Evaluation of laser and LED phototherapy for the treatment of canine acral lick dermatitis and Staphylococcus pseudintermedius in vitro
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University
By
Amy H. Schnedeker, D.V.M.
Graduate Program in Comparative and Veterinary Medicine
The Ohio State University
2017
Master’s Examination Committee:
Dr. Lynette Cole, Advisor
Dr. Sandra Diaz
Dr. Gwendolen Lorch
Dr. Joshua Daniels
Dr. Paivi Rajala-Schultz
i
Copyright by
Amy H. Schnedeker
2017
i
Abstract
Staphylococcus pseudintermedius is the most common cause of bacterial skin infections in dogs. Methicillin-resistant infections have become more common and are challenging to treat. Blue light phototherapy may be an option for treating these infections.
The objective of this study was to measure the in vitro bactericidal activity of
465-nm blue light on methicillin-susceptible Staphylococcus pseudintermedius (MSSP) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). We hypothesized that irradiation with blue light would kill MSSP and MRSP in a dose-dependent fashion in vitro as previously reported for methicillin-resistant Staphylococcus aureus (MRSA).
In six replicate experiments, each strain (MSSP: n=1), (MRSP ST-71 [KM1381]: n=1) and (MRSA [BAA-1680]: n=1) were cultivated on semisolid media, irradiated using a 465-nm blue light phototherapeutic device at the following cumulative doses: 56.25,
112.5, and 225 J/cm2 and incubated overnight at 35oC. Controls were not irradiated.
Colony counts (CC) were manually performed. Descriptive statistics were performed and treatment effects assessed using the Mann-Whitney-Wilcoxon rank-sum test. Bonferroni- corrected rank sum tests were performed for post-hoc analysis when significant differences were identified.
ii There was a significant decrease in CC with blue light irradiation at all doses for
MRSA (P=0.0006) but not for MSSP (P=0.131) or MRSP (P=0.589).
Blue light phototherapy significantly reduced CC of MRSA, but not of MSSP or
MRSP. The mechanism for the relative photosensitivity of the MRSA isolate is unknown, but is hypothesized to be due to an increased concentration of porphyrin in S. aureus relative to S. pseudintermedius, which would modulate blue light absorption.
Canine acral lick dermatitis (ALD) is a frustrating, chronic disease that can have numerous primary causes including pruritic and painful diseases. Conventional therapy generally consists of systemic antibiotics and anti-anxiety medications. Low-level laser therapy (LLLT) is an alternative therapy that has been used to treat conditions associated with inflammation and pain.
The objective of this study was to determine whether the use of LLLT combined with conventional therapy would result in a significant decrease in the licking of the ALD lesion than conventional therapy alone. We hypothesized that the combination of LLLT and conventional therapy would result in a >50% reduction in licking visual analog score
(LVAS) than conventional therapy alone.
Dogs were randomly assigned to two groups. The treatment group received LLLT with blue and red light-emitting diodes (LEDs), while control group had sham therapy
(laser off). Treatments were three times weekly for two weeks, then twice weekly for two weeks for a total of 10 visits. All dogs received systemic antibiotics and trazodone.
Descriptive statistics were performed (mean, standard deviation).
There was an overall decrease in LVAS in both control and treated dogs.
Treatment dogs had a 20% greater decrease in LVAS compared to control dogs. iii The use of LLLT as a non-invasive treatment resulted in an additional decrease in licking behavior in dogs with ALD. LLLT should be considered as an adjunctive treatment in the management of dogs with ALD.
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Dedication
To my parents: Thank for your love and unending support. Without you both I would not
have the work ethic and drive to accomplish all that I have.
To my husband Jayme: Through all these stressful years we have finally made it to the end, but I hope this is just the beginning. Thank you for everything, I would not have made it to this point and become the person I am today without you by my side.
v
Acknowledgements
I would like to thank my advisor, Lynette Cole, for all her help and guidance throughout this entire residency. You have always pushed me to be my best and your strong mentorship is the reason I was able to accomplish my goals. This thesis, and our mutual love for Cavies, will always tie us together. Thank you to my committee members, Sandra Diaz, Wendy Lorch, Joshua Daniels and Paivi Rajala-Shultz. Your friendship and support will be forever appreciated.
