Irradiation-induced hair graying in mice: an experimental model to evaluate the effectiveness of interventions targeting oxidative stress, DNA damage prevention, and cellular senescence

Hair graying, also known as canities or achromotrichia, is a natural phenomenon associated with aging and is influenced by external factors such as stress, environmental toxicants, and radiation exposure. Understanding the mechanisms underlying hair graying is an ideal approach for developing interventions to prevent or reverse age-related changes in regenerative tissues. Hair graying induced by ionizing radiation (γ-rays or X-rays) has emerged as a valuable experimental model to investigate the molecular pathways involved in this process. In this review, we examine the existing evidence on radiation-induced hair graying, with a particular focus on the potential role of radiation-induced cellular senescence. We explore the current understanding of hair graying in aging, delve into the underlying mechanisms, and highlight the unique advantages of using ionizing-irradiation–induced hair graying as a research model. By elucidating the molecular pathways involved, we aim to deepen our understanding of hair graying and potentially identify novel therapeutic targets to address this age-related phenotypic change.


Introduction
Hair graying, also known as canities or achromotrichia, is a common age-related phenomenon characterized by the loss of pigmentation in hair strands [1,2].It is a result of a decrease in melanin production and distribution within the hair follicles [1][2][3][4].Hair graying is a widespread occurrence, affecting both men and women as they grow older.Epidemiological studies have shown that the prevalence of hair graying increases with age, with a substantial proportion of individuals experiencing some degree of graying by their 50s or 60s.While hair graying is predominantly associated with chronological aging, there is considerable variation, both inter-individual and based on genetic background, in the onset and progression of graying [5,6].Hair graying is a multifaceted process influenced by a combination of genetic, environmental, and age-related factors [7][8][9][10][11][12].
In addition to the physiological changes associated with hair pigmentation that can inform on the molecular and cellular basis for aging, hair graying can have significant psychological and emotional impacts.For many individuals, the graying of hair is not merely a cosmetic concern but is intertwined with their self-perception and body image.Hair graying can lead to feelings of embarrassment, selfconsciousness, and a perceived loss of attractiveness.These emotional problems associated with hair graying can have a detrimental effect on individuals' self-esteem and overall well-being.Particularly for women, gray hair is one of the key physical traits that fuels age-related discrimination [13][14][15][16].
Therefore, understanding the mechanisms underlying hair graying and exploring potential interventions can not only address questions on the biological aspects of aging but also provide means for improving the emotional and psychological health of affected individuals.
In addition to the natural process of aging, several external factors have been identified as potential contributors to hair graying [8,10,17,18].These factors include environmental exposures, stress, and certain medical treatments.Among these factors, radiation exposure (IR) has gained significant attention due to its potential to induce DNA damage, oxidative stress, and cellular senescence in melanocytes.Understanding the mechanisms through which radiation exposure influences hair graying can provide valuable insights into developing strategies to prevent or reverse the effects of DNA damage associated with both radiation exposure and aging on hair pigmentation.
In recent years, there has been a growing body of preclinical evidence suggesting that the plasticity of aging can be harnessed, as demonstrated by the ability of various anti-aging treatments to delay or even reverse age-related changes in various organs, including the skin and fur of rodent models [19][20][21].Various medications that cause hair repigmentation to occur in humans suggest that this plasticity is not limited to rodents [22,23].In addition, animal models of induced hair graying have emerged as particularly useful tools for evaluating the effectiveness of diverse anti-aging treatments aimed at reversing hair graying and promoting hair rejuvenation [24].These models allow researchers to explore the potential of different interventions and therapies in a controlled setting, providing valuable insights into the feasibility and efficacy of anti-aging strategies for restoring hair pigmentation.
In this comprehensive review, we explore the current evidence surrounding ionizing-irradiation-induced hair graying.By synthesizing existing research, we aim to shed light on the mechanisms underlying this phenomenon, thereby contributing to our broader understanding of hair graying due to aging and exposure to external stressors, including radiation.Additionally, we highlight the advantages of using ionizing-irradiation-induced hair graying as an experimental model, specifically to address the role of cellular senescence and senolytics in hair Vol.: (0123456789) graying, providing a roadmap for future investigations of potential therapeutic interventions.

Hair graying: mechanisms and pathways
The process of hair pigmentation is complex and involves a delicate balance of various cellular and molecular mechanisms [25,26].Here, we provide a brief overview of cellular mechanisms that have been causally linked to hair graying [1,2,10,27,28].

