA-p-galactosidase activity, was screened for in the DRG of young (10-16 weeks) or aged (21 -24 months) male and female mice. An increase in SA-p-galactosidase activity was found in aged compared to young DRG, indicating increased senescence of cells in the DRG with age (Figure 1 A). Based on morphology, a majority of DRG cells with SA-p-galactosidase activity were sensory neurons (Figure 1 A).

[0103] Although SA-p-gal activity is an indicator of senescence, its presence alone is insufficient for determining a senescent phenotype which can be heterogeneous (34). The expression of two major drivers of senescence, Cdknla (p21CIP1 / WAF) and Cdkn2a (p16INK2A), was therefore assessed in young and aged tissues. (17, 22, 53) As it is known that the Cdkn2a transcript has two variants producing different protein products, it was verified that the specific variant which produces p16INK2Awas in fact expressed by DRG neurons (Figure 7). When quantified in young mice, a baseline expression of p16 was detected in 6% of DRG neurons (Figure 1 B & C). Further, approximately 20% of DRG neurons expressed p21 in young uninjured mice (Figure 1 B & C). These p21 + and p16+ cell populations were non-overlapping in young uninjured mice (data not shown). In aged mice, significantly increased numbers of both p21 + and p16+ neurons were detected compared to young mice, indicating enhanced senescence in DRG neurons with age (Figure 1 B & C). As deleterious senescent cells are associated with pro-inflammatory SASP, senescent markers were further co-localized with downstream SASP factor and cytokine IL6. The aged DRG displayed a significant increase in the number of IL6+ as well as double positive p21 +IL6+ neurons, but not p16+IL6+ neurons, when compared to the young DRG (Figure 1 C & D). Neither young nor aged DRG had notable numbers of triple positive p21 +p16+I L6+ neurons (Figure 1 D). Interestingly, of all IL6-expressing neurons, a larger fraction expressed one or both senescence markers p21 and p16 in aged DRG (aged: 62% vs young: 33%) (Figure 1 E). Additionally, enhanced IL6 protein levels were detected in the plasma of aged versus young mice (Figure 1 F). These collective results indicate that senescent primary sensory neurons accumulate in the mouse DRG with age and express pro-inflammatory mediator and SASP factor IL6.

[0104] Example 2 - Senescent neurons accumulate in the DRG following peripheral nerve injury in young and aged mice

[0105] Investigated next was whether direct peripheral injury to primary sensory neuron axons would increase senescence within the DRG of young adult mice. Spared nerve injury (SNI) was performed in young mice (8-16 weeks old), in which two of the three distal branches of the sciatic nerve are transected and ligated with a suture (Figure 2A). Lumbar DRGs were collected at multiple time points following injury to evaluate senescence (Figure 2A). Gene expression of a panel of senescent markers and SASP factors was evaluated by qPCR in whole lumbar DRGs. A significant increase in senescent markers p21 and p16, and multiple SASP factors post-injury including IL6, IL1 p, and Ccl2 in the SNI DRG, was found at 3-weeks post-injury compared to controls (Figure 2B). To localize the cellular source of senescent marker expression in the DRG following SNI, assessed was the mRNA for p21 and p16 in young male and female mice. The majority of both p21 and p16-expressing cells were found to be neurons based on morphology post-injury (Figure 20). Neuronal expression of these senescent markers was quantified at acute (7-day) and chronic (3-week and 7-week) time points following SNI to assess the induction and longevity of senescent neurons in the DRG after injury (Figure 2A). Numbers of p21 + neurons increased significantly at the early 7-day time point post-injury in young mice and remained significantly increased when compared to the uninjured DRG (Figure 2D, left). In contrast, a gradual increase in the number of p16+ neurons was found over time following injury which was significant at 3-weeks post-SNI and peaked at the chronic 7-week post-injury time point compared to the young uninjured DRG (Figure 2D, right). These findings are in concordance with other studies which found p16 to be a marker of ‘chronic’ senescence. These data support that nerve injury drives senescence in neurons in the DRG of young mice into chronic stages, and that there is heterogeneity of senescent neuron phenotype over time following injury.

[0106] Given that senescent marker expression can be induced in DRG neurons in young mice following injury, and that aged mice already carry a higher senescent burden at baseline, it was hypothesized that there would be an additive effect of age and injury on the senescent burden in aged mice following injury. To test this, SNI surgery in aged mice (20-22mo old) was performed followed by assessment of senescence markers in the DRG (Figure 2A). The number of neurons expressing senescent marker p21 or p16 were then quantified following the time course post-SNI by RNAscope (Figure 2A). Aged DRG neurons increased in expression of p21 and p16 postinjury (Figure 2E). Similar to young mice, a significant increase in the number of p21+ neurons was found at 7-days and 3 weeks post-injury in aged mice, which reverted to aged uninjured baseline levels by 7-weeks post-injury (Figure 2F, left). In contrast to young mice, p16+ neurons were significantly increased in the aged DRG at the early 7-day time point, and remained increased throughout the time course (Figure 2F, right). In addition, a significant increase in the number of neurons co-expressing both p21 and p16 was found in young and aged mice starting at 3-weeks after injury compared to uninjured controls. Furthermore, there was a significant increase in p21 +p16+ neurons in injured aged compared to injured young DRG, suggesting that aged DRG have greater numbers of senescent cells transitioning to a deep senescent state compared to young DRG (Figure 2G).

[0107] To detect pro-inflammatory associated neuronal senescence within the DRG, the numbers of neurons that co-express any combination of p21 , p16, and downstream SASP factor IL6 were quantified at 3-weeks post-SNI by RNAscope (Figure 2H), as both young and aged mice have significantly higher numbers of senescent marker-expressing neurons at this time point. It was found that young mice accumulated significant numbers of neurons co-expressing p21 and IL6 in the DRG at 3-weeks post-injury (Figure 2I). Additionally, while increased baseline numbers of p21 +IL6+ cells were found in aged mice compared to young, these numbers were not found to further increase after injury in aged mice (Figure 2I), suggesting a heterogeneity of senescent marker induction after injury dependent on age. Further, while an increase in p16+IL6+ neurons post-injury was not observed (data not shown), an increase in triple positive p21+p16+IL6+ neurons in both young and aged mice, albeit in low percentages out of total DRG neurons was detected (Figure 2J). Further, a majority (-65%) of all IL6+ neurons were found to express either p21 , p16, or both of these senescent markers in the DRG 3-weeks post SNI in young or aged mice (Figure 2K). Collectively, these results indicate that nerve injury can induce senescence in neurons which are the major cellular source of IL6 in the young and aged DRG. Example 3 - ATF3+ injured neurons co-express senescent markers following peripheral nerve injury in young and aged mice

