Critically Appraised Topic on Low-Level Laser Therapy (LLLT) in Dogs: An Advisable Treatment for Skin Diseases?

Simple Summary Low-level laser therapy (LLLT) is a therapeutic technique with reported regenerative, anti-inflammatory, antibacterial, and analgesic effects. In the last few years, LLLT has been used in dogs for the management of different skin lesions and diseases. This study reports a literature review using the critically appraised topic (CAT) method to determine the canine skin diseases for which LLLT is an advisable treatment. Only primary clinical prospective studies were considered. A meticulous literature search revealed 19 significant clinical trials, and these were critically analyzed. The evaluation of the best accessible evidence in July 2022 suggests that LLLT can be a promising and effective adjunctive treatment in combination with systemic antibiotic therapy for canine interdigital pyoderma and canine deep pyoderma. Furthermore, the use of LLLT is not recommended as a therapy for pedal pruritus secondary to canine atopic dermatitis. In other canine skin diseases, there is a possible helpful effect of LLLT; however, the evidence for its use is not currently convincing. Abstract Low-level laser therapy (LLLT) is a therapeutic option that stimulates cellular function through intracellular photobiological and photochemical reactions, promoting better tissue repair and an anti-inflammatory, antibacterial, and analgesic effect. Previous studies in human and veterinary medicine have shown the clinical efficacy of LLLT in many fields. In this study, the literature was reviewed using the critically appraised topic (CAT) method to determine the canine skin diseases for which LLLT is an advisable treatment. A meticulous literature search revealed 19 significant clinical trials, which were critically analyzed. The evaluation of the best accessible evidence in July 2022 suggests that fluorescence biomodulation (FBM), a type of LLLT, can, in combination with systemic antibiotic therapy, be a promising and effective adjunctive treatment for canine interdigital pyoderma and canine deep pyoderma. Furthermore, the evidence suggests that the use of LLLT is not recommended as a therapy for pedal pruritus secondary to canine atopic dermatitis. For other canine skin diseases included in the CAT, although LLLT appears to be a promising treatment, there is not yet good scientific evidence to recommend its use.


Introduction
Low-level laser therapy (LLLT) is a noninvasive, easy-to-apply therapeutic option, with minimal side-effects. It uses photons at diverse wavelengths via a nonthermal mechanism to affect biological activity [1]. The use of LLLT is increasing in human and veterinary medicine; LLLT has been studied in a number of species, and a variety of clinical uses in veterinary medicine have recently been reviewed [2][3][4]. However, to date the precise biochemical mechanism of LLLT is not totally understood [1]. LLLT does not exploit thermal or ablative mechanisms but instead stimulates cellular function. The photons emitted by the laser or LED are absorbed by the mitochondrial chromophores (particularly cytochrome c-oxidase) or by the chromophores contained in the photoconverting substrate (applied prior to exposure to the light source), stimulating oxidative phosphorylation to

Results
The literature search in PUBMED identified 19 articles consistent with the desired characteristics, relevant to the clinical question and compliant with the inclusion criteria in the study. Other databases, such as Google Scholar, Web of Science, CAB Abstract, and Agricola were also searched, but no other relevant articles were found in addition to those previously identified on PUBMED. The selected articles, all written in English, consisted of prospective clinical studies, published from 1999 to 2022 (Table 1). Dogs assigned into three groups: (1) control group (C) managed with irrigated saline and without PBM (n = 7); (2) L1 group with irrigated saline together with PBM radiation at 830 nm (n = 7); (3) L2 group with irrigated saline together with SPMW-PBM radiation (n = 7). Wound healing estimated on wound size decrease as a percentage of wound zone every 2nd day for 15 days employing image analysis software.
A significant difference in the percentage of wound area reduction was recorded between the C and PBM groups at the end of the study (15 days). A consistent decrease in wound size was observed in both PBM and non-PBM groups. The percentage of wound area reduction was significantly different between the PBM and non-PBM groups on day 7 (p < 0.05). [12] Acute traumatic wounds FBM  Two case reports of two aged mixed-breed dogs FBM therapy began 5 days after the initial presentation in both dogs. The wound was then covered with a bandage to avoid contamination. The whole process was duplicated once a week until wound healing.