Melanogenesis and pigmentation
Melanogenesis plays a crucial role in hair pigmentation [25,26].The activity of melanocytes within the hair follicles and the transfer of melanin to hair shafts are essential for maintaining normal hair color.Melanocytes are specialized pigment-producing cells located in the hair follicles, which are responsible for the synthesis and distribution of melanin.Melanin exists in two main forms: eumelanin, which produces brown to black colors, and pheomelanin, which produces yellow to red colors.The balance between these two types of melanin, as well as the amount of melanin produced, determines the specific hair color observed.The process of melanogenesis is regulated by a complex interplay of genetic, epigenetic, cellular, and molecular mechanisms [25,26,29].Key proteins involved in melanin synthesis include tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TRP2) [25,26].These enzymes catalyze the conversion of the amino acid tyrosine into melanin precursors, such as dopaquinone and dopachrome, which eventually lead to the production of mature melanin pigments.The biochemical reaction of melanin synthesis occurs within the melanosome, an intracellular organelle specialized for pigment production, and requires other proteins to promote and regulate melanosome maturation.This includes, but is not limited to, the formation of premelanosome-based proteinaceous fibrils as the scaffold for melanin deposition and the shuttling of ion channels to the melanosome membrane for intralumenal deacidification required for melanocyte enzyme function [30,31].The activity of melanocytes is regulated by various signaling pathways and factors.One crucial regulator is the microenvironment within the hair follicle, including factors secreted by adjacent cells, such as keratinocytes and dermal papilla cells [32][33][34][35][36]. Signal molecules, such as melanocyte-stimulating hormone (MSH), stem cell factor (SCF), endothelin-1, and WNT, are involved in modulating melanocyte function and melanogenesis during hair growth [37][38][39][40][41][42][43].Mature melanosomes are transferred from melanocytes to the cortical and medullary keratinocytes of the hair shaft, imparting color to the hair.Melanosome transfer and distribution to skin and hair keratinocytes require various transport proteins and structural components, but the precise mechanisms are still debated.
Disruptions in melanogenesis and pigmentation processes can lead to hair graying.With aging, the number and function of melanocytes decrease, resulting in reduced melanin production and distribution [1,2,27,32,44].Preclinical studies, primarily in rodents, suggest that an important cause for hair graying is the defective self-maintenance of melanocyte stem cells [27].The resulting decline in melanin content leads to a loss of hair color and the appearance of gray or white hair.Genetic factors, environmental exposures, oxidative stress, and inflammation can also influence melanocyte function and melanin synthesis, contributing to premature hair graying.

Human versus mouse hair graying
Both human and mouse hair undergoes cyclical regeneration.Anagen represents the growth phase of the hair follicle and is when the hair is formed.During anagen, melanocyte stem cells divide to give rise to melanocyte precursors that colonize the bulb of the hair as it extends proximally.Upon differentiation, these melanocytes deposit pigmented melanosomes into the matrix keratinocytes that create the elongating hair shaft.These differentiated matrix melanocytes comprise the hair follicle pigmentary unit (HFPU).Within anagen hairs in mouse, melanocyte stem cells reside within the hair bulge, a region anatomically distinct from the hair bulb [45].Melanocytes and melanocyte stem cells are positioned similarly in human anagen hair follicles, but these follicles also harbor a population of amelanotic melanocytes, presumed melanocyte stem cells, in the outer root sheath and periphery of the hair bulb [3,46,47].After the cessation of anagen, the lower portion of the hair follicle, including the hair bulb keratinocytes and its differentiated melanocytes, regresses in a programmed apoptotic process known as catagen [48, Vol:.(1234567890) 49].From catagen, hairs transition into telogen, a nonproliferative and quiescent stage [50,51].At telogen, only undifferentiated melanocyte stem cells are retained within the hair follicle.
Major differences exist between mouse and human hair cycling, particularly when considering human scalp hairs.The length of the growth phase for scalp hair is generally between 0.5 and 7 years but ranges significantly between individuals and between hairs on the head of the same individual [52].The resting phase (which presumably includes catagen and telogen) can last 4-10 months.Using averages from an individual study of male subjects, this equates to human scalp hairs spending 68% of their time in anagen [52].In mouse, this is reversed with hairs progressing through anagen quickly, ~ 13 days or ~ 16% of the average mouse hair cycle [53].These differences are particularly meaningful when considering the etiology of hair graying in mouse versus human.Since the discovery of melanocyte stem cells in mouse [27], hair graying has been largely attributed to a stem cell defect.With age and in acute mouse models of hair graying, depletion of melanocyte stem cells is a common feature and in many cases is preceded by their premature and ectopic differentiation into melanocytes in the hair bulge [27,54].However, the short hair growth phase in mouse means that hair bulb melanocytes in this species are only required transiently to pigment the hair shaft.This leaves almost no opportunity for the HFPU to manifest timedependent changes in their function in this model system.Conversely, human hairs that experience continuous growth over years can acquire bulb melanocytes with dysmorphic features, reduced dendricity, and positive staining for apoptosis markers [55].This indicates local depletion of bulb melanocytes as a mechanism for graying in humans.While both melanocyte stem cells and hair bulb melanocytes decline in number with age in humans, it has been suggested that defects in stem cell-based regeneration of the HFPU are secondary to the decline of bulb melanocytes, and it is the latter that drives the progressive hair graying phenotype in humans.
