Abstract
Objective
To develop an ordinal hair density score (HDS), determine its inter-rater agreement, and use it in a trial of photobiomodulation as a sole treatment for alopecia X.
Methods
A 5-level ordinal HDS system was developed. Four blinded veterinary dermatologists independently graded a 50-image reference set using the HDS. Inter-rater agreement was assessed using the quadratic-weighted Fleiss κ, Brennan-Prediger, and Gwet AC2 coefficients. A double-blind, randomized, controlled trial was performed using a convenience sample of alopecia X dogs recruited based on inclusion and exclusion criteria over 16 months. Photoconverter gels were applied on both alopecic sides of each patient once per week for 8 weeks. One randomly chosen side was exposed to excitatory light (active treatment) but not the other (sham). Skin biopsies were taken from the center of each treated side before and at the end of the study. The images of active and sham sides acquired before the study, at day 50, and at the end of the study were graded using the HDS.
Results
Inter-rater agreement coefficients were greater or equal to 0.81. Seven dogs were enrolled, but 1 withdrew after day 50. Hair density score evolved over time in both sides, but the OR of improved HDS increased with time only for the 3 central HDS grades. Histopathology revealed no notable differences between sides and across time.
Conclusions
The HDS seems valid and useful in assessing the effect of photobiomodulation on the exposed areas of our alopecia X patients.
Clinical Relevance
This novel, easily applicable scale may facilitate the therapeutic monitoring of alopecia in dogs.
Alopecia X is a skin disorder affecting the hair follicle growth. It is characterized by a noninflammatory bilaterally symmetric hair loss commonly occurring in double-coat and dense undercoat breeds.1,2 The diagnosis relies on a coherent history, compelling examination findings, and exclusion of infectious conditions and endocrinopathies, such as hypothyroidism and hyperadrenocorticism.3 Histopathological examination reveals a predominance of hair follicles in kenogen or telogen over anagen follicles and, in most cases, hyperpigmentation of the epidermis.4,5 The underlying pathogenesis is still not fully understood, but a recent study6 aiming to identify its genetic basis has highlighted the potential role of mitochondrial gene mutation in the condition.6
Different therapies have been described in the literature, with a variability in the response as well as types of side effects. Reported treatments include sterilization, growth hormone, melatonin, mitotane, trilostane, medroxyprogesterone, deslorelin, and microneedling, and some cases can exhibit spontaneous remission.1,3 There is, however, no gold standard for care, opening the door to alternative treatments.1 Photobiomodulation (PBM), which includes low-level laser therapy, has been used for decades in human and veterinary medicine in clinical fields, such as dermatology, oncology, dentistry, neurology, surgery, and ophthalmology.7,8 Studies7,9 have already shown the potential of PBM on alopecia in human and animal models through its stimulatory effect on mitochondrial activity, cell proliferation, and other targets and possibly its ability to increase the fraction of hair follicles in anagen. A study10 testing the clinical efficacy of PBM has shown promising effects on hair regrowth in dogs with different types of noninflammatory alopecia. Recently, a study11 protocol aiming to compare the efficacy of melatonin and PBM, either alone or combined, for the treatment of alopecia X has been published, but no results have been reported yet.
Various methods have been used in research to quantify hair density and alopecia.12,13 To determine whether a treatment is working, a noninvasive, reliable, inexpensive, and rapid method is required.14 To the authors’ knowledge, there is no established standardized scoring system for hair loss in dogs. Ordinal outcomes (ie, monotonically ranked categories that are ordered hierarchically such that the distance between any 2 categories is not necessarily equal or even meaningfully quantifiable) are increasingly used in randomized, controlled trials.15 Among other favorable characteristics, ordinal scales easily represent multiple patient states and increase the statistical power as compared to the dichotomous scale.15 However, the statistical analysis of ordinal outcomes is more complex and requires greater care to avoid misleading results.15
The present study tested 2 hypotheses. First, the ordinal scale used in this study is robust enough to reproducibly detect changes in canine hair density when used by different raters. Second, considering the favorable effect of PBM on mitochondrial activity and hair follicle cycle, the ordinal scale is sensitive enough to detect hair density changes of alopecia X lesions repeatedly exposed to fluorescent light energy (FLE). We tested these hypotheses by accomplishing 3 objectives: (1) developing an ordinal hair density scoring system, (2) determining the inter-rater agreement (IRA) of veterinary dermatologists when evaluating hair density with this scoring system, and (3) using this scoring system in a double-blind, randomized, sham-controlled trial of FLE exposure on otherwise untreated alopecia X skin. The OR of the statistical analysis of ordinal data will be used to assess the sensitivity to changes in hair density over time and the effect of FLE therapy.