Wound closure and wound healing were fulfilled after 9 and 16 weekly treatments, respectively, with a total re-epithelization of the skin. The small degree of wound contraction did not restrict the free movements and apparently did not disturb the dogs (no signs of suffering or tendency to self-trauma).
[ Dogs randomly assigned to one of two study groups. Group A: LLLT on the right paw and placebo on the right paw; Group B: placebo on the left paw and LLLT on the left paw. The principal investigator and owners were unaware of the group designations. Each dog experienced three laser sessions per week over the course of weeks 1 and 2, two laser sessions per week in weeks 3 and 4, and no laser treatments in week 5. At weeks 0, 2, 4, and 5, dogs assigned a score (localized canine atopic dermatitis severity score-LCADSS) by the principal investigator and a score (localized pruritic visual analog score-LPVAS) by the owner, and cytology assessed. The primary outcome assessment was a >50% reduction from baseline of the LCADSS and LPVAS.
There were no significant dissimilarities in LCADSS or LPVAS between LLLT and placebo treatments between weeks 0 and 5. However, LCADSS and LPVAS significantly decreased from week 0 at weeks 2, 4, and 5 in both LLLT and placebo groups. One lesion randomly allocated as control (treated with a 0.0584% hydrocortisone aceponate spray), and one or more other lesions managed with LLLT daily for 5 days. Lesions clinically scored before treatment (D0), at the end (D4), 16 days after the last laser treatment (D20), and after 2 months (D65).
There was a statistically significant difference at D4 and D20 between treated and control groups; in the treated group over time, there was a statistically significant advancement between D0, D4, and D20. Lesion recurrence was not present in more than 50% of the treated lesions at D65. No adverse reactions were recorded. Dogs randomly assigned to three groups: group QW with a topical LED-illuminated gel (LIG) once weekly; group BW with LIG twice weekly; group C with enrofloxacin and silver sulfadiazine twice daily. The estimation protocol (T0 to T5) considered clinical assessment (OTIS-3 index scoring system; pruritus severity scale; pain severity score; aural temperature), cytological scoring system, and quali-quantitative bacteriologic evaluation.
All groups achieved improvement during the study. The greatest clinical score reduction appaired in Group BW. BW obtained a clinically relevant effect level at T3, QW reached it at T4, and C did not reach it. No differences between groups were noted in the reduction in CFU/mL (T0-T5). RCT: randomized controlled trial; PBM: photobiomodulation, FBM: fluorescence biomodulation; PDT: photodynamic therapy; SPMW: simultaneous superpulsed and multiple wavelengths.These studies met the inclusion criteria and addressed the clinical question but had very dissimilar study designs. The scientific quality of each study was analyzed employing the following parameters to establish the risk-of-bias assessment of treatment efficacy [29], as summarized in Table 2: -Levels of evidence: assigned according to the previously identified criteria for therapeutic studies [30,31]. Briefly, level IA = systematic review (with homogeneity) of randomized control trials (RCTs); level IB = individual RCT (with narrow confidence intervals); level IC = all or none study; level IIA = systematic review (with homogeneity) of cohort studies; level IIB = individual cohort study; level 2C = "outcomes" ecological studies; level IIIA = systematic review (with homogeneity) of case-control studies; level IIIB = individual case-control study; level IV = case series (or poorquality cohort and case-control study); level V = expert opinion without explicit critical appraisal or based on physiology bench research or "first principles" [31]. Group size: score: 0 (<10 subjects), 1 (10-20 subjects), 2 (21-40 subjects), 3 (>40 subjects).
To emphasize the overall strength of chosen studies, we evaluated conclusive, highly suggestive, and suggestive studies for each dermatological pathology treated ( Adverse effects: score 0 = none; score 1 = yes, mild or rare (<10%); score 2 = yes, moderate or common (≥10%); score 3 = yes, common and moderate or severe. -Number of administrations carried out: score 1 = ≤5 administrations; score 2 = 6-10 administrations; score 3 = >10 administrations. -Efficacy of the treatment: WCG = only treatment group without control group; NS = no statistical difference between treatment group and control group; SD = statistical difference between treatment and control group.