[0108] Hypothesized next was that senescence was primarily induced in injured neurons in the DRG. To test this, dual immunohistochemistry / RNAscope was performed to detect senescent markers in ATF3-positive injured neurons within lumbar L3 / L4 DRGs, and their numbers were quantified over time following injury in young and aged mice. (54, 55) First, very few ATF3+ neurons were detected in uninjured young or aged animals (Figure 3A). Post-injury, ATF3- positive neurons were increased to -44% in the young DRG and -35% in the aged DRG neurons (Figure 3A). There was a gradual decline in number of ATF3-positive neurons by 7-weeks post injury with -20% of neurons ATF3-positive in young DRG and -22% ATF3+ in aged DRG, which remained elevated compared to uninjured DRG (Figure 3A). p21 and / or p16 RNA expression was then quantified in ATF3-positive DRG neurons, and it was strikingly found that the majority of all ATF3-positive neurons co-expressed p21 and / or p16 senescent markers in both young and aged mice at all time points post-injury (Figure 3B, arrows & 3C-D). The fraction of p16- expressing ATF3-positive neurons increased over time following injury in both young and aged mice, to a similar degree, suggesting that injured neurons progress into a p16-senescent state over time, independent of age (Figure 3C-D). Further, it was found that non-injured (ATF3- negative neurons) also expressed senescent markers (Figure 3B below asterisks). An increase in the proportion of p21 or p16 expressing neurons were found to be ATF3-negative, which either increased or remained stable over time after injury in young or aged mice (Figure 3E-F). These results suggest either non-injured neurons senesce, or neurons which downregulated ATF3 remain senescent in the DRG of nerve-injured mice.

[0109] Example 4 - Trpyl + nociceptors express senescent markers and SASP factor IL6 in the DRG in young and aged mice

[0110] To characterize the subtypes of primary sensory neurons which express senescent markers after nerve injury, analyzed were the cell diameters of neurons that co-expressed senescent markers along with SASP factor IL6, as cell diameter is indicative of DRG subtype populations. Previously it was reported that the distribution of small-, medium-, and large-diameter DRGs does not change with age. (29) Found in the present study was that the majority of p21 +IL6+ neurons measured in the range of 16-30pm in size, with a mean diameter of 22|jm in young and 20pm in aged mice (Figure 4A). In comparison, young p16+IL6+ neurons were found to be slightly larger in diameter with a majority in the range of 21 -35pm with mean diameter of 29pm, while in aged mice the diameter of p16+IL6+ neurons was smaller with a mean diameter of 24pm (Figure 4A). In either case, young or aged senescent neurons were rarely of large diameter (Figure 4A).

[0111] Given that the majority of neurons expressing senescent markers were small diameter, it was theorized that these senescent neurons may be Trpvl +, as this ion channel is widely expressed in small diameter neurons classified as nociceptors (47, 56). Therefore, DRG cells were co-labeled for Trpvl , p21 , and p16 transcript expression to determine this senescent neuron subpopulation. First, it was found that a majority of Trpvl + neurons co-labeled with p21 , with few co-labeled with p16 in young mice following injury (data not shown). This finding is in agreement with the finding that the p21 +IL6+ neurons were smaller in mean diameter (~22 m) compared to the larger diameter p16+IL6+ neurons. Due to the high co-localization of p21 in Trpvl + DRG neurons after injury, these cells were co-labeled with downstream SASP factor IL6 to validate this p21 +IL6+ neuronal population following SNI (Figure 4B). In uninjured young mice, 19% Trpvl +p21 + neurons were detected with only 2% co-expressing SASP factor IL6, which markedly increased after injury to 48% Trpvl +p21 + with 15% co-expressing IL6 (Figure 4C). In uninjured aged mice, 50% Trpvl +p21 + neurons were detected with 6% co-expressing IL6, a 3-fold increase in IL6-expression compared to young uninjured DRG (Figure 4C & 4D). Similar to young mice after injury, an expansion of the aged Trpvl + population co-expressing p21 + and IL6+ was found after injury compared to uninjured aged DRG (uninj: 6% vs SNI: 13%) (Figure 4D). Taken together these data support enhanced Trpvl + nociceptor senescence with age and following injury, with an increased fraction which co-express IL6 after injury in both young and aged mice.

[0112] Example 5 - Pharmacological elimination of senescent cells ameliorates pain behaviors in aged mice

[0113] Given that Trpvl + nociceptors and neighboring cells within the DRG express senescent markers and SASP factor IL6 after nerve injury in young and aged mice (Figure 4B, inset asterisks), and that senescent neurons contain high-responding and nociceptor-like populations, it was next hypothesized that senescent DRG neurons may exhibit hyperexcitability and clearance of these cells would improve pain behaviors induced by SNI. Whole-cell electrophysiological recordings were performed in ex vivo DRG from aged mice, followed by single-cell qPCR to identify neuronal markers of senescence, nerve injury, and cytokine expression (Figure 5A). We demonstrated excitability from current injection protocols recorded in a neuron found to express p16 (senescence), ATF3 (injury), and Tubb3 (neuronal) (Figure 5B) and from a p21 +, ATF3+, IL6+ (SASP / cytokine), and Tubb3-expressing neuron (Figure 5C).

[0114] We next treated mice with ABT263, a potent and peripherally restricted senolytic which promotes apoptosis of senescent cells. (26, 57, 58) Mice were treated using ABT263 (1 OOmg / kg, p.o. in veh) or vehicle (60% Phosol40PG, 30% PEG400, 10% Ethanol, p.o.) at 3 weeks post-SNI, a time-point when senescent neurons have accumulated in young and aged mice (Figure 5D). Treatment with ABT263 successfully targeted DRG neurons as a significant increase was found in the number of cleaved caspase-3 (CC3) positive (i.e. apoptotic) neurons in the DRG of ABT263-treated mice as compared to vehicle-treated mice with SNI (Figure 5E). Additionally, the fraction of CC3-positive neurons that co-express both senescence markers p16 and p21 was increased in ABT263-treated mice, indicating senescent neurons were targeted by ABT263 (Figure 5J).ln aged male and female mice with ABT263, found was a gradual improvement in mechanical allodynia that was sustained in the weeks following the 10-day treatment, compared to vehicle treated mice (Figure 5F). In addition, both aged mice showed a significant improvement in weight bearing immediately after (Day 16) and 2 weeks after (Day 39) senolytic treatment (Figure 5G, respectively). When young mice were treated, a significant but transient reduction in mechanical threshold was found during the second 5-day treatment window of ABT263 when compared to vehicle treated mice (Figure 5H). Despite this transient allodynia effect, improved weight bearing on the injured hindlimb was observed in young mice immediately after senolytic treatment (Figure 5I, Day 16), which persisted to 2 weeks after senolytic treatment (Figure 5I, Day 29). These results indicate senescent neurons can be targeted by senolytics and result in improved behaviors associated with pain outcomes in a nerve injury model.