Methods
Hair density score development and IRA testing
As no veterinary alopecia grading system has been published yet, we developed an ordinal scale system based on features of 2 existing human alopecia scoring systems, the Severity of Alopecia Tool (SALT) and the Alopecia Areata Investigator Global Assessment (AA-IGA) scale.16,17 Both systems quantify the relative surface area of hair loss as a percentage, grouping the alopecic surface area values into 5 (AA-IGA) or 6 (SALT) categories. In both systems, the first and last categories represent the extreme values (ie, 0% hair loss and either 100% [SALT] or ≥ 95% [AA-IGA]). The remaining range of values is distributed either evenly (SALT) or unevenly (AA-IGA) across the other categories. Based on the clinical experience of the dermatologists (CCB, GB, CDJ, and NP) authoring this study, we decided (1) that the grading system should measure hair density, not hair loss; (2) to keep the bare skin category (ie, 0% density) only because a full coat density is difficult to establish in the clinical setting without prealopecic data; and (3) to evenly distribute the remaining percentage of density values across 4 other categories because an uneven distribution may lead to errors when the category represents a narrow range of hair density values greater than 0%.
To test the reliability of this scale, we built an alopecia X reference image set containing 50 photographs of affected areas, which were afterwards rated independently by the 4 dermatologists certified by the American College of Veterinary Dermatology working at the CENTREDMVET referral centers (Québec, Canada). Then, the scores given by each rater were used in estimating the IRA as described in the Statistical analysis section.
Effect of FLE therapy on alopecia X
To test the sensitivity to change of our hair density score (HDS) and its reliability when used for the purpose of assessing therapeutic efficacy, we performed a randomized, double-blind, sham-controlled clinical trial with the PHOVIA System (Vetoquinol N-A Inc) on privately owned dogs. Our reporting of this trial adhered as closely as possible to the PetSORT guidelines.18
Ethics
This study was approved by the CENTREDMVET bioethical committee (approval No. 2023-01#). The owners were informed of the experimental process and signed consent forms prior to participation.
Animals
As we had no previous knowledge of the potential effects of PBM on alopecia X and how these would distribute in an ordinal system, we were unable to calculate a sample size. Therefore, we recruited all eligible cases presented at the Montreal, Blainville, and Saint-Hubert referral centers of CENTREDMVET from July 2023 through October 2024. Seven unrelated, privately owned dogs diagnosed with alopecia X were enrolled. To rule out other conditions, dermatological examinations, including trichograms, skin scrapings, Wood lamp examinations, and cytological evaluations, were performed. In addition, we screened for other potential disorders by means of physical examination and blood analysis (ie, serum chemistry, CBC, thyroid-stimulating hormone, total T4 quantification, urinalysis, and urine cortisol-to-creatinine ratio).
Inclusion and exclusion criteria
Inclusion criteria consisted of dogs with nonpruritic, noninflammatory, bilaterally alike alopecia, excluding the head and distal limbs; absence of infection on dermatological examination; histopathological findings consistent with alopecia X; and absence of systemic clinical signs.
Exclusion criteria consisted of dogs with photosensitivity due to any condition or taking drugs/products known to induce photosensitivity reactions; dogs with known skin hypersensitivity; dogs receiving systemic or topical corticosteroids at the time of presentation or having received such therapy within the previous 2 months; dogs with skin diseases or other underlying diseases associated with nonpruritic hair loss (eg, hyperadrenocorticism and hypothyroidism); dogs receiving trilostane, deslorelin, growth hormone, mitotane, or medroxyprogesterone acetate within the previous 3 months; dogs sterilized within the past 6 months; dogs receiving treatment with oral melatonin within the previous 2 months; and pregnant or lactating bitches.