Thank you to my resident-mates Stephanie Abrams, Melanie Hnot and Holly
Roberts. You all kept me levelheaded, smiling and laughing. Thank you to Deb Crosier for all the cute cat videos and cat noses that made the days go by easier.
vi
Vita
May 2004………………………………………..B.S. Animal Science, Cornell University
May 2013……………………………………….D.V.M., University of Minnesota
July 2014 to present……………...... …………...Graduate Teaching and Research
Associate, The Ohio State University
Fields of Study
Major Field: Comparative and Veterinary Medicine
Studies in Dermatology
vii
Table of Contents
Abstract…………………………………………………………………………...……….ii
Dedication………………………………………………………………………...…...…..v
Acknowledgements………………………………………………………………..……...vi
Vita…………………………………………………………………………………....…vii
List of Tables………………………………………………..……………………...….…xi
List of Figures………………………………………….………………………………..xiii
CHAPTER 1 Introduction…………………………………………………..……….....….1
CHAPTER 2 Literature Review...... 4 2.1 Acral lick dermatitis………………..…………………………………………....…….4 2.1.1 Disease Pathophysiology………………….……………………..……...…..4 2.1.1.1 Primary Etiologies……………………………………………...…4 2.1.1.2 Differential Diagnoses…...……………………………………...... 6 2.1.1.3 Predisposing Factors……………….……………………………...8 2.1.1.4 Perpetuating Factors……………………...……….…………...... 8 2.1.2 Clinical Signs………………………………………..…………………....…9 2.1.3 Diagnostic Approach…………………..………………………………...... 9 2.2 Treatment………………………………………………………………………….....12 2.2.1 Antibiotics………………………………………………………………….12 2.2.2 Topicals…………..………………………………………………………...13 2.2.3 Treatment of Pruritic Disease……………………………...…...……….....13 2.2.3.1 Glucocorticoids……………...…………………………...14 2.2.3.2 Janus-kinase Inhibitors……………………………...... ….14 2.2.3.3 Monoclonal Antibody Therapy…………………….…….15 2.2.4 Anti-anxiety Medications...... ………………………...15 2.2.4.1 Serotonin Antagonist and Reuptake Inhibitor….....……...15 2.2.4.2 Tricyclic Antidepressants...... …………………………16 2.2.4.3 Selective Serotonin reuptake Inhibitors (SSRIs)...... …….17 2.2.4.4 Narcotic Antagonists...... ………………………………...18 viii 2.2.5 Laser Therapy...... ……………………...18 2.2.6 Radiation Therapy...... ……………….….19 2.2.7 Surgery...... ……………...20 2.2.8 Electronic Stimulation...... ………………………………………21 2.2.9 Orgotein...... ………………………………………………………………21 2.2.10 Acupuncture...... ……………………………………………………...22 2.3 Prognosis...... ……………………………………………………………...22 2.4 Photostimulation...... ……………………………………………………...22 2.4.1 Low-Level Light Therapy (LLLT)………………………....…...…………22 2.4.1.1 Classification...... …………………………………....22 2.4.1.2 Mechanism of Action………….….……....……………………...23 2.4.2 Uses of LLLT……….………...... ……...... …………………...... 24 2.4.2.1 Human Medicine……………………..………...... …………..24 2.4.2.2 Veterinary Medicine……………………………………………..27 2.4.3 Light Emitting Diodes...... ………….………………………….29 2.4.3.1 Definition………………...... ……………………...29 2.4.3.2 Classification...... 30 2.5 Photodynamic Therapy……………...... …………………………………….33
CHAPTER 3 In vitro bactericidal activity of blue light (465-nm) phototherapy on methicillin-susceptible and methicillin-resistant Staphylococcus pseudintermedius…………………………………………………………………..….…35 3.1 Abstract…………………………...………………………………………….35 3.2 Introduction………………….....….…………………………………………36 3.3 Materials and Methods……………………………....……………………….38 3.3.1 Bacterial Isolates and Culture…………………………….………..38 3.3.2 Blue Light Therapy……...... ……………………………………...39 3.3.3 Statistical Analysis…………………...... ………………………….40 3.4 Results...... …………………………………………………………….40 3.4 Discussion...... ,41