Discussion
Despite the variety of published articles, in the international literature, there are few prospective clinical studies conducted in vivo that provide good scientific evidence to evaluate the effectiveness of LLLT as a treatment for canine skin diseases.
On the basis of the results obtained from this CAT, only three of the 19 studies were considered conclusive. The study of Marchegiani et al. [21] on canine interdigital pyoderma is one of the three, with a total score of 8, characterized by good methodological quality and scientific evidence. For this reason, FBM can be recommended for the therapy of interdigital pyoderma in dogs. This prospective randomized blinded clinical study on 36 dogs evaluated the effect of a LED lamp with a photoconverter gel system, used for 2 min twice weekly until clinical resolution, in combination with systemic antibiotics on clinical manifestations of canine interdigital pyoderma. This was compared to dogs treated with antibiotics alone as control group. A statistically significant decrease was noted in measured parameters for the treatment group compared to the control group. The mean time to lesion resolution was 4.3 weeks in treatment group and 10.4 weeks in control group.
The second study [23] was performed by the same authors and with the same experimental design, but on 35 dogs affected by deep pyoderma, regardless of body location. The total score was 8, and the study was characterized by good methodological quality and scientific evidence. It was a prospective randomized blinded clinical trial evaluating the effect of a LED lamp with a photoconverter gel system, used for 2 min twice weekly until clinical resolution in combination with systemic antibiotics on clinical manifestations of canine deep pyoderma. This was compared to control dogs treated with antibiotics alone. A statistically significant decrease was recorded in the measured parameters for the treatment group compared to the control group. The mean time to resolution of lesions was 5.7 weeks in the treatment group and 11.7 weeks in the control group. For this reason, FBM can be recommended for the therapy of deep pyoderma in dogs.
It is interesting to note that, in both these trials, the duration of the course of systemic antibiotic therapy was significantly reduced if FBM was administered as an additional treatment.
The study by Stich at al. [18] is the third study with good methodological quality and scientific evidence that was rated with "conclusive evidence", with a total score of 8. This study demonstrated that the use of PBM is not beneficial as a treatment for pedal pruritus secondary to canine atopic dermatitis. This was a prospective, randomized, double-blinded, intraindividual study (with each dog serving as their own placebo control) on 30 clientowned dogs with symmetrical pedal pruritus secondary to canine atopic dermatitis. PBM was not effective as a localized treatment; only 38% of patients treated with PBM had a reduction of more than 50% in pruritus or lesion scores for the treated paw compared to baseline values, and there was no significant difference in scores between the paws of individual dogs treated with PBM and placebo laser. Scores decreased significantly for untreated paws, as well as in the treatment group, and the authors postulated that this improvement probably represented a placebo effect (the principal investigator and the owners were informed that one paw was being treated with PBM), although it is also possible that PBM caused a systemic effect for both treated and untreated paws in the same subject [18].
With regard to the other canine skin diseases investigated in this critically appraised topic, all LLLT methods were promising in many of the included studies. In fact, in most of the articles analyzed, LLLT led to an improvement in symptoms of treated subjects and often to their complete resolution [10][11][12]16,17,20,22,[24][25][26][27][28]. In four studies (three in surgical or surgically created wounds [13][14][15] and one [19] in acral lick dermatitis), the LLLT did not show any significant clinical efficacy in the treated subjects. However, all clinical studies, unlike the studies by Stich et al. [18] and Marchegiani et al. [21,23], were characterized by insufficient scientific evidence for an incomplete and inappropriate methodology, as listed below. Indeed, it is important to distinguish the clinical outcome of treated subjects with the scientific-based evidence of a study, which is the purpose of a CAT. In the field of LLLT used on dogs with skin diseases, many of the studies published were unfortunately clinical cases series, thus strongly limiting their scientific evidence.