[0115] Example 6 - Human DRG neurons express senescent markers and accumulate in number during aging

[0116] To validate the translational potential of senescent cell subpopulations as a target in humans, assessed next was whether human DRG neurons express senescent markers. As it is difficult to obtain human DRG tissues with confirmed injury / damaged nerves, assessed was whether senescent markers increase with age. L4 DRG were collected from young (33yr) and aged (65yr) female human donors, followed by surveying of senescent marker expression in the DRG using RNAscope to detect p16 and p21 RNA expression. Similar to mice, human DRG neurons very clearly expressed these markers (Figure 6A & 6B). In addition, a greater percentage of aged human DRG neurons expressed senescent markers compared to young DRG neurons (p21+’. 24% young vs 48% aged; p16+ 36% young vs 42% aged; p21+p16+'. 10% young vs 40% aged) (Figure 6C). Further, greater numbers of IL6+ neurons were found in aged compared to young human DRG (36% young vs 57% aged) (Figure 6D). Of this IL6-expressing neuronal population in the DRG, an increased overall fraction expressing either p21 or p16 in the aged DRG was found compared to young DRG, with a striking increase in triple positive cells (IL6+p21+p16+: 23% young vs 48% aged) (Figure 6E). When total neurons co-expressing senescent markers and IL6 were counted, an increased fraction of neurons co-positive for p21 +IL6+, p16+IL6+, and triple positive for p21 +p16+IL6+, were found in aged compared to young DRG, suggesting an increase in SASP-producing senescent cells with age (Figure 6F). The assessment of senescent markers increase with age has been extended by analyzing DRG from an addition young person (32 yrs old) and an additional old person (65 yrs old) (data not shown). This additional data also shows that DRG senescence increases with age. Lipofuscin accumulation within cells can also serve as an additional marker of accumulated cellular stress and senescence. (59, 60) It was observed that human DRG neurons had a number of lipofuscin filled cells with >75% of the neuron filled (Figures 8A-8B), occluding the RNAscope signal in these cells and precluding further analysis of additional senescent marker expression. When quantified, found was an increased fraction of DRG neurons filled with lipofuscin with age in the human DRG, indicative of additional senescence in the aged human DRG (9.5% aged vs 1.5% in the young, Figures 8A-8B). Assessed next was whether injured (ATF3+) human sensory neurons existed in the young and aged human DRG (61 ). A limited number of ATF3+ neurons were detected in either young or aged human L4 DRG tissues which was not different between subjects (Figure 6G). These ATF3+ cells were co-labeled with senescent markers p21 and p16. Strikingly, the majority of ATF3+ neurons expressed p21 , with a greater percent expressing p21 in aged (92%) compared to young (23%) human DRG.

[0117] Analyses were extended to additional human DRG across ages and associated with pain states. Two publicly available human DRG RNAseq datasets were re-analyzed (Figure 10). Upon re-analyzing a single-cell RNAseq dataset (66), an age-related increase in the percentage of DRG neurons expressing p16 as well as p21 was detected when comparing three human DRG samples (ages 23, 56, and 61 years old) (Figure 10A-10B). Further, in this dataset, the coexpression of ATF3+ neurons with p21 and p16, as well as the combination p21 +p16+, by human DRG neurons was confirmed, which also showed an age-related increase in number for all subtypes (Figure 10C). In a separate re-analysis of a bulk RNA-sequencing dataset generated using excised DRG collected from patients with or without at-level pain (67), a significant correlation between increased CDKN2A (p16) expression and age was found (Figure 10D, Spearman correlation, coefficient= 0.612, p=0.00321 ). A significant association of SenMayo gene expression specifically in “pain” DRG samples compared to the “no pain” condition was also discovered a (t-test, p=0.0182) (Figure 10E). These data together replicated the findings of an age-related increase of senescent neurons in the human DRG as well as demonstrated an association between pain and senescence marker expression.

[0118] Analyzed next was neuron diameter, and similar to the mouse DRG, senescent markerexpressing human neurons were largely of small diameter in both young and aged DRG (Figure 9). Finally, assessed / verified was whether human TRPV1 + nociceptors had increased senescent marker expression in the aged versus young DRG. TRPV1 + nociceptors were labeled along with senescent markers p21 and p16. First, it was found that -68% of all DRG neurons expressed TRPV1 in young human L4 DRG and -55% were TRPV1 + in aged human L4 DRG (Figure 6I, left). Of these TRPV1 + neurons, a higher fraction co-expressing either p16 or p21 was found in aged versus young DRG (Figure 6I, right boxed & 6J). Further, a shift was observed in TRPV1 + nociceptor expression of senescent markers p21 and p16, with a majority of TRPV1 + neurons (81%) expressing one or both markers in the aged DRG compared to young DRG, where 60% express one or more senescent markers (Figure 6J & 6K). These data collectively suggest that DRG neurons, including injured ATF3+ and TRPV1 + neurons, have a more prominent senescent profile with increased age in the human DRG.

[0119] Example 7 - FOXO4-DRI Treatment

[0120] Young and aged mice developed profound mechanical allodynia after SNI as evidenced by a drop in mechanical threshold from pre-injury baseline that was significantly improved after treatment with the senolytic, FOXO4-DRI (***p<0.001 vs. age-matched vehicle by ANOVA). See Figure 1 1 A.

[0121] Mice maintained responsivity to VonFrey filaments within the expected range. Senolytic treatment did not result in any change in latency to a 50°C hot plate (Fig. 1 1 B) or 4°C cold plate (Figure 11 C), suggesting preservation of sensory function after senolytic treatment.

[0122] Methods

[0123] Animals

[0124] All procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care and the Institutional Animal Care and Use Committee in accordance with American Veterinary Medical Association guidelines and the International Association for the Study of Pain. Adult male and female mice 10-16 weeks old were housed 2-5 per cage maintained on a 12-hour light / dark cycle in a temperature-controlled environment with ad libitum access to food and water. Young mice used in this study: wild type C57BL / 6J mice (Jax stock #00664). Aged mice used in this study: wild type C57BL / 6J mice (National Institute of Aging). Aged mice were pre-screened for abnormal masses and cataracts.

[0125] Human samples

[0126] Human lumbar L4 DRG tissues were obtained from one white female donor (age 33) who died from head trauma, one Hispanic or Latino female donor (age 32) who died from head trauma, one male donor whose ethnicity was not reported who died from head trauma (age 65), and one white female donor (age 65), who died from stroke. Human post-mortem DRG were obtained in collaboration with Donor Network West.

[0127] Quantitative Real Time RT-PCR (qPCR)

[0128] DRG samples were collected, the tissue was homogenized and placed in TRIzol Reagent (Invitrogen, #15596018). RNA was isolated using miRNeasy® Mini kit (Qiagen, Cat No. 217004). The concentration and purity of RNA samples were determined using NanoDrop 2000 (Thermo Fisher Scientific). RNA was reverse transcribed using Superscript™ VILO™ cDNA Synthesis Kit (Cat No 1 1754-050). qPCR analysis was performed with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, #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, 1 min at 60°C. Dissociation analysis was performed at the end of each run to ensure specificity. Relative quantification of gene expression was performed via 2AAC(T)method. Spared Nerve Injury

[0129] To perform SNI surgery, mice were anesthetized with isoflurane and a small incision is made over the left thigh and blunt dissection is 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 are then ligated using an 5-0 nylon suture (ETHILON™ ref#1668G) and these nerves are then axotomized using small-sized spring scissors. The sural nerve is left intact (the “spared nerve”). The incision is then closed with surgical staples. Following surgery, mice are 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.