CHAPTER 4 Low-level laser therapy as an adjunctive treatment for canine acral lick dermatitis: A randomized, double-blinded, sham-controlled study……………...... …....51 4.1 Abstract………………………………....…………………………………....51 4.2 Introduction…………………………………………...... …………………....52 4.3 Materials and Methods…………………………………...…………………..54 4.3.1 Animals...... ………………………………….....55 4.3.2 Study Design...... …………………………………………..56 4.3.3 Laser Treatment……………………………………...... ….…....59 4.4 Results.……………………………...... ……….……....60 4.4.1 Signalment and History.…………………………………….....…...60 4.4.2 Diagnostic Tests.…………………...... ………………...61 4.4.2.1 Deep Skin Scrapings and Surface Cytology.….………....61 4.4.2.2 Deep Bacterial and Dermatophyte Culture...... ……….....61 4.4.2.3 Histopathology...... ……………………….....62 ix 4.4.2.4 Radiography...... …………………………………..63 4.4.3 Side effects...... 63 4.4.4 LVAS...... 63 4.4.5 Overall Lesion Size...... 63 4.4.6 Lesion Thickness...... 64 4.4.7 Composite Ulcer Size...... 64 4.5 Discussion...... 65
CHAPTER 5 Conclusions and Future Directions………………………………….....….87
Bibliography.……………………………………………………………………...... 92
x
List of Tables
Table 1. Properties of the Blue Light Phototherapeutic Device (MR4 ACTIVet PRO™,
Multi Radiance Medical®; Solon, OH, USA)...... ……..44
Table 2. Median Colony Counts and Percent Reduction for Control Group and Treatment
Groups (Methicillin-Susceptible Staphylococcus pseudintermedius, Methicillin-Resistant
Staphylococcus pseudintermedius [KM1381] and Methicillin-Resistant Staphylococcus aureus [BAA-1680]) after Irradiation with 465-nm Blue Light...... 45
Table 3: Median, Interquartile Range and Range of Colony Counts for Control Groups and Treatment Groups (Methicillin-Susceptible Staphylococcus pseudintermedius,
Methicillin-Resistant Staphylococcus pseudintermedius [KM1381] and Methicillin-
Resistant Staphylococcus aureus [BAA-1680]) after Irradiation with 465-nm Blue
Light...... 46
Table 4: Historical information for all dogs with acral lick dermatitis...... 72
Table 5: Antimicrobial susceptibility test results of Staphylococcal isolates from deep tissue acral lick dermatitis lesions in eight dogs...…………….....……………………....73
Table 6. Summary of histopathologic findings in nine cases of acral lick dermatitis.…………...... ………....74
Table 7. Licking Visual Analog Scale (LVAS) of the acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser) groups over 10 visits. V1 is the baseline after which treatment or sham was started. V1 through V6 occurred three times weekly and V7 to V10 occurred two times weekly for two weeks...... 76 xi Table 8. Lesion size (cm2) of acral lick dermatitis for all dogs in control (sham laser) and treatment (laser) groups over 10 visits...... 77
Table 9: Composite ulcer size (cm2) of acral lick dermatitis for all dogs in control (sham laser) and treatment (laser) groups over 10 visits...... 78
xii
List of Figures
Figure 1. The 465-nm blue light therapeutic device (MR4 ACTIVet PRO™ device, Multi
Radiance Medical®; Solon, OH, USA) irradiating a 35mm petri dish containing tryptic soy agar (TSA) plated with a bacterial isolate...... 47
Figure 2. a. MSSP, b. MRSP (KM1381), c. MRSA (BAA-1680). Box plots showing the bacterial colony counts at each dose (56.25, 112.5, 225 J/cm2) for the control (not irradiated) and treatment groups (irradiated with 465-nm blue light) for MSSP (a), MRSP
(KM1381) (b), and MRSA (BAA-1680) (c). Black circles and open triangles represent the colony counts of each individual plate (gray squares are outliers). The control and treatment groups were compared within each bacterial isolate using a Mann-Whitney-
Wilcoxon rank sum test (SPSS IBM version 24; Armonk, NY, USA). When a significant difference was identified, Bonferroni-corrected rank sum tests (SPSS IBM version 24;
Armonk, NY, USA) were performed at each dose for post-hoc analysis. Statistical significance was set at P<0.05. There was a significant difference in CC for MRSA between treatment and control groups at each dose (P=0.006). MSSP = methicillin- susceptible Staphylococcus pseudintermedius, MRSP = methicillin-resistant
Staphylococcus pseudintermedius, MRSA = methicillin-resistant Staphylococcus aureus...... 48