It is not possible to conclusively recommend or not recommended the use of LLLT for management of surgical wounds and incisions, because the relevant studies [13][14][15][16][17], although all randomized and blinded studies, were rated only with "highly suggestive or suggestive evidence" mainly due to the small number of subjects treated and the lack of adequate follow-up. For acute traumatic and chronic wounds, two studies [10,12] were not randomized or blinded and, therefore, categorized with "inconclusive evidence", while another study [11], randomized but not blinded, was rated as "suggestive evidence".
In the case of otitis externa, one study, not blinded or randomized, was categorized with "inconclusive evidence" [25], while the other, randomized but not blinded, had "suggestive evidence" [26]. For acral lick dermatitis [19] and sterile pyogranulomatous pododermatitis [20], the studies were categorized with "highly suggestive or suggestive evidence", while, for perianal fistulas, the study was graded as "inconclusive evidence", due to the lack of a control group [24]. In noninflammatory alopecia, the study was graded as "inconclusive evidence", due to a lack of randomization and blinding [27]; for bacterial skin infection associated with calcinosis cutis [28], the study was rated as "inconclusive evidence" due to the lack of a control group, randomization, and blindness.
A separate case is the very recent study by Marchegiani et al. [22], an update on FBM for the treatment of interdigital furunculosis. The trial tested the once-weekly administration on 12 dogs affected by interdigital pyoderma by comparing the results obtained with those of a previous study of 2019 [21] with identical structure (inclusion/exclusion criteria, blinding scheme, scoring system, etc.) but twice-weekly administration. Unlike the 2019 study, which was "conclusive", this new study was categorized only as "highly suggestive", due to the lack of randomization and the fewer subjects treated. This, therefore, suggests that once-weekly administration of FBM for interdigital furunculosis cannot be recommended at the moment.
In all evaluated studies, PBM, FBM, and PDT proved to be noninvasive, easy to deliver in unsedated dogs, and without side-effects during the investigation period.
Further research in this field is indicated to increase our understanding of this new therapeutic option in the veterinary dermatological field. Generalizable, in vivo, randomized, double-blind, and controlled studies are required on a sufficiently large scale.
The subject inclusion criteria, the group randomization process, and the blinding procedure should always be described in detail in future studies. Moreover, to allow the comparison of results, all new studies evaluating the efficacy of LLLT as a treatment for canine dermatological diseases should follow the same clinical criteria for the inclusion of subjects, use the same clinical score when monitoring healing, and follow up subjects after treatment for at least 12 months, so as to be able to identify any relapses.
Through further studies, it would also be interesting to investigate the potential of LLLT to reduce the use of antibiotics, a potential advantage that emerged from the studies by Marchegiani et al. [21,23], in which duration of systemic antibiotics was reduced following concurrent FBM. The antimicrobial effect of LLLT has not yet been totally proven, although some human in vitro dentistry studies have highlighted this aspect [33,34]. The use of antibiotics is a very hotly debated topic with regard to the development of antimicrobial resistance, which is considered a global public health crisis that threatens our ability to successfully treat bacterial infections [35].
Further studies are also required to draft standardized protocols, which are currently inadequate, relating to the optimal parameters of the therapeutic lasers to be used and the posology for the treatment of each pathology. Factors such as spot size, wavelength, energy density, power density, pulse structure, total energy, total power, delivery mode (contact, point, and wide beam), the duration of the treatment, and the treatment intervals might influence the success of the LLLT. It is evident that, to obtain positive results with LLLT, each of these dosimetric parameters must be controlled within a limited range of values [36].

Conclusions
In this critically appraised topic on the use of LLLT as a treatment for canine skin diseases, good scientific evidence was identified only for the recommendation of fluorescence biomodulation (FBM) for management of canine interdigital pyoderma and canine deep pyoderma, in combination with systemic antibiotic therapy.
LLLT has the potential to be a promising treatment for many canine skin diseases. However, additional valid and generalizable clinical studies, with good scientific evidence, are required to investigate its actual efficacy and potential antimicrobic effect, as well as to produce scientifically validated standardized therapeutic protocols.