In humans, the only gene with an established association with progressive, age-related hair graying is interferon regulatory factor 4 (IRF4).The intronic SNP rs12203592 serves a melanocyte-specific enhancer element that controls the expression of IRF4, and IRF4 protein cooperates with MITF to regulate the expression of the key melanogenic enzyme gene Tyr.The rs12203592 SNP is also associated with lighter hair color in humans and conditional knockout of Irf4 in the melanocyte lineage in mice results in notably lighter coat color [71,72].Despite its role in pigmentation, it is currently unclear how IRF4 contributes to the age-related graying phenotype.
In addition to genetic factors, epigenetic modifications have also been implicated in the regulation of hair graying.Epigenetic modifications, including DNA methylation, histone modifications, and non-coding RNA molecules, are known to regulate critical cellular processes of aging [73] and likely also contribute to hair graying.Epigenetic changes can affect the activity of genes involved in melanogenesis and melanocyte function, among others [29].Epigenetic modifications can be influenced by various factors that are known to modulate aging processes, including environmental exposures, lifestyle choices, and exposure to a wide range of stressors [29].Further research is needed to uncover additional genetic and epigenetic factors associated with hair graying and to elucidate the specific mechanisms through which they influence melanocyte function and melanin production.By expanding our knowledge of the genetic and epigenetic basis of hair graying, we can gain a deeper understanding of this process and potentially identify targets for interventions aimed at preventing or reversing hair graying.

Mechanisms for hair graying: neuronal regulation
The role of neuronal mechanisms in hair graying has garnered attention in recent research exploring the complex interplay between the nervous system and hair follicles [12,74].Empirical and anecdotal evidence has associated increased level of stress with premature hair graying [75].Recent experimental evidence provide proof-of-concept showing that, in mouse models, acute stress leads to hair graying via a mechanism that involves exhaustion of melanocyte stem cells [12,74].Dermatomes, which are specific regions of skin innervated by a single spinal nerve, have been implicated in focal dysregulation of hair pigmentation (e.g., in segmental vitiligo [76,77]).
Neuronal signaling from the central nervous system to the hair follicles can influence melanocyte function and melanin production [12].Studies suggest that neuronal factors, including neurotransmitters and neuropeptides, play a role in modulating melanocyte activity and regulating hair pigmentation [78][79][80].Disruptions in the neural pathways and impaired communication between the nervous system and hair follicles may contribute to the loss of melanocyte function and premature hair graying.Importantly, there is proteomic evidence that hair graying associated with psychological stressors and its underlying mechanisms are potentially reversible [75].Further research is needed to elucidate the precise mechanisms underlying the role of dermatomes and neuronal signaling in hair graying and how much these mechanisms ultimately contribute to human hair graying.
Mechanisms for hair graying: oxidative stress, DNA damage, and mitochondrial dysfunction Age-related oxidative stress, DNA damage, and mitochondrial dysfunction have emerged as significant factors in the process of hair graying [55,81].Oxidative stress directly affects melanocytes within the hair follicles [55,[81][82][83].Reactive oxygen species (ROS) can cause damage to cellular components, including DNA, proteins, and lipids, and induce gene expression changes leading to cellular dysfunction and decreased melanin production [82,83].Accumulating evidence suggests that increased ROS production and impaired antioxidant defense systems contribute to melanocyte dysfunction and reduced melanin production.In comparison to pigmented hairs, melanocytes in human gray hairs exhibit high levels of 8OHdG, a free radical-induced DNA lesion that serves as a biomarker of oxidative stress.Gray hairs also exhibit a higher prevalence of the mitochondrial common deletion, an indication of mitochondrial oxidative damage, along with reduced catalase, methionine sulfoxide reductase and hydroxy-radical scavenging activity, and accumulation of hydrogen peroxide (H 2 0 2 ) and oxidized amino acids.One direct target of this prooxidant environment is likely the melanogenic enzyme tyrosinase; H 2 0 2 -mediated oxidation of tyrosinase abolishes its activity [55,84,85].Sources of reactive oxygen species can be endogenous or exogenous.Superoxide anion and H 2 0 2 are both generated during melanin synthesis, and, despite this process being sequestered to the melanosome, these ROS are thought to contribute to melanocytes succumbing to oxidative challenge.UV and ionizing irradiation, toxins (like smoking), chemicals, and the inflammatory process are all external sources of ROS [86][87][88].Melanin itself scavenges ROS and while cellular pigmentation is protective against UV and H 2 0 2 -induced damage, a melanocyte's ability to balance its antiand pro-oxidant properties is essential for preventing pathogenesis.