At the time of recruitment (ie, 2 weeks before the start of the study [d0]), owners were instructed to use neither oral treatments that could affect hair density (mentioned in the exclusion criteria) nor any kind of topicals (eg, gel, cream, ointment, spray, shampoo/conditioner, and medication). This was also prohibited during the study duration. Failure to respect this would result in exclusion.
Test materials
The PHOVIA System comprises a hand-held photobiomodulation device, a Carbopol-based amorphous carrier hydrogel and a second gel containing proprietary chromophore molecules. The device delivers an incoherent blue light with a power density ranging from 55 to 129 mW/cm2 at a 5-cm distance from the skin, and produces peak excitation wavelengths ranging from 440 to 460 nm. The gels are homogenized together before application on the targeted skin area. The chromophore molecules act as a topical photoconverter that, when exposed to the excitation wavelengths, emit photons at different wavelengths (400 to 700 nm) within the spectrum of visible light, which, in turn, induce the modulatory effects on the cells underneath.19
Clinical trial protocol
During the baseline period, all diagnostic procedures were performed. At the end of the experimental d0, finishing immediately prior to treatment administration, a baseline image was acquired. Then, an animal health technician tossed a coin to randomize the side to receive the active treatment (AT) and sham treatment (ST). On each side, a 2-mm-thick layer of photoconverter gel was applied on the skin with a spatula. Then, the AT side received a 2-minute FLE exposure, whereas the ST was left unexposed because the excitation wavelength is key to the biomodulatory photon emission by the chromophore molecules. Starting on day (d)1, once a week for 8 consecutive weeks (ie, d1, d8, d15, d22, d29, d36, d43, and d50), the patients received 2 consecutive treatments on each affected side over the 50-cm2 area delimited by the circumference of the lamp. The identity of the AT and ST sides were undisclosed to the owners and the HDS raters. To ensure the consistency of the localizations of the treated areas, we used anatomical landmarks to place plastic stencils and took photographs. The gel was then gently removed with gauze soaked in saline solution for both sides. The process was repeated once in the same session approximately 2 minutes afterwards. Protective eyewear provided by the company was worn during the procedure, and the dog’s eyes were covered by a towel.
Assessment of HDS change following FLE
The photographs of each patient’s treated side taken on d0, d50, and d78 were examined independently by the 4 dermatologists. Similarly to the reference image set, they used the HDS to grade the hair densities in a treatment side–blinded manner (ie, they withdrew prior to treatment administration to ensure their blinding to patient identity and treated sides). In addition, they qualified (null, marginal, minor, moderate, and major) the posttreatment recoveries from alopecia at d50 and d78 relative to d0.
Biopsy collection and histological examinations
A pretreatment (d0) biopsy was performed for each treated side to confirm the clinical diagnosis of noninflammatory alopecia by means of histological analysis. Each sample was taken at the center of the areas that would receive either AT or ST. Additional biopsies were taken from both areas at d78, approximately 1 cm away from the initial biopsy site, to assess potential histological changes relative to d0. Following a standard preparation procedure, the biopsy sites were anesthetized locally by injecting 1 mL of a 1:1 mixture of lidocaine (Lurocaine 2%; Vetoquinol; 10 mg/site) and sodium bicarbonate (Sodium Bicarbonate Injection 8.4%; Omega Laboratories; 42 mg/site). Ten minutes after, the biopsy was obtained using 6-mm punches. The sites were closed with resorbable sutures. Following tissue fixation in 10% buffered formalin, the samples underwent tissue processing and paraffin embedding and were sectioned into 3-µm-thick sections. The slides were then stained with hematoxylin, phloxine, and saffron following standard procedures.