[0130] Senolytic Administration

[0131] 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. Young and aged mice were dosed by oral gavage (p.o.) at 100 mg / kg daily for 5 days, followed by 2 rest days and a second 5-day daily dosing. FOXO4-DRI was injected intrathecally (i.t.) to mice at 10pg dissolved in sterile saline for 5 consecutive days starting at 3- weeks post SNI injury.

[0132] Histology

[0133] Tissue preparation

[0134] 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 RNAIater solution at RT (ThermoFisher), then frozen in OCT on dry ice, and stored at -80°C. Mouse DRG was sectioned at 14pm and mounted onto SuperFrost Plus glass slides and stored at -80°C until histology protocols performed. Nerve endings were trimmed and 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 to avoid thawing and sectioned at 20pm onto SuperFrost Plus glass slides and stored at -80°C until histology protocols performed.

[0135] RNAscope / immunohistochemistry dual labeling

[0136] Fluorescent in situ hybridization using the RNAscope Multiplex V2 Kit (ACD, #323100) was performed in combination with immunohistochemistry to detect senescent marker, cytokine, and DRG subtype marker mRNA (p21 , p16, IL6, Tprvl ) and protein markers (ATF3), respectively. Briefly, DRG tissues were isolated and processed as described above in tissue preparation section. Dorsal root ganglion (DRG) sections (14pm) were mounted on glass slides and dried 1 - hr at room temperature and transferred to -80°C for storage. On Day 1 of RNAscope, slides were submerged in 10% formalin and incubated for 20 min at 4°C. Slides were washed with 1 XPBS and dehydrated in EtOH as described in the ACD RNAscope protocol. Sections were incubated for 10 min in RNAscope® Hydrogen Peroxide solution, washed in Millipore water, and incubated in RNAscope® ProteaselV for 10 min (Mm-IL6 probe, #315891 ) at room temperature. Probes were hybridized at 40°C for 2hrs and stored overnight in 5X SSC buffer at room temperature. On Day 2 RNAscope, slides were incubated in Amp1 , Amp2, and Amp3 solutions as recommended by the ACD RNAscope protocol and HRP-C1 was used to develop HRP signal followed by incubation with Opal™ or Vivid™ dye reagents (1 :1000, Akoya Biosciences) and finally HRP Block. Slides were then washed in 1 XPBS and blocked with 10% Normal Donkey Serum, 0.3 % Triton-X 100 for 1 h at room temperature. Slides were shielded from light and incubated with Rabbit anti-ATF3 (1 :200) in 1 % normal donkey serum and 0.3 % Triton X-100 in 1 XPBS at 4°C overnight. Slides were washed 3X in 1XPBS for 5 min each, incubated with AlexaFluor secondary antibodies (anti-rabbit-A488), and mounted with Fluoromount G with DAPI (ThermoFisher, #00- 4959-52). Images of the DRG were captured using a Keyence BZ-X810 fluorescent microscope (Keyence) with a 40X objective. Negative control probes (ACD, #321838) were used to assess background levels of RNAscope signal. In order to quantify mRNA expression in DRG neurons, DAPI was used to determine glial-neuronal boundary to carefully associate RNAscope puncta with neurons, as was the focus of this study. We captured 8-12 DRG images per mouse. Neurons expressing IL6 were counted as positive if 5 or more bright puncta were detected throughout the cytoplasm of the neuron. Neurons expressing p16 were counted as positive if 10 or more puncta were detected throughout the cytoplasm of the neuron. Signal for p21 and Trpvl always exceeded 20 puncta and were counted as positive. Human DRG neuron cut offs for positive counted cells were as follows: IL6 >10puncta, p16 >15puncta, p21 >15puncta, Trpvl >15puncta.

[0137] Immunohistochemistry

[0138] For cleaved caspase-3 staining, mice were transcardially perfused as described and DRG tissues were extracted and frozen in OCT. DRG were sectioned at 14pm and mounted on SuperFrost Plus glass slides and dried 1 -hr at room temperature and transferred to -80°C for storage. Slides were then blocked with 10%Normal Donkey Serum, 0.3 % Triton-X 100 for 1 h at room temperature. Rabbit Cleaved-caspase-3 primary antibody (1 :200, Cell Signaling Technology) was incubated overnight at 4°C. Slides were incubated with secondary antibody, anti-rabbit Alexa-555, for 2hr in the dark. Slides were mounted with Fluoromount G with DAPI and imaged on a Keyence BZ-X810 fluorescent microscope. Primary antibody controls (noprimary conditions) were used throughout to validate immuno-positive signal.

[0139] SA-B-aalactosidase activity assay

[0140] Mice were perfused with PBS. L3-L5 dorsal root ganglion (DRG) was extracted, rinsed in RNAIater and PBS, and then mounted onto OCT. DRG were sectioned at 14mm onto glass slides. Slides were removed from the freezer, and 1 X of fixative solution provided by Senescence p-Galactosidase Staining Kit (9860S, Cell Signaling Technology kit) was added to the slides for 15 minutes. Slides were rinsed in PBS, and a wax barrier was drawn around the edges of the sections. Fresh p-Galactosidase Staining Solution staining solution at pH 6.1 was added to the slides and then incubated at 37 °C for 22 hours. p-Galactosidase Staining Solution was removed and slides were rinsed twice in PBS and twice in distilled water before mounting and imaging. Imaging was done in FIJI Imaged, where the area of the DRG neurons was outlined, and the percentage of positive p-Gal signal in the area was acquired. Sections (5-10) were analyzed per mouse. The total area analyzed per group was not significantly different from each other (data not shown).

[0141] ELISA assay

[0142] 1 ml_ syringes were coated with heparin and used to withdraw ~0.8ml of blood from mice anesthetized mice. To extract the plasma, the blood was centrifuged at 2000 RCF at 4°C for 10 minutes. The supernatant was removed, aliquoted, and stored at -80°C until used. IL-6 plasma concentration levels were assessed by following the manufacturer's instructions (ThermoFisher, Cat. No.: KMC0061 ). Samples were tested in duplicate and diluted 1 :2. The final concentration was corrected for the dilution factor. A spectrophotometer (add model) was used to calculate optical density (OD) values at 450 nm. The data was analyzed with Boosterbio’s 5PL regression model and subtracting the blank well’s OD value from the sample’s OD values (elisa-data- analysis-online at bosterbio.com; biology-research-tools).