Figure 3. Representative culture plates of MSSP (a-d), MRSP (KM1381) (e-h) and
MRSA (BAA-1680) (i-l) irradiated with 56.25, 112.5, and 225 J/cm2 465-nm blue light.
Control plates (a,e,i) were not irradiated. MSSP = meticillin-susceptible Staphylococcus xiii pseudintermedius, MRSP = meticillin-resistant Staphylococcus pseudintermedius, MRSA
= meticillin-resistant Staphylococcus aureus……...... ……………………………..50
Figure 4. Owner Licking Visual Analog Scale (LVAS)…………………...... ……..79
Figure 5. Investigator Licking Visual Analog Scale (LVAS). Once the owner has placed a mark on the LVAS form, this scale is applied to get a numeric value for the licking behavior level…………………...... …..80
Figure 6. Mean Licking Visual Analog Scale for dogs in control (laser sham) and treatment (laser) group...... 81
Figure 7. Examples of acral lick dermatitis lesions in dog 4 in the control group (sham laser) at enrollment (a) and study completion (b) and dog 2 in the treatment group (laser) at enrollment (c) and study completion (d). Red lines represent outline of overall lesion, yellow lines represent outline of ulcers...... 82
Figure 8: Mean lesion size (cm2) of acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser)...... 83
Figure 9: Mean lesion size (cm2) of acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser) group with dog 5 data removed...... 84
Figure 10: Mean composite ulcer size (cm2) for dogs in control (laser sham) and treatment (laser).
...... 85
Figure 11: Mean composite ulcer size (cm2) of acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser) group with dog 5 data removed...... 86
xiv
Chapter 1
Introduction
Low-level laser therapy (LLLT) is an alternative therapy used to treat a multitude of conditions that require stimulation of healing and relief of inflammation, pruritus and pain.1 It is a form of phototherapy that uses either coherent light sources (lasers) or non- coherent light sources consisting of filtered lamps or light-emitting diodes (LEDs) or a combination of both.1 While the mechanism of action of LLLT is poorly understood, it is thought to be through the absorption of visible red light (620-690 nm) and near-infrared light (720-1260 nm) by mitochondrial chromophores, particularly cytochrome c oxidase, which is located within the mitochondria.1 This leads to production of adenosine triphosphate (ATP),1 which alters factors associated with cell proliferation, survival, tissue repair, and regeneration.2
LEDs produce a narrow spectrum of light in an incoherent manner, where the light is randomly spread out once emitted from a light source.3 LEDs have different depth of penetration based on their wavelength and can affect cellular metabolism by triggering intracellular photobiochemical reactions.3 Red LEDs (630-700-nm) are used to target dermal structures and have been used for treatment of wound healing and photodamage.3
Blue LED light (400-470-nm) has both anti-inflammatory and antibacterial properties and penetrates to the level of the epidermis, so is best used for superficial conditions.3
Irradiation with blue LED light leads to photoexcitation of bacterial porphyrins, singlet 1 oxygen production and eventually bacterial destruction.4 These reactive oxygen species have an antimicrobial effect on the bacteria, but are not detrimental to the host cells.5
Blue light phototherapy has been shown to be bactericidal against Staphylococcus species in vitro. A dose-dependent bactericidal effect was observed on Staphylococcus aureus, with the greatest reduction of bacteria (62%) at the highest dose of 15 J/cm2 using 470- nm blue light.6 However, for methicillin-resistant Staphylococcus aureus (MRSA), optimal doses of 470-nm blue light phototherapy have been reported to be between 55 to