Statistical analysis
Inter-rater agreement of HDS—The IRA of HDS gradings of the reference image set and of the images taken during the trial were analyzed separately using a series of SAS/IML functions programmed in SAS, version 9.4 (SAS Institute Inc).20 Fleiss-Cohen quadratic weights accounting for the distances among raters’ scores were used in all chance-corrected agreement coefficient calculations.21 For each dataset, we computed the Fleiss generalized κ, the Brennan-Prediger (BP) agreement coefficient, and the AC2 coefficient of Gwet and their associated SEs and limits of their 95% CIs. The latter were benchmarked using the interpretative scale of Altman.20
To explore the robustness of our HDS during the clinical trial, the IRA coefficients of the patient’s HDS grades were calculated separately for each treated side and day combination.
Effect of FLE on alopecia X—The time courses of HDS and the clinical improvement scores were analyzed separately in SAS, version 9.4 (SAS Institute Inc), each with a generalized linear mixed model (GLMM) for repeated measures. Both ordinal outcomes were modeled using a multinomial distribution and the cumulative logit link function.22 The proportional odds assumption on which this model relies was verified afterwards by dichotomizing the ordinal score at every threshold of the scoring system (ie, 0 vs ≥ 0, ≤ 1 vs ≥ 1, etc) and reanalyzing each of them with the same repeated-measures GLMM but this time using a binary distribution and logit link.15 In the case of HDS, treatment, time, and the time X treatment interaction were used as fixed-effect factors. The patient and rater were considered random-effect factors with subject-specific HDS intercepts and time courses in each treated side. A first-order autoregressive covariance matrix was used to model the dependencies of HDS values over time both for the treated sides nested within patient and for the rater nested within treated side and patient.
The clinical improvement scores were modeled using treatment and hair density score as fixed factors. Patient and rater were used as random factors. A diagonal covariance matrix was used to model the dependencies of improvement scores both for the treated sides nested within the patient and for the raters nested within treated side and patient.
Results
Hair density score development and IRA testing
The following 5-level ordinal visual scale was defined to quantify hair density over the affected areas: (1) null [0%], (2) marginal (0% to 25%], (3) mild (25% to 50%], (4) moderate (50% to 75%], and (5) major (75% to 100%] (Supplementary Table S1). Figure 1 depicts the agreement of HDS grades given by each pair of raters examining the reference image set. When comparing the HDS grades given by raters #1, #2, and #4, a majority were either on or within 1 unit away from the agreement line, suggesting smaller discrepancies between each pair. Rater #3 had larger discrepancies relative to the other 3 raters as suggested by the uneven distributions of HDS grades around the agreement lines. Considering their respective 95% CIs, all 3 quadratic-weighted, chance-corrected IRA coefficients were close to or above 80% (Table 1). Of note, the 95% CI of Fleiss κ was roughly twice as large as those of the BP and Gwet AC2 coefficients. Because the lower bounds of the 95% CIs of the first 2 coefficients were lower than 0.8, our scoring system had either good or very good IRA according to the Altman benchmarking system.
Pairwise agreement of the raters’ scores. The bubble plots illustrate the frequencies of pairs of hair density scores (HDSs) given by each unique pair of the 4 board-certified veterinary dermatologists to the reference image set (n = 50). Overall, partial agreements most often lie within 1 score unit away from perfect agreement (diagonal line), and rater #3 shows the most discordance. Diagonal, perfect agreement between the pair of raters; bubbles, recorded pairs of scores; number to the right of the bubble, frequency of the pair of scores; bubble size and color, visual cues of frequency.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.01.0034
Inter-rater agreement (IRA) coefficients.
| Altman benchmarking range | ||||||||
|---|---|---|---|---|---|---|---|---|
| IRA coefficient | Estimate | LCB | UCB | VG | G | M | F | P |
| Fleiss κ | 0.809 | 0.727 | 0.891 | 0.589 | > 0.999 | 1 | 1 | 1 |
| Gwet AC2 | 0.876 | 0.832 | 0.921 | > 0.999 | 1 | 1 | 1 | 1 |
| BP coefficient | 0.836 | 0.789 | 0.882 | 0.940 | 1 | 1 | 1 | 1 |
BP = Brennan-Prediger. F = Fair (0.2 to 0.4]. G = Good (0.6 to 0.8]. LCB = Lower confidence bound. M = Moderate (0.4 to 0.6]. P = Poor [−1 to 0.2]. UCB = Upper confidence bound. VG = Very good (0.8 to 1].