[0143] Electrophysiology

[0144] For ex vivo recording preparations, animals were deeply anesthetized with a ketamine / xylazine bolus (0.2 ml of 37.5 and 0.25 mg / ml in sterile saline, respectively). Mice were transcardially perfused with a sucrose-based dissection solution (containing in mM: 250 sucrose, 2.5 KCI, 25 NaHCO3, 1 NaH2PO4, 6 MgCI2, 0.5 CaCI2, and 25 glucose). All extracellular solutions in contact with live tissue were bubbled with a 95% 02 / 5% CO2 gas and chilled on ice, then decapitated. The vertebral column and sciatic nerves were isolated and placed in dissection solution. The DRG with attached nerves and dorsal roots were manually freed from the bone and muscle 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 allow for digestion of the perineurium. DRG recordings were performed in a chamber (RC-26GLP; Warner Instruments) within an upright microscope with platform (Nikon Eclipse FN1 ) and secured with a platinum wire-based anchor, and tissue was constantly perfused with carbogenated artificial cerebrospinal fluid (aCSF; composition in mM: 125 NaCI, 2.5 KCI, 25 NaHCO3, 1 .0 NaH2PO4, 1.0 MgCI2, 2.0 CaCI2, and 25 glucose). For in vitro (culture) preparations, coverslips were preincubated with either IL6 (50 ng / mL with carrier, prepared from 100 g / 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 then removed from wells and transferred directly to the recording setup, and aCSF containing the same concentration of either IL6 or control BSA was applied at 29-32°C for the duration of the recording. Coverslips were not recorded for more than one hour following their transfer to the recording chamber. For ex vivo preparations, the nerve end of the preparation was secured in a suction electrode attached to a stimulus isolator (A365, World Precision Instruments). Neurons were visualized with infrared differential interference contrast illumination. Recordings were done with patch pipettes 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 MfT and internal patch solution as follows (in mM): 120 potassium gluconate, 20 KCI, 165 2 MgCI2, 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 (pCIamp 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, gap- free recordings were taken for 2 minutes (to measure spontaneous firing and membrane potential), then depolarizing current steps were applied from resting membrane potential in 50 pA steps (to measure rheobase, action potential [AP] threshold and AP latency) and finally, stepwise current pulses were injected from resting membrane potential to measure evoked firing frequency and Ih current (-300-1400 pA in 50 pA steps, 400 ms duration). Ih is reported as current density, which was computed as Ih current (amplitude in pA) / cell size (diameter in pm). 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) to determine gene presence or absence in the sample. Of note, glial fibrillary acidic protein (GFAP) was present in several of our samples, thereby precluding us from determining the definitive source of p16, p21 or IL6 (i.e. DRG neurons or glial cells); nonetheless, our results reflect the state of the microenvironment in which the DRG was sampled. We intentionally recorded from mainly small- to medium-sized DRG to increase the chances of sampling senescence marker- expressing neurons. Uniform Manifold Approximation and Projection (UMAP; python implementation from Imcinnes / umap at github.com) was performed to use machine learning to integrate and connect the highdimensional 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)) I (max(p) - min(p)) where p is a vector containing all measurements of a given parameter.,.

[0145] Neuron diameter analysis

[0146] Fluorescent TIFF images taken from RNAscope experiments which labeled p21 , p16, and IL6 RNA were used to measure diameters of neurons in both mouse and human DRG. Cells were individually labeled and categorized for the co-expression of markers p21 , p16, with IL6. Using Fiji software, the scale (pm) was appropriately set based on objective used in image. The longest end-to-end cell diameters, with line placed through the center of each neuron, were drawn using the line segment tool. The line segment was then measured using ‘Measure’ as an output in pm unit and recorded. Neurons per category of RNA co-expression were then binned into 10pm segments and the percentage of neurons which fell into each pm bin were displayed as a percentage of total neurons analyzed in each subgroup.

[0147] Behavioral testing - Mechanical nociception assays

[0148] 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, the 50% withdrawal mechanical threshold scores were calculated for each mouse and then averaged across the experimental groups. Mechanical nociception testing was performed at 3-weeks post SNI at Days: 1 , 3, 8, 12, 15, 19, 29, 39 post senolytic treatment start.

[0149] Unweighting

[0150] 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. The duration of 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. Unweighting was measured post-senolytic or vehicle treatment in SNI young and aged mice. 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. Transcriptomic Analysis of existing datasets

[0151] Single nucleus and single cell RNA sequencing datasets were acquired from several sources as described in paragraphs below. Transcriptomic data was processed in python 3.10 using scanpy-1 .10.1 , anndata-0.10.7, numpy-1 .26.4, scipy-1 .1 1 .4, pandas-2.2.2, statsmodels- 0.14.2 software packages. Differential gene expression significance was calculated using the Wilcoxon rank-sum test, as provided by the SciPy software package. P-values were FDR corrected using the Benjamini / Hochberg method using the StatsModels package. Log2fold change was calculated as the Iog2 of the ratio of the per-gene mean expressions. A gene is considered differentially expressed between two populations if the FDR corrected ranksums p- value is less than .05, and the Iog2fold change is greater than .6 or less than -.6. 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 only 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, singlecell and single- nucleus data was logl p transformed and centered to the per-gene mean and scaled to the per- gene variance 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 per- gene zero centering and variance scaling was performed for each annotated cell type from Renthal, et. al, 2019 independently. Scoring performed on the North et. al, 2019(67) bulk rna sequencing data was mean centered and variance scaled similarly but across the entire dataset. Gene-set scoring was performed using the ScanPy score genes method, as initially implemented in Seurat. Scores were assessed for individual cells. For analysis of Yu et al. 2023(66) we aligned raw count matrices from GEO Accession GSE249746. Deposited data had 1 ,136 cells. Data was of generally high quality. 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) 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.

[0152] References

[0153] 1 . Canta A, Chiorazzi A, Carozzi VA, Meregalli C, Oggioni N, Bossi M, et al. Age-related changes in the function and structure of the peripheral sensory pathway in mice. Neurobiol Aging. 2016;45:136-48. 2. Hofer SM, Berg S, Era P. Evaluating the interdependence of aging-related changes in visual and auditory acuity, balance, and cognitive functioning. Psychol Aging. 2003;18(2):285- 305.

[0154] 3. Millecamps M, Shi XQ, Piltonen M, Echeverry S, Diatchenko L, Zhang J, et al. The geriatric pain experience in mice: intact cutaneous thresholds but altered responses to tonic and chronic pain. Neurobiol Aging. 2020;89:1-11 .

[0155] 4. Vincent K, Dona CPG, Albert TJ, Dahia CL. Age-related molecular changes in the lumbar dorsal root ganglia of mice: Signs of sensitization, and inflammatory response. JOR Spine. 2020;3(4):e1124.

[0156] 5. Wang S, Albers KM. Behavioral and cellular level changes in the aging somatosensory system. Ann N Y Acad Sci. 2009;1170:745-9.