Damage to nuclear and mitochondrial DNA is a direct result of oxidative stress, and faulty mechanisms to mitigate this damage can lead to hair graying.ATM, a protein coded from the ataxia-telangiectasia mutated gene that senses and transduces DNA damage, is expressed specifically by human bulb melanocytes and positively correlates to pigmentation status; in hairs from middle aged individuals, lightly pigmented bulb melanocytes have reduced ATM in comparison to heavily pigmented bulb melanocytes.Human patients with ataxia-telangiectasia, a progeroid or accelerated-aging syndrome, experience several cutaneous progerias including hair graying.Similarly, ATM-deficient mice are significantly more sensitive to oxidative challenge.ATM knockout mice acquire a robust gray hair phenotype in response to low-level ionizing irradiation (3 Gy) because of melanocyte stem cell differentiation and depletion from the stem cell niche [83,89,90].Gene expression studies on pigmented and non-pigmented hair follicles reveal downregulation of additional DNA repair mechanisms, namely a whole suite of nucleotide excision repair (NER) family genes, including ERCC3.Specific knockdown in melanocytes of ERCC3, a core DNA helicase subunit essential for unwinding DNA after damage for NER, reduces tyrosinase expression and activity [91].Patients with Werner's syndrome, 90% of which are attributed to gene mutations in the RecQ helicase gene WRN that functions in double strand break repair, also exhibit gray hair along with generalized progeria [92].
Age-related mitochondrial dysfunction can contribute to oxidative stress and further exacerbate melanocyte dysfunction and hair graying [2,[93][94][95].Mitochondrial oxidative stress promotes the genesis of mtDNA damage in aging, and aging is associated with a decrease in mtDNA content and mitochondrial number.Modifications of the POLG gene, the alpha subunit of the mitochondrial DNA polymerase gamma, lead to a range of aging phenotypes, including hair graying.In mice, gray hair is observed in both PolG mtDNA-mutator mice that produce elevated mtDNA mutations due to disruption in the protein's proofreading-exonuclease activity and PolG mtDNA-depleter mice that incur mitochondrial DNA depletion through a dominant negative mechanism [93,[96][97][98].Interestingly, repletion of mtDNA in mtDNA depleter mice reverses many of their cutaneous aging phenotypes.This indicates that mtDNA depletion phenotypes are not necessarily permanent and thus amenable to therapeutics that restore mtDNA content levels.Interestingly, temporary transitions between pigmented and non-pigmented states occur naturally along individual human hairs, and proteomic analysis of white and black human hair follicles shows the upregulation of key genes involved in mitochondrial energy metabolism (CPT1A, ACOT7, SOD1, CFL1, PGK1) [75].Altogether, these studies suggest active metabolic remodeling involving mitochondria is critical in the graying process, and there may be a key threshold where reversibility of these changes remains possible.
Understanding the role of oxidative stress, DNA damage, and mitochondrial dysfunction in hair graying provides insights into potential therapeutic targets aimed at reducing ROS levels, enhancing antioxidant defenses, and preserving melanocyte function.Mitigating oxidative stress through activation of the skin's own antioxidant defense system via master regulator NRF2 provides one such example of promising strategies for preventing or reversing hair graying [99,100].

Historical perspective on radiation-induced effects
The association between radiation exposure and its effects on hair pigmentation has been recognized for almost a century.In the 1920s, interested in the biological action of X-rays, Hance and Murphy followed up an incidental observation of hair graying in rabbits during a study of X-ray dosage.Using mice, they observed that hairs with direct exposure to X-ray exhibit a pattern of delayed regrowth and emerge white.The amount of whitening correlated with length of X-ray exposure, and patches of hair that were completely white immediately after exposure remained white after a second round of hair regrowth [112].In humans, significant observations were made in individuals exposed to ionizing radiation during medical treatments and nuclear accidents.One notable historical event was the atomic bombings of Hiroshima and Nagasaki in 1945 [103].Survivors of the bombings, known as hibakusha, experienced various health effects, including changes in hair pigmentation.Many hibakusha reported a rapid onset of hair graying following exposure to high doses of radiation [103].These observations provided early evidence of the connection between radiation exposure and hair graying.

Clinical observations in radiation therapy patients
In addition to the atomic bombings, the field of radiation therapy also played a crucial role in understanding the effects of radiation on hair pigmentation.Radiation therapy is a widely used treatment modality for various types of cancer.While it is highly effective in targeting and eliminating cancer cells, it can also have unintended effects on healthy tissues [113][114][115][116][117][118][119][120][121], including hair follicles [122].Patients receiving radiation therapy for various cancers often experience localized hair graying in the irradiated areas.Clinical records and case studies documented the incidence and severity of radiation-induced hair graying [102,123], providing valuable insights into the effects of radiation on hair pigmentation.
Patients undergoing radiation therapy for head and neck cancers, brain tumors, or other malignancies in close proximity to the scalp frequently experience localized hair graying within the radiation field.The severity and extent of hair graying can vary depending on the radiation dose, fractionation, and treatment duration.Clinical observations reveal that the onset and progression of radiation-induced hair graying can occur over different timeframes.In some cases, rapid hair graying within weeks or months after initiating radiation therapy has been reported.Alternatively, a delayed onset of hair graying may manifest months or even years after the completion of radiation treatment.These variations in timing suggest that the effects of radiation on hair pigmentation may involve complex interactions between acute and chronic cellular responses to radiation exposure as well as the functional state of the hair follicle during exposure.It is important to note that radiation-induced hair graying typically occurs within the irradiated field, leading to distinct patterns of graying based on the radiation treatment site.For example, patients receiving radiation therapy for brain tumors may experience graying in specific regions of the scalp corresponding to the radiation beam entry and exit points.This localized graying provides clinical evidence of the direct impact of radiation on hair follicles in the irradiated area.