Four board-certified veterinary dermatologists independently graded the reference image set (n = 50) using the hair density score. The quadratic-weighted Fleiss generalized κ, the BP, and the Gwet AC2 IRA were computed as well as the lower and upper bounds of their 95% CIs. The latter were benchmarked using Altman system. All estimated IRA coefficients are above 80%, but only the LCB of AC2 exceeded the 0.8 threshold value. The cumulative probabilities of belonging to a specific agreement benchmarking category, assuming a Gaussian distribution, are presented under the Altman benchmarking range. Reading the line from left to right, the lowest cumulative probability value above 95% indicates which qualifier best describes the IRA level.
Patient characteristics
The signalment data of the dogs included in the study and the locations of AT and ST are reported (Table 2). One patient withdrew after the last treatment, at d50, due to the owner’s perceived worsening of the alopecia on the untreated zones. No adverse effects were noted during the study.
Characteristics of study participants.
| Treatment side | |||||||
|---|---|---|---|---|---|---|---|
| No. | Breed | Age (y) | Sexa | Age at onset (y) | Area | Sham | Active |
| 1 | Chow Chow | 3.3 | M | 1.5 | Neck | R | L |
| 2 | Pomeranian | 2.7 | F | 2 | Thigh | L | R |
| 3 | Pomeranian | 4.9 | M | 3.5 | Thorax | R | L |
| 4 | Pomeranian | 6.2 | F | 4.5 | Thigh | L | R |
| 5 | Pomeranian | 3.2 | M | 2.5 | Thorax | R | L |
| 6 | Pomeranian | 3.8 | F | 2 | Thigh | L | R |
| 7 | Pomeranian | 4.2 | M | 3.5 | Thorax | R | L |
F = Female. L = Left. M = Male. R = Right.
All patients are neutered.
Effect of FLE treatment on HDS
The time courses of the individual ranges of HDS stratified by treated sides, which encompass all raters’ observations, are illustrated (Figure 2). The d0 HDS highly varied among patients, but each treatment side had almost identical grade distributions. The median individual HDS values for the ST side remained within the range of d0 scores given by the raters for all but 1 patient, whereas on the AT side, the median individual HDS values tended to increase after d0 in 4 of 7 patients.
Time course of HDS of patients exposed to active photobiomodulation therapy (AT) or sham photobiomodulation therapy (ST). Seven alopecia X patients were evaluated independently by 4 board-certified veterinary dermatologists at the start of the study (d0) and at day (d)50 and d78. The HDS tends to increase more readily on the AT than the ST side. Poorly responding patients are located at the extremes of the HDS range. Markers, median individual score at the time of visit; whiskers, associated range of individual scores; gray color, patients with extreme HDS values; other colors, patients with midrange HDS values. The individual median scores are connected by hatched lines and are slightly moved apart to avoid overlapping. Vertical reference lines, start and end of exposure to the AT and ST.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.01.0034
The statistical model for ordinal outcomes revealed that time had a significant effect on the odds of reaching higher HDS grades in dogs with alopecia X (P = .019), but statistically nonsignificant effects were recorded for treatment (P = .43) and for the time X treatment interaction (P = .50). However, the analysis of the dichotomized scores using repeated-measures GLMM for binary outcomes refuted the proportional odds assumption of the ordinal data analysis. When testing the lowest 2 (≤ 25%) versus the highest 3 (> 25%) HDS grades, only time significantly increased the odds of reaching a higher HDS grade (P = .029). When testing the lowest 3 (≤ 50%) versus the highest 2 (> 50) HDS scores, time (P = .002) and treatment (P = .0006) significantly increased the odds of a higher HDS grade. Testing the extreme HDS grades against the remaining ordinal grades (ie, 0% vs > 0% and ≤ 75% vs > 75%) yielded either a statistically nonsignificant effect or did not reach a solution. Attempts to analyze the HDS data using a repeated-measures, generalized nonlinear mixed model for ordinal outcomes with nonproportional odds failed to reach a solution because the model was too complex for this small-sized database.