[0157] 6. Weyer AD, Zappia KJ, Garrison SR, O'Hara CL, Dodge AK, Stucky CL. Nociceptor Sensitization Depends on Age and Pain Chronicity(1 ,2,3). eNeuro. 2016;3(1 ).

[0158] 7. Singh SP, Guindon J, Mody PH, Ashworth G, Kopel J, Chilakapati S, et al. Pain and aging: A unique challenge in neuroinflammation and behavior. Mol Pain.

[0159] 2023;19:17448069231203090.

[0160] 8. Cruz-Almeida Y, Aguirre M, Sorenson HL, Tighe P, Wallet SM, Riley JL, 3rd. Age differences in cytokine expression under conditions of health using experimental pain models. Exp Gerontol. 2015;72:150-6.

[0161] 9. Chung HY, Cesari M, Anton S, Marzetti E, Giovannini S, Seo AY, et al. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev. 2009;8(1 ):18- 30.

[0162] 10. Chung HY, Kim DH, Lee EK, Chung KW, Chung S, Lee B, et al. Redefining Chronic Inflammation in Aging and Age-Related Diseases: Proposal of the Senoinflammation Concept. Aging Dis. 2019;10(2):367-82.

[0163] 11 . Dos Santos NL, Lenert ME, Castillo ZW, Mody PH, Thompson LT, Burton MD. Age and sex drive differential behavioral and neuroimmune phenotypes during postoperative pain. Neurobiol Aging. 2023;123:129-44.

[0164] 12. Arbat-Plana A, Bolivar S, Navarro X, Udina E, Alvarez FJ. Massive Loss of Proprioceptive la Synapses in Rat Spinal Motoneurons after Nerve Crush Injuries in the Postnatal Period. eNeuro. 2023;10(2).

[0165] 13. Shaffer SW, Harrison AL. Aging of the somatosensory system: a translational perspective. Phys Ther. 2007;87(2):193-207.

[0166] 14. Borghesan M, Hoogaars WMH, Varela-Eirin M, Talma N, Demaria M. A Senescence- Centric View of Aging: Implications for Longevity and Disease. Trends Cell Biol.

[0167] 2020;30(10):777-91.

[0168] 15. van Deursen JM. The role of senescent cells in ageing. Nature. 2014;509(7501 ):439-46. 16. Jeon OH, David N, Campisi J, Elisseeff JH. Senescent cells and osteoarthritis: a painful connection. J Clin Invest. 2018;128(4):1229-37.

[0169] 17. Fielder E, von Zglinicki T, Jurk D. The DNA Damage Response in Neurons: Die by Apoptosis or Survive in a Senescence-Like State? J Alzheimers Dis. 2017;60(s1 ):S107-s31.

[0170] 18. Jurk D, Wang C, Miwa S, Maddick M, Korolchuk V, Tsolou A, et al. Postmitotic neurons develop a p21 -dependent senescence-like phenotype driven by a DNA damage response. Aging Cell. 2012;11 (6):996-1004.

[0171] 19. Chinta SJ, Woods G, Rane A, Demaria M, Campisi J, Andersen JK. Cellular senescence and the aging brain. Exp Gerontol. 2015;68:3-7.

[0172] 20. Muralidharan A, Sotocinal SG, Yousefpour N, Akkurt N, Lima LV, Tansley S, et al. Longterm male-specific chronic pain via telomere- and p53-mediated spinal cord cellular senescence. J Clin Invest. 2022;132(8).

[0173] 21 . Gorgoulis V, Adams PD, Alimonti A, Bennett DC, Bischof O, Bishop C, et al. Cellular Senescence: Defining a Path Forward. Cell. 2019;179(4):813-27.

[0174] 22. Stein GH, Drullinger LF, Soulard A, Dulic V. Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Mol Cell Biol. 1999;19(3):2109-17.

[0175] 23. Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, et al. Naturally occurring p16(lnk4a)-positive cells shorten healthy lifespan. Nature. 2016;530(7589):184-9.

[0176] 24. Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, et al. Clearance of p16lnk4a-positive senescent cells delays ageing-associated disorders. Nature. 2011 ;479(7372):232-6.

[0177] 25. Bussian TJ, Aziz A, Meyer CF, Swenson BL, van Deursen JM, Baker DJ. Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature. 2018;562(7728):578-82.

[0178] 26. Kirkland JL, Tchkonia T. Senolytic drugs: from discovery to translation. J Intern Med. 2020;288(5):518-36.

[0179] 27. Musi N, Valentine JM, Sickora KR, Baeuerle E, Thompson CS, Shen Q, et al. Tau protein aggregation is associated with cellular senescence in the brain. Aging Cell. 2018;17(6):e12840.

[0180] 28. Herdy JR, Traxler L, Agarwal RK, Karbacher L, Schlachetzki JCM, Boehnke L, et al. Increased post-mitotic senescence in aged human neurons is a pathological feature of Alzheimer's disease. Cell Stem Cell. 2022;29(12):1637-52. e6.

[0181] 29. Schwab N, Taskina D, Leung E, Innes BT, Bader GD, Hazrati LN. Neurons and glial cells acquire a senescent signature after repeated mild traumatic brain injury in a sexdependent manner. Front Neurosci. 2022;16:1027116. 30. Wang J, Lu Y, Carr C, Dhandapani KM, Brann DW. Senolytic therapy is neuroprotective and improves functional outcome long-term after traumatic brain injury in mice. Front Neurosci. 2023;17:1227705.

[0182] 31 . Paramos-de-Carvalho D, Martins I, Cristovao AM, Dias AF, Neves-Silva D, Pereira T, et al. Targeting senescent cells improves functional recovery after spinal cord injury. Cell Rep. 2021 ;36(1 ):109334.

[0183] 32. Du J, Cheng N, Deng Y, Xiang P, Liang J, Zhang Z, et al. Astrocyte senescence-like response related to peripheral nerve injury-induced neuropathic pain. Cell Mol Biol Lett. 2023:28(1 ):65.

[0184] 33. Fuentes-Flores A, Geronimo-Olvera C, Girardi K, Necunir-lbarra D, Patel SK, Bons J, et al. Senescent Schwann cells induced by aging and chronic denervation impair axonal regeneration following peripheral nerve injury. EMBO Mol Med. 2023:e17907.

[0185] 34. Cohn RL, Gasek NS, Kuchel GA, Xu M. The heterogeneity of cellular senescence: insights at the single-cell level. Trends Cell Biol. 2023;33(1 ):9-17.

[0186] 35. Demaria M, Ohtani N, Youssef SA, Rodier F, Toussaint W, Mitchell JR, et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell. 2014;31 (6):722-33.

[0187] 36. Wilkinson HN, Hardman MJ. Cellular Senescence in Acute and Chronic Wound Repair. Cold Spring Harb Perspect Biol. 2022;14(11 ).