Severity of radiation-induced hair graying can also vary among individuals.Factors such as the radiation dose, treatment techniques, individual susceptibility, and genetic predisposition may influence the extent and rapidity of hair graying.Hair graying induced by radiation therapy is often permanent, and the affected hair typically does not regain its original pigmentation even after the completion of treatment.
Beyond the aesthetic concerns, radiation-induced hair graying can have significant psychosocial implications for patients.Changes in physical appearance, particularly those associated with cancer treatments, can impact an individual's self-image, self-esteem, and quality of life.Hair graying may act as a visual reminder of the patient's cancer journey, potentially leading to emotional distress, body image issues, and reduced overall well-being.Therefore, the psychological impact of radiation-induced hair graying should not be underestimated, and supportive care measures should be considered for patients experiencing these changes.Clinical observations of hair graying in radiation therapy patients have contributed to our understanding of the effects of radiation on hair pigmentation.These observations highlight the need for further research to elucidate the underlying mechanisms and develop interventions to prevent or mitigate radiationinduced hair graying.By understanding the cellular and molecular processes involved, it is possible to explore strategies to protect hair follicles from radiation damage and potentially preserve hair pigmentation during cancer treatments.

Experimental models and methodologies
The dominant experimental models for evaluation of hair graying are in vivo mouse models and human hair follicle explants.Research involves visual inspection and quantification of the extent and severity of hair depigmentation.Various assessment methods have been used, including macroscopic observation, digital imaging, and quantitative measurements of hair pigmentation using specialized software.These approaches allow researchers to quantify the percentage of gray or white hairs, assess the rate of hair graying, and compare different experimental groups or treatment conditions.Histological analysis of the hair follicles is also a valuable technique in studying radiation-induced hair graying.It is possible to examine the histological features of the hair follicles, including the number and morphology of melanocytes, melanin content, follicular structure, and the presence of apoptotic or senescent cells.Techniques like immunohistochemistry and in situ hybridization enable the visualization and characterization of specific cellular Vol:.(1234567890) markers associated with changes in hair pigmentation.Recently, omics analyses including RNA sequencing and LC-MS/MS-based proteomics have elevated our understanding to the level of entire signaling pathways and molecular cascades that are reprogrammed in concert with hair pigmentation changes.
Animal studies conducted throughout the twentieth century provide much of the basis for our current understanding of the molecular and cellular mechanisms involved in radiation-induced hair graying.Experiments using laboratory animals, including mice, hamsters, and non-human primates, exposed to ionizing radiation demonstrated changes in hair pigmentation consistent with human observations [27, 104-107, 109, 110].These animal models allowed for controlled radiation exposure to dissect the timing, dosage, and frequency required to induce the phenotype [100,[124][125][126][127].These observations provide the foundation for using irradiation-induced hair graying as an experimental model to study the mechanisms underlying this phenomenon and its relationship to aging [80], as well as employing this model to test therapeutic approaches to reverse hair graying [24,111].
Mouse models, in particular, have been used extensively to investigate radiation-induced hair graying [107,108,110,128] (Fig. 1).Mice offer several advantages, including their relatively short lifespan, ease of handling, and well-characterized hair follicle cycling.Experimental protocols typically involve exposing the dorsal skin of the mice to a low dose of ionizing irradiation using specialized radiation sources.Sources of ionizing radiation to induce hair graying experimentally include γ-rays from radioactive sources like cesium-137 and X-rays generated from a non-radioactive X-ray tube [129].In the last two decades, the technology of X-ray irradiators has matured to provide parallel efficiency and improved safety in comparison to radioactive sources, and irradiators using cesium-137 fallen out of favor as regulatory agencies mitigate global security risks associated with easily obtainable radioactive substances [130,131].Thus, ~ 5 Gy delivered by an X-ray irradiator is commonly used.Importantly, this dose is sublethal and insufficient to ablate bone marrow, yet effective for inducing gray hair in mouse.
As early as the 1950s, it was acknowledged that radiation-induced graying in mouse depends on irradiation dose and dose rate, type and size of hair (zigzag hairs are most sensitive), and stage of the hair cycle.These early studies also provided the basis for interrogating differences in the DNA damage response between the stem and non-stem components of the regenerative pigmentary tissue system.In mouse, telogen hair follicles are notably more susceptible to IR-induced hair graying in comparison to anagen follicles.Because telogen follicles contain only quiescent melanocyte stem cells and no amplifying or differentiated melanocytes, this suggests heterogeneity in the cellular response to IR and a dependency on cell state (quiescence) and differentiation status (stem).More recently, lineage mapping using a melanocyte lineage-specific reporter gene (DCT-H2B-GFP) and selective depletion of non-quiescent melanocyte stem cells confirmed that it is the quiescent stem cells in the hair bulge that are most highly radiosensitive.In response to IR, quiescent melanocyte stem cells acquire irreparable DNA damage but do not undergo apoptosis.Rather, irradiated stem cells prematurely differentiate without self-renewal [90,138].Quiescent cells rely on non-homologous end joining rather than homologous recombination for repairing DNA double-stranded breaks and is speculated to be the reason for quiescent melanocyte stem cell radiosensitivity.The use of genetic mouse models further shows that IR-induced melanocyte stem cell differentiation is not reliant on p53, p19ARF, or p16INK4a and is distinct from senescence.Advanced non-invasive methods to isolate telogen follicles from mouse skin using suction provide additional feasibility for investigating the molecular mechanism that drives stem cell differentiation due to DNA damage [139].