Inter-rater agreement of HDS during the trial
Chance-corrected IRA coefficients of HDS in alopecic patients were calculated separately for each treated side and measurement time. The agreement coefficients and associated 95% CIs largely overlapped the 95% CIs of the coefficients determined using the reference image set (Figure 3). The 95% CIs of the Fleiss κ coefficients were again much larger than the ones of the BP and Gwet AC2 coefficients.
Inter-rater agreement (IRA) of HDS of dogs exposed to photobiomodulation therapy over time. Four board-certified veterinary dermatologists independently assessed the AT and ST sides of each patient at d0, d50, and d78. Three IRA coefficients (Fleiss generalized κ, Brennan-Prediger [BP], and Gwet AC2) and their associated 95% CIs were calculated. The corresponding IRA and 95% CI of the reference image set are used as a reference level to detect potential effects of time and treatment on the patient’s IRA. The overlap of the 95% CIs of both sources of data suggests that the HDS scale is robust to either variable. Circles, estimated IRA for a given time and treatment side; whiskers, 95% CI of the coefficient; green color, AT side; red color, ST side; horizontal reference lines, estimated agreement coefficient (Ref) and bounds of the 95% CI (± 95% CI) for the reference image set; vertical reference lines, start and end of exposure to the treatments.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.01.0034
Effect of HDS on perceived clinical improvement
The majority (91.3%) of clinical improvement scores given on d50 and d78 ranged from “null” to “mild,” but their distributions noticeably differed across treated sides. Indeed, 75% of ratings given to the ST were either “null” or “marginal,” whereas 56% of ratings given to the AT side received these grades. The likelihood of perceiving clinical improvement tended to increase with HDS, but the overall effect of HDS on clinical improvement score did not reach statistical significance (P = .099). Moreover, the effect of the AT on the likelihood of perceived clinical improvement was nonsignificant (P = .57).
Histopathology
The histological analysis of the initial samples revealed results consistent with alopecia X across all 7 patients (Figure 4). Follow-up histological analysis in 6 patients showed no substantial differences between both sides.
Photomicrograph of skin biopsies obtained for patient #1 comparing the findings of the treatment area at d0 (A) and at d78 (B). Notice the absence of notable difference between sides at both time points. Orthokeratotic hyperkeratosis (*) can be observed with epidermal atrophy (arrowheads). Atrophic follicles with an excessive amount of trichilemmal keratin (thin arrow) and dilated infundibula filled with a large amount of keratin (thick arrow) are observed. No inflammatory infiltration of the hair follicle is observed. Hematoxylin, phloxine, and saffron stain. Scale bars = 400 µm.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.25.01.0034
Discussion
This study aimed to investigate 2 hypotheses. First, that an ordinal scale would be sensitive enough to reproducibly detect changes in canine hair density when used by different raters, and second, that the ordinal scale would be sensitive enough to detect significant hair density changes following FLE exposure in alopecia X patients.7,9 To the extent of the authors’ knowledge, ours is the first attempt to develop an HDS targeted toward small animals and the first trial on the use of incoherent PBM as the sole intervention for alopecia X patients.