[0188] 37. Beck J, Horikawa I, Harris C. Cellular Senescence: Mechanisms, Morphology, and Mouse Models. Vet Pathol. 2020;57(6):747-57.

[0189] 38. Farr JN, Fraser DG, Wang H, Jaehn K, Ogrodnik MB, Weivoda MM, et al. Identification of Senescent Cells in the Bone Microenvironment. J Bone Miner Res. 2016;31 (11 ):1920-9.

[0190] 39. Minamino T, Orimo M, Shimizu I, Kunieda T, Yokoyama M, Ito T, et al. A crucial role for adipose tissue p53 in the regulation of insulin resistance. Nat Med. 2009;15(9):1082-7.

[0191] 40. Sapieha P, Mallette FA. Cellular Senescence in Postmitotic Cells: Beyond Growth Arrest. Trends Cell Biol. 2018;28(8):595-607.

[0192] 41 . Moreno-Blas D, Gorostieta-Salas E, Pommer-Alba A, Mucino-Hernandez G, Geronimo- Olvera C, Maciel-Baron LA, et al. Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy. Aging (Albany NY). 2019;11 (16) :6175-98.

[0193] 42. Riessland M, Kolisnyk B, Kim TW, Cheng J, Ni J, Pearson JA, et al. Loss of SATB1 Induces p21 -Dependent Cellular Senescence in Post-mitotic Dopaminergic Neurons. Cell Stem Cell. 2019;25(4):514-30.e8.

[0194] 43. Kang C, Xu Q, Martin TD, Li MZ, Demaria M, Aron L, et al. The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4. Science. 2015;349(6255):aaa5612. 44. Palmer A, Epton S, Crawley E, Straface M, Gammon L, Edgar MM, et al. Expression of p16 Within Myenteric Neurons of the Aged Colon: A Potential Marker of Declining Function. Front Neurosci. 2021 ;15:747067.

[0195] 45. Krames ES. The dorsal root ganglion in chronic pain and as a target for neuromodulation: a review. Neuromodulation. 2015;18(1 ):24-32; discussion

[0196] 46. Khoutorsky A, Price TJ. Translational Control Mechanisms in Persistent Pain. Trends Neurosci. 2018;41 (2):100-14.

[0197] 47. Mickle AD, Shepherd AJ, Mohapatra DP. Sensory TRP channels: the key transducers of nociception and pain. Prog Mol Biol Transl Sci. 2015;131 :73-118.

[0198] 48. von Hehn CA, Baron R, Woolf CJ. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron. 2012;73(4):638-52.

[0199] 49. Gaudet AD, Popovich PG, Ramer MS. Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury. Journal of neuroinflammation. 2011 ;8:110.

[0200] 50. Goncalves Dos Santos G, Delay L, Yaksh TL, Corr M. Neuraxial Cytokines in Pain States. Frontiers in immunology. 2019;10:3061.

[0201] 51 . Fang D, Kong LY, Cai J, Li S, Liu XD, Han JS, et al. lnterleukin-6-mediated functional upregulation of TRPV1 receptors in dorsal root ganglion neurons through the activation of JAK / PI3K signaling pathway: roles in the development of bone cancer pain in a rat model. Pain. 2015; 156(6) :1124-44.

[0202] 52. Hernandez-Segura A, Nehme J, Demaria M. Hallmarks of Cellular Senescence. Trends in cell biology. 2018;28(6):436-53.

[0203] 53. Herrup K, Yang Y. Cell cycle regulation in the postmitotic neuron: oxymoron or new biology? Nat Rev Neurosci. 2007;8(5):368-78.

[0204] 54. Tsujino H, Kondo E, Fukuoka T, Dai Y, Tokunaga A, Miki K, et al. Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: A novel neuronal marker of nerve injury. Mol Cell Neurosci. 2000;15(2):170-82.

[0205] 55. Laedermann CJ, Pertin M, Suter MR, Decosterd I. Voltage-gated sodium channel expression in mouse DRG after SNI leads to re-evaluation of projections of injured fibers. Mol Pain. 2014;10:19.

[0206] 56. Shiers S, Klein RM, Price TJ. Quantitative differences in neuronal subpopulations between mouse and human dorsal root ganglia demonstrated with RNAscope in situ hybridization. Pain. 2020;161 (10):2410-24.

[0207] 57. Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S, et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 2008;68(9):3421 -8.

[0208] 58. Zhu Y, Tchkonia T, Fuhrmann-Stroissnigg H, Dai HM, Ling YY, Stout MB, et al. Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors. Aging Cell. 2016;15(3):428-35. 59. Jung T, Bader N, Grune T. Lipofuscin: formation, distribution, and metabolic consequences. Ann N Y Acad Sci. 2007;1 119:97-11 1 .

[0209] 60. Georgakopoulou EA, Tsimaratou K, Evangelou K, Fernandez Marcos PJ, Zoumpourlis V, Trougakos IP, et al. Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues. Aging (Albany NY). 2013;5(1 ):37-50.

[0210] 61 . Linda H, Skbld MK, Ochsmann T. Activating transcription factor 3, a useful marker for regenerative response after nerve root injury. Front Neurol. 201 1 ;2:30.

[0211] 62. Melo Dos Santos LS, Trombetta-Lima M, Bjl E, Demaria M. Cellular senescence in brain aging and neurodegeneration. Ageing Res Rev. 2023:102141 .

[0212] 63. Sah E, Krishnamurthy S, Ahmidouch MY, Gillispie GJ, Milligan C, Orr ME. The Cellular Senescence Stress Response in Post-Mitotic Brain Cells: Cell Survival at the Expense of Tissue Degeneration. Life (Basel). 2021 ;1 1 (3).

[0213] 64. von Zglinicki T, Wan T, Miwa S. Senescence in Post-Mitotic Cells: A Driver of Aging? Antioxid Redox Signal. 2021 ;34(4):308-23.

[0214] 65. Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through a tragedy: mitotic catastrophe. Cell Death Differ. 2008;15(7):1 153-62.

[0215] 66. Frade JM, Ovejero-Benito MC. Neuronal cell cycle: the neuron itself and its circumstances. Cell Cycle. 2015;14(5):712-20.

[0216] 67. Hernandez-Ortega K, Quiroz-Baez R, Arias C. Cell cycle reactivation in mature neurons: a link with brain plasticity, neuronal injury and neurodegenerative diseases? Neurosci Bull.

[0217] 2011 ;27(3):185-96.

[0218] 68. Ohtani N. The roles and mechanisms of senescence-associated secretory phenotype (SASP): can it be controlled by senolysis? Inflamm Regen. 2022;42(1 ):11 .

[0219] 69. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. Journal of neuroscience methods. 1994;53(1 ):55-63.

[0220] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:1 . A method of treating pain in a subject in need thereof, the method comprising administering to the subject a senolytic in an amount effective to treat the pain.