Because of the robust graying induced by IR at telogen, mouse models have been used predominately to dissect the effects of IR on melanocyte stem cells.As a complementary alternative, explants of human anagen hair follicles are an effective model to reveal IR's impact on melanogenesis and maintenance of terminally differentiated melanocytes.Anagen hair follicles grown ex vivo continue to grow and produce pigmented hair shafts.Follicles respond quickly to IR perturbation, and within 3 days after exposure (5-8 Gy), matrix melanocytes are reduced in number and exhibit reduced melanin content; a small proportion upregulate the DNA damage marker H2AX or the senescent marker P16.Undifferentiated melanocytes within the ORS and proximal hair bulb (presumed melanocyte stem cells) react to IR (5-8 Gy) similarly to that observed in mice by prematurely upregulating the differentiation markers TRP1 and TYR and producing ectopic pigment.These responses altogether mimic those observed in white hair follicles obtained from aged individuals suggesting that irradiation can model aspects of aging in the HFPU [111].
The experimental model of IR-induced hair graying in mice offers several advantages.Dosage and timing of radiation exposure can be precisely controlled, allowing researchers to study dose-dependent effects and to investigate the threshold doses required to induce hair graying and the kinetics of hair graying [140].It is possible to employ multiple time points post-irradiation to assess the temporal progression of hair graying and monitor the cellular and molecular changes over time.The mouse model also provides researchers with a self-controlled experimental design.By comparing the irradiated areas with nonirradiated control areas in the same animals, researchers can effectively control for individual variations and genetic differences among the subjects minimizing confounding factors and strengthening the ability Fig. 1 Representative images of γ-irradiated mice with and without senolytic treatment.Male transgenic 3-MR mice (p16-3MR mice [132,133]) that carry a trimodal fusion protein (3MR) under the control of the p16 Ink4a promoter were subjected to clinically relevant whole brain irradiation protocol (5 Gy twice weekly for a total cumulative dose of 40 Gy) at 3 months of age [134].Radiation was administered using a 137 Cesium gamma irradiator (GammaCell 40, Nordion International).A Cerrobend® shield was utilized to minimize exposure outside the brain.After 3 months, to eliminate senescent cells, one group received the senolytic drug ABT263 (Navitoclax) via oral gavage, 50 mg/kg dissolved in phosal PG:PEG400:ethanol (60:30:10)) [133,135,136] for 5 days and for 2 cycles with a 2-week interval between cycles.3MR contains the herpes simplex virus thymidine kinase, which allows the selective elimination of p16-positive senescent cells by administering the prodrug ganciclovir [137].Thus, to deplete senescent cells, another group received ganciclovir (i.p. 25 mg/ kg/daily in PBS) for 5 days and for 2 cycles with a 2-week interval between cycles.Animals were photographed 3 months after the initiation of senolytic treatment to attribute observed hair graying specifically to radiation exposure.Further, the IR-induced hair graying model allows for reproducible and standardized protocols, ensuring consistency across different studies.
Animal studies also provide opportunities for intervention and therapeutic exploration [24].Previous studies have explored the use of antioxidants, anti-inflammatory agents, and other compounds to mitigate the effects of radiation on hair pigmentation.For example, a recent study demonstrated the efficacy of the combination treatment with cyclosporine A and minoxidil, along with RT175, a non-immunosuppressive immunophilin ligand in this model [24].These studies have shown promising results in preventing or reducing hair graying in irradiated mice, highlighting this model as a potential screening tool to identify novel biological pathways involved in melanocyte stem cell and HFPU rejuvenation in response to genotoxic stress [24].The ability to genetically manipulate specific genes or signaling pathways in mice further enables our ability to gain mechanistic insight into key players in this process.
The use of in vitro cell culture models has also contributed to our understanding of radiation on melanocyte function.Melanocyte cell lines or primary melanocyte cultures can be irradiated and subsequently assessed for changes in melanin production, cellular senescence markers, and gene expression profiles.These in vitro models allow for controlled experiments and provide insights into the molecular pathways underlying radiation-induced changes in melanocyte function and hair graying.