To investigate the first hypothesis, a hair density scoring system based on a 5-level ordinal scale was developed, and the IRA of mutually blinded veterinary dermatologists was assessed. The findings from this study indicate that the HDS has the capacity to distinguish the patients and detect changes in hair density between the AT and ST sides over time. Moreover, the statistical analysis demonstrated that our scoring system had good to very good IRA. These results concur with the reported usefulness of blinded expert evaluation of alopecic area photographs in assessing the response to treatment in clinical settings and its reproducibility and interobserver agreement.13,23 The larger CI of the Fleiss κ when compared to the BP and Gwet AC2 coefficients results from the sensitivity of κ coefficients to the distributions of scores awarded by raters.24 In addition, Fleiss κ is less robust and less sensitive to change than the other 2 measures of agreement, which has already been reported in the medical literature.25 In small animals, the lack of an established standardized scoring system for hair density is evident. The closest scoring system available for dogs is incorporated in the Canine Atopic Dermatitis Extent and Severity Index-4, but it combines the evaluation of excoriations and alopecia together.26 To evaluate alopecia and hair density as a standalone parameter, scoring systems and standardized tools can both be used.27 Although the use of tools can provide more proximate measurement, visual scoring systems are of recognized utility as they offer a faster, affordable, reliable, and more intuitive option that can allow clinicians and owners to more readily monitor changes over time.27 Consequently, there seems to be a great need for a scoring system such as the one developed in this trial. We can conclude from these results that this visual scoring system, HDS, seems to allow raters to estimate different degrees of alopecia in a localized area with a good IRA.
To verify the hypothesis of reproducible sensitivity to changes in hair density, we used this newly developed HDS to assess the effects of FLE exposure on otherwise untreated alopecia X patients. The ordinal scoring system was sensitive to change as evidenced by a statistically significant positive effect of time on the odds of improved HDS grades (P = .019). However, we were unable to reject the null hypothesis on the effect of the PHOVIA System on hair density. The time X treatment interaction was not statistically significant (P = .41), implying that the odds of improved HDS grades did not change at a significantly faster rate in the AT side relative to the ST side. We must, however, consider these results with caution as the proportional odds assumption implicit to the ordinal outcome statistical model did not hold true, implying that at least one of the estimated effects and associated statistical significances of time, treatment, or time X treatment of the ordinal outcomes model does not accurately represent its actual effect on each of the HDS values.
The analysis of the dichotomized score data and inspection of Figure 3 suggest that the effects of time, treatment, and their interaction were limited to the patients whose HDS grades were not at the extremes of the ordinal scoring range. Relative to d0 values, the median HDS of the patients tended to increase between d50 and d78 on the AT side, whereas a similar increase was noticeable only at d78 on the ST side. It is conceivable that the patients with the severest alopecia (ie, HDS = 1) had the most pronounced and widespread follicular arrest and mitochondrial dysfunction and therefore had limited ability to respond to PBM.6,28 On the other end, the patients with the slightest degree of alopecia severity (ie, HDS = 5) had little margin of improvement, and the fur coat may hide the density changes occurring during the course of therapy. This finding may help in narrowing the inclusion criteria for further trials where our HDS is used. Moreover, the likelihood of perceiving clinical improvement tended to increase with HDS but did not reach statistical significance. This could be associated with multiple variables, such as the interindividual variability in treatment progress, the few assessments that received scores of 3 or 4 (8.65% of the total), and the volunteer withdrawal of 1 patient from the 7 patients recruited.
Different treatment modalities for alopecia X have been described in the literature, with a variability in their success rates going from 40% to 100% in inducing either partial or complete hair regrowth.2,28–33 The findings in our study provide preliminary evidence of the effect of FLE on alopecic areas: the clinical consequences should be investigated further. No adverse effect was recorded throughout the treatments and follow-up period. The efficacy and safety of FLE has already been investigated in veterinary medicine, and results have been demonstrated in multiple dermatological conditions, such as wounds, interdigital furunculosis, perianal fistula, otitis externa, and pyoderma.19,34–39 To date, there is only 1 study10 conducted on the efficacy of PBM on hair regrowth in dogs afflicted with different types of noninflammatory alopecia, which showed improvement in all animals (n = 7). It is worth mentioning that in the aforementioned study, another PBM device was used that produces 3 different wavelengths emerging simultaneously from 21 foci (470 nm, 685 nm, and 830 nm). It is possible that the higher wavelengths, which penetrate further into the skin layer, can stimulate the follicle seated deeper and thus bring more improvement.7 Further studies comparing the responses with different PBM devices would be interesting.