2. The method of claim 1 , wherein the senolytic is ABT263, FOXO4-DRI, dasatinib, quercetin, fisetin, A1331852, A1155463, procyanidin C1 , curcumin, catechin, ABT737, panobinostat, apigenin, epigallo-catechin-gallate (EGCG), piperlongumine, 17-DMAG, ansamycin, resorcinol, luteolin, enzastaurin, MK1903, D- -hydroxybutyrate (DBHB), nicotinic acid, rapamycin, or nicotinamide.

3. The method of claim 1 or 2, wherein a single senolytic is administered to the subject as the only active agent to treat the pain.

4. The method of claim 1 or 2, wherein a single senolytic is administered to the subject in combination with a second non-senolytic active agent to treat the pain.

5. The method of claim 1 or 2, wherein two or more senolytics are administered to the subject to treat the pain.

6. The method of claim 5, wherein the two or more senolytics are independently selected from ABT263, FOXO4-DRI, dasatinib, quercetin, fisetin, A1331852, A1155463, procyanidin C1 , curcumin, catechin, ABT737, panobinostat, apigenin, epigallo-catechin-gallate (EGCG), piperlongumine, 17-DMAG, ansamycin, resorcinol, luteolin, enzastaurin, MK1903, D-p- hydroxybutyrate (DBHB), nicotinic acid, rapamycin, and nicotinamide.

7. The method of claim 6, wherein the two or more senolytics comprise dasatinib and quercetin.

8. The method of any one of claims 1 -7, wherein the senolytic is a peripherally restricted senolytic.

9. A method of treating pain in a subject in need thereof, the method comprising administering to the subject a senostatic in an amount effective to treat the pain.

10. The method of claim 9, wherein the senostatic targets the mTOR pathway.11 . The method of claim 10, wherein the senostatic that targets the mTOR pathway is rapamycin or RAD001 .

12. The method of claim 9, wherein the senostatic targets the ataxia-telangiectasia mutated (ATM) pathway.

13. The method of claim 12, wherein the senostatic is metformin.

14. The method of any one of claims 9-13, wherein a single senostatic is administered to the subject as the only active agent to treat the pain.

15. The method of any one of claims 9-13, wherein a single senostatic is administered to the subject in combination with a second non-senostatic active agent to treat the pain.

16. The method of any one of claims 9-13, wherein two or more senostatics are administered to the subject to treat the pain.

17. The method of any one of claims 1 -16, wherein the senolytic or the senostatic is conjugated to an antibody that binds to a protein present on the cell surface of a senescent cell.

18. The method of claim 17, wherein the antibody is an anti- Dipeptidyl peptidase-4 (DPP4) antibody, an anti-Decoy receptor 2 (DcR2) antibody, or an anti-EphA2 antibody.

19. The method of any one of claims 1 -16, wherein the senolytic or the senostatic is conjugated to a small interfering RNA (siRNA) or antisense oligonucleotides (ASOs) that binds an anti-apoptotic regulator.

20. The method of any one of claims 1 -19, wherein the senolytic or the senostatic is administered orally.21 . The method of any one of claims 1 -19, wherein the senolytic or the senostatic is administered parenterally.

22. The method of claim 21 , wherein the senolytic or the senostatic is administered by transforaminal, intravenous, transdermal, intramuscular, subcutaneous, or intrathecal administration.

23. The method of claim 22, wherein the transforaminal, transdermal, intramuscular or intrathecal administration is at the location of the pain.

24. The method of any one of claims 1 -22, wherein the pain is chronic inflammatory pain.

25. The method of claim 23, wherein the chronic inflammatory pain is injury-induced pain.

26. The method of any one of claims 1 -22, wherein the pain is surgery-induced pain.

27. The method of claim 26, wherein the surgery-induced pain is post-fracture pain, orthopedic injury, or complex orthotrauma.

28. The method of any one of claims 1 -22, wherein the pain is complex regional pain syndrome (CRPS) type 1 or type 2.

29. The method of claim 28, wherein the pain is CRPS type 1 .

30. The method of claim 28, wherein the pain is CRPS type 2.31 . The method of claim 28, wherein the pain is reflex sympathetic dystrophy or causalgia.

32. The method of any one of claims 1 -22, wherein the pain is neuropathic pain.

33. The method of claim 32, wherein the neuropathic pain is mononeuropathy, nerve injury- associated pain, post-herpetic neuralgia, small fiber neuropathy, polyneuropathy, nerve entrapment such as carpal tunnel syndrome, neuroma, chemotherapy-induced peripheral neuropathy, phantom limb pain, or thoracic outlet syndrome.

34. The method of any one of claims 1 -22, wherein the pain is diabetic peripheral neuropathy.

35. The method of any one of claims 1 -22, wherein the pain is age-related pain, and wherein the subject is a human subject at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 years of age.

36. The method of any one of claims 1 -22, wherein the pain is chronic back pain.

37. The method of claim 36, wherein chronic back pain is lumbar radiculopathy, discogenic back pain, or spinal stenosis.

38. The method of any one of claims 1 -22, wherein the pain is pain from osteoarthritis.

39. The method of any one of claims 1 -22, wherein the pain is associated with fibromyalgia or benign hypermobility, wherein the pain is a widespread pain condition, or wherein the pain is musculoskeletal pain syndrome.

40. The method of any one of claims 1 -22, wherein the pain is associated with endometriosis or vulvodynia, or wherein the pain is chronic pelvic pain.41 . The method of any one of claims 1 -22, wherein the pain is neck pain.

42. The method of claim 41 , wherein the neck pain is associated with cervical radiculopathy, cervical stenosis, or cervicogenic headache.

43. The method of any one of claims 1 -22, wherein the pain is associated with sacroiliac joint dysfunction.

44. The method of any one of claims 1 -43, wherein the subject is a human subject at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at least 70 years of age.

45. The method of any one of claims 1 -44, wherein the subject has been diagnosed as having the pain prior to the administering.

46. The method of any one of claims 1 -44, comprising diagnosing the subject as having the pain, and administering the senolytic or senostatic to the subject subsequent to the diagnosis to treat the pain.

47. A kit comprising: a senolytic or a senostatic; and instructions for administering the senolytic or the senostatic to treat pain in a subject in need thereof.

48. The kit of claim 47, wherein the senolytic is ABT263, FOXO4-DRI, dasatinib, quercetin, fisetin, A1331852, A1155463, procyanidin C1 , curcumin, catechin, ABT737, panobinostat, apigenin, epigallo-catechin-gallate (EGCG), piperlongumine, 17-DMAG, ansamycin, resorcinol,luteolin, enzastaurin, MK1903, D-p-hydroxybutyrate (DBHB), nicotinic acid, rapamycin, or nicotinamide or wherein the senostatic is rapamycin, RAD001 , or metformin.

49. The kit of claim 47 or 48, comprising the senolytic or the senostatic present in a single unit dosage.

50. The kit of claim 47 or 48, comprising the senolytic or the senostatic present in two or more unit dosages.

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