Exploring the irradiation (IR) model to investigate the role of cellular senescence in hair graying and graying reversal While the precise mechanisms through which radiation exposure contributes to hair graying are still being elucidated, the evidence suggests that radiationinduced DNA damage and oxidative stress affecting both melanocyte stem cells and differentiated melanocytes directly play crucial roles and mimics the aging phenotype.However, less attention has been given to the role of the niche as a contributing factor to the decline of the pigmentary system after IR, or in age-related hair graying.Significant evidence implicates the niche in both melanocyte stem cell support and HFPU maintenance.For instance, exogenous growth factors can prevent hair graying caused by repeated depilation [141], and mutations conditionally affecting hair follicle stem cells (i.e., Col17a1) can also lead to disruptions in melanocyte stem cell maintenance and cause premature gray hair [142].In growing human hairs, hair follicle keratinocytes especially those differentiating within the inner root sheath, more so than melanocytes, exhibit markers of sustained oxidative stress and apoptosis potentially creating their own stressful microenvironment as part of natural hair cycling [83].
It is likely that the keratinocyte-melanocyte relationship also plays an important role in the context of IR-induced hair graying.IR slows hair regrowth after depilation and reduces the colony-forming capacity of follicular keratinocyte stem cells.Reconstitution of hairs using irradiated host skin and nonirradiated donor melanocyte stem cells results in gray hair, suggesting irradiated hair follicle keratinocytes are insufficient to support or can act as mediators of melanocyte susceptibility to IR [143].In reference to the latter, how hair follicle keratinocytes negatively impact melanocyte stem cells or the HFPU in the context of IR is unknown, but one understudied aspect of radiation exposure that has not been directly implicated in hair graying is cellular senescence.Cellular senescence is a state of irreversible growth arrest characterized by altered cellular function and the secretion of pro-inflammatory molecules known as the senescence-associated secretory phenotype (SASP) [144][145][146][147][148][149].The interplay between cellular senescence and hair graying likely involves complex molecular pathways that include cells of the melanocyte lineage along with senescing cells within the niche.
In several contexts, cellular senescence is an intrinsic aspect of melanocyte pathology, particularly in vitiligo and melanoma [111][112][113][114]. Senescent epidermal melanocytes have also been implicated in driving skin aging [150].However, the contribution of senescent melanocytes to hair graying in response to radiation or aging remains unclear.In mice, the dominant response by melanocyte stem cells in response to IR is not senescence.Rather, melanocyte stem cells prematurely differentiate, and this process occurs even in mice that do not express P16.Differentiating/ed melanocytes in anagen hair bulbs of mice are radioresistant, and exposure to IR does not induce significant hair graying in the time frame in which pigmented Vol.: (0123456789) hair shafts are produced [90].In human scalp hairs, on the other hand, senescent hair matrix melanocytes are observed with both IR and aging, suggesting that this is a characteristic unique to human hair bulb melanocytes and is perhaps attributable to the much longer time period these cells remain functional during the lengthy anagen hair growth period in humans [111].Outside of melanocytes, and in both mouse and human, radiation exposure induces robust cellular senescence within the hair follicle similar to that observed in aged hair follicles [111,139].Because SASP molecules secreted by senescent cells can influence neighboring cells, including melanocytes, senescent keratinocytes may contribute to the chronic inflammation observed in hair follicles and further influence the hair-graying process.In mice, although the initial insult that causes hair graying does not depend directly on senescent melanocyte stem cells or melanocytes, repeated depilation shows that irradiated mice continue to gray across multiple hair cycles, and whether senescent cells participate in this progressive process is unknown.Cellular senescence is recognized as a fundamental process in aging and age-related diseases [144][145][146][147][148][149].Understanding the role of cellular senescence in hair graying, including its potential induction by radiation exposure, provides valuable insights into the underlying mechanisms of how senescence augments the decline of regenerative tissues.
Exposure to ionizing radiation can induce cellular senescence through various mechanisms.DNA damage resulting from radiation exposure can activate cellular stress response pathways, such as the DNA damage response (DDR) pathway [151].DDR signaling triggers cell cycle arrest and can lead to the induction of cellular senescence [152,153].Strategies aimed at preventing DNA damage, modulating cellular senescence or the SASP, and eliminating senescent cells (senolytic treatments) may offer promising avenues for future research and the development of interventions to prevent premature development of age-related pathologies [154][155][156][157] including hair graying.As an example, Fig. 1 depicts γ-irradiated mice in two different senolytic models, administration of ganciclovir to p16-3MR mice [132,133], and administration of ABT/Navitoclax to wild-type mice.In this study, the primary focus was to investigate the impact of wholebrain irradiation on cerebrovascular outcomes [133].However, changes in hair color were also recorded as a secondary outcome during the course of the study.These preliminary data showed that systemic senolytic treatment initiated 3 months post-irradiation did not reverse or curtail hair graying in irradiated mice (Fig. 1).This suggests that senescence, be it intrinsic or extrinsic to the melanocyte lineage, may not contribute to the continued hair graying observed in irradiated mice over time, but additional studies should be performed to solidify this finding.When using the mouse model, understanding the dynamics of hair cycling and hair cycle stage during irradiation and treatment is critical to interpreting the experimental outcomes.Repeated depilation in mice has the added benefit of addressing senescence chronologically in the same animals and allows for direct assessment of stem cell regenerative potential.While the mouse model is fantastic for investigating the effects of perturbation or therapeutics on melanocyte stem cell function, it is limited in its ability to investigate longterm effects of IR or age on differentiated hair follicle melanocytes.Thus, expanding studies of senolytics to explants of human anagen hair follicles serves as an important and complementary approach if we want to understand senolytics in the context of human hair graying.It is also important to note that various senolytic regimens may have side effects that can confound the results.For example, previous studies showed that defective self-maintenance of melanocyte stem cells promotes hair graying, and this process is accelerated dramatically with Bcl2 deficiency, which causes selective apoptosis of melanocyte stem cells [27].In the field of geroscience, Bcl2 inhibitors (e.g., ABT263/Navitoclax) are often used as senolytic treatments [133,135,154,155].However, administration of ABT263 to mice results in regional hair graying at the site of the injection (Ungvari and coworkers, unpublished observation, 2021), probably due to the aforementioned side effects of the drug.Thus, future studies should employ specific senolytic treatments without side effects that potentially impact hair color.