As more studies on the pathogenesis of alopecia X continue to emerge, there seems to be a consensus toward a genetic basis. Transcriptome studies6,40 have revealed a downregulation of key regulator genes of the Wnt and Shh pathways that are involved in the hair cycle, and whole-genome sequencing has identified the increased number of mitochondrial gene mutations in affected dogs. It is well known that mitochondria play an important role in the cell cycle and in the control of cell proliferation.41 Its role in alopecia is still being investigated, but recent findings in androgenic alopecia suggest a contribution of altered mitochondrial function of the hair follicle dermal papilla.42 The basis of the use of PBM in our study relies on suspected stimulation at the mitochondria level, but the proposed mechanism of actions of PBM on alopecia in humans are numerous: stimulation of the mitochondria in the bulge stem cells associated with a burst of intracellular reactive oxygen species; activation or reprogramming of macrophages promoting a less inflammatory environment; inhibition of apoptosis by different mechanisms, such as the upregulation of antiapoptotic proteins; and the action of opsins on prolonging the anagen phase in ex vivo hair follicles.9,43
Our results show an increase in the HDS in AT and ST sides in certain patients, which could be associated with the cyclical nature of the condition and thus a spontaneous improvement.3 The chromophore or the gel itself that was applied on the ST sides could also have penetrated the first layer of the epidermis and activated the hair follicle by different pathways, such as mechanical stimulation during gel removal, fluorescence from ambient light, or even the presence of urea peroxide in the carrier gel.28,44 Information on the chromophore itself is proprietary, and thus it is difficult to draw any conclusions on that matter. As for the mechanical stimulation associated with the removal of the gel, precautions were taken so as to not cause trauma on the skin, so it seems unlikely.
This study presents multiple limitations. First, the small number of subjects recruited and their variability in baseline alopecia severity contributed to the inability to draw significant conclusions on the efficacy of the treatment. Second, all participating raters are board-certified dermatologists working in the same institution, and therefore it is conceivable that the IRA of our scale may not be inferred safely to other types of users (ie, other specialists, generalists, residents, interns, or students). Further studies (eg, multicentric) evaluating the reliability of the scale with a larger group of more diverse raters are necessary to further validate the usefulness of this scale.45 Thirdly, the choice of the ST was made to minimize the impact of the chromophore-containing gel; however, some could argue that other categories of controls could have been added (1 without any gel nor FLE, 1 with only the carrier gel without the chromophore solution). Fourthly, the study design did not include long-term follow-up, and the choice of the protocol length was made based on protocols of FLE studies, but these were mainly conducted on infectious and inflammatory conditions, which is not the case in alopecia X. Indeed, hair growth is influenced by the interactions of complex intrinsic and extrinsic factors. In healthy dogs, rate of daily hair growth is known to vary anywhere from 0.04 to 0.40 mm based on the breed, age, type, and location of the body.1 However, there have been only a few studies1,46 evaluating the regrowth rate after clipping, which is estimated to be around 3 or 4 months in short-coated breeds and up to 18 months in long-coated breeds. Unfortunately, there have not been longitudinal studies on the subject for alopecia X, which renders it difficult to determine the ideal length of follow-up in treatment-oriented trials. Fifthly, a standard histopathological analysis was performed to evaluate changes in hair follicles; however, the evaluation of morphometric parameters could have allowed the identification of more subtle differences. Finally, we could not completely exclude Cushing syndrome in our patients as the preferred test is usually low-dose dexamethasone suppression, but the results of the urinary cortisol-to-creatinine ratios conducted, along with the absence of clinical signs or other anomalies, made it less likely.47
This study has demonstrated the capability of an HDS to assess alopecia in dogs, but additional research evaluating the accuracy and repeatability is required to validate this new scoring system. It also provides preliminary evidence of the effect of FLE on the alopecia X–affected areas, which warrants further investigation. Further studies with larger sample sizes and longer follow-up periods would be warranted to better assess the efficacy of FLE for this condition.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors thank Elyane Dugas, Fannie Nantais-Desrochers, and Valerie Fleury-Gravel for their meticulous technical work in this trial.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
This study was partially funded by the Resident Research Grant from the Canadian Academy of Veterinary Dermatology. Photoconverter gel was kindly provided by Vetoquinol N-A Inc.
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