Future Directions and Conclusions
In conclusion, IR-induced hair graying serves as a valuable experimental model to study the intricate processes underlying hair graying, with a particular focus on the potential role of radiation-induced oxidative stress, DNA damage, and cellular senescence.By unraveling the molecular pathways involved, this research contributes to our broader understanding of hair graying in aging and sheds light on potential therapeutic targets for preventing or reversing this aesthetic change.These studies can also answer basic biological questions regarding the role of the niche and cellular senescence in tissue regeneration in response to genotoxic stress and delineate short-and long-term effects of radiation on stem and differentiated cell function.Further investigations in this field, using a combination of both mouse and human models, are warranted to unlock the full potential of this experimental model and translate the findings into clinical applications.
Long-term studies exploring the reversibility of radiation-induced hair graying with novel antiaging interventions, including specific senolytic treatments, are also warranted.In humans, repigmentation of gray or white hair is a well-documented phenomenon and can occur both naturally along an individual hair shaft or in response to chemicals and medications [22].One dramatic example includes complete gray hair reversal as a side effect of cancer therapy (e.g., anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (anti-PD-L1) therapy for lung cancer [23]).Preclinical models of hair graying, like the IR-induced models described above, can help elucidate mechanisms by which hair repigmentation occurs.This may include mechanisms that directly mitigate the effects of irradiation (e.g., enhancing antioxidant levels) but can also include gain-offunction mechanisms unrelated to the initial insult (e.g., promoting repopulation of the hair bulb from inactive melanocyte stem cells).The application of multiomics technologies is anticipated to offer novel insights into the biology of hair aging, ultimately aiding in the identification of specific targets for the development of effective therapeutic interventions [158].A recent study found that caloric restriction, an anti-aging dietary regimen, significantly prolongs the time it takes for hair to turn gray in response to IR-induced DNA damage in mice [159].The research revealed that caloric restriction extends the resting phase of hair follicles by keeping hair follicle stem cells in a quiescent state, which temporarily prevents the depletion of melanoblasts and delays hair graying [159].These promising preclinical findings underscore the need for further investigation into the effects of antiaging dietary regimens, potentially in combination with other treatments, in human subjects.Beyond identifying therapeutic targets for preventing or treating hair graying in both radiation-exposed individuals and the general aging population for the purpose of aesthetics, the melanocyte stem cell, HFPU, and IR-induced hair graying provide a powerful tool to investigate and test mechanisms associated with regenerative tissue decline with aging and the effects of pro-longevity interventions during the progression of stem cell differentiation. .The authors express their gratitude for Unity Biotechnology, Inc. for providing the p16-3MR mouse model.The funding sources had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the American Heart Association, or the Presbyterian Health Foundation.The 3.5 version of ChatGPT, developed by OpenAI, proved to be a useful tool in refining our writing and enhancing the clarity of our work.

Acknowledgements
This work was supported by grants from the Oklahoma Center for the Advancement of Science and Technology, the National Institute on Aging (RF1AG072295, R01AG055395, R01AG068295; R01AG070915, K01AG073614, K01AG073613, R03AG070479), the National Institute of Neurological Disorders and Stroke (R01NS100782), the National Cancer Institute (R01CA255840), the Oklahoma Shared Clinical and Translational Resources (U54GM104938) with an Institutional Development Award (IDeA) from NIGMS, the Presbyterian Health Foundation, the Reynolds Foundation, the Oklahoma Nathan Shock Center (P30AG050911), and the Cellular and Molecular GeroScience CoBRE (P20GM125528).Project no.TKP2021-NKTA-47 implemented with the support provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development, and Innovation Fund, financed under the TKP2021-NKTA funding scheme; by funding through the National Cardiovascular Laboratory Program (RRF-2.3.1-21-2022-00003)provided by the Ministry of Innovation and Technology of Hungary from the National Research, Development, and Innovation Fund; project no.135784 implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the K_20 funding scheme and the European University for Well-Being (EUniWell) program (grant agreement number: 101004093/ EUniWell/EAC-A02-2019 / EAC-A02-2019-1)