220 J/cm2 resulting in anywhere from 90.4 to 100% reduction of MRSA, with denser bacterial cultures requiring higher doses. 7-9
Staphylococcus pseudintermedius is a common commensal and opportunistic pathogen of the skin of dogs and is the most common cause of bacterial skin infections.10
In recent years, methicillin-resistant S. pseudintermedius (MRSP) infections have become much more prevalent.11 With the emergence of MRSP, the number of oral antimicrobial drugs to which bacterial isolates are susceptible is limited often leading to pharmacotherapeutic choices that have profound side effects or are reserved for human infections.12 Topical antimicrobial therapy has been shown to be effective for treatment of some MRSP infections,13 but these treatments can be time consuming and inconvenient for owners.
Since blue light phototherapy has been shown to be effective in killing bacteria associated with human Staphylococcal infections, it may be a useful adjunctive or sole therapy option for Staphylococcal infections in dogs, specifically methicillin-resistant infections. Therefore, the objective of our study was to determine the in vitro bactericidal activity of 465-nm blue light on methicillin-susceptible Staphylococcus pseudintermedius 2 (MSSP) and MRSP. We hypothesized that irradiation with 465-nm blue light would suppress growth of MSSP and MRSP in vitro in a dose dependent manner, as previously reported for MRSA.7,9
Canine acral lick dermatitis (ALD) is a disease that manifests as excessive, compulsive licking at a focal area on a distal limb resulting in a proliferative, ulcerative, and alopecic lesion that has secondary deep infection.14,15 There are numerous primary causes of ALD including allergic disease, hypothyroidism16, orthopedic abnormalities, neuropathies, trauma and psychogenic.17 Some of these causes are considered pruritic
(e.g. allergic disease), which can manifest as licking, however, some are not (e.g. osteoarthritis) and the licking behavior in those diseases may be due to pain.14-16,18
Conventional therapy for ALD includes systemic antibiotics for the deep bacterial infection19 and a systemic behavior-modifying medication (i.e., trazodone, fluoxetine, clomipramine).15,20-23 Non-conventional treatment options include surgery,24 radiation,25 cryosurgery,26 acupuncture,27 and electrostimulation28; all have variable efficacy and numerous side effects.
As the ALD lesion is a direct result of excessive licking from pain and/or pruritus and often infected, LLLT (delivered by laser and blue and red LED) may be an effective therapy for ALD, regardless of the underlying condition.29-31 We aimed to determine whether the use of LLLT combined with conventional therapy would result in a significant decrease in the licking of the ALD lesion by the dog when compared to conventional therapy alone. Our hypothesis was that the combination of LLLT and conventional therapy for canine ALD would result in a >50% reduction in licking than conventional therapy alone. 3
Chapter 2
Literature Review
2.1 Acral lick dermatitis (ALD)
2.1.1 Disease pathophysiology
2.1.1.1 Primary Etiologies
Canine ALD is a disease that manifests as excessive, compulsive licking at a focal area on a distal limb. This disease is most common in dogs, but has been reported in other species such as humans,32 a rabbit,33 a jackal34 and a dairy cow.35 There are numerous primary causes of canine ALD. Foremost among these are allergic diseases, which include cutaneous adverse food reaction (CAFR) and atopic dermatitis (AD).15,18,36
Excessive licking is a manifestation of the allergic pruritus. Historical information about the pruritic disease may be helpful in determining the relationship between allergy and the development of lick granulomas.15 CAFR has been related to the development of
ALD lesions and may be the result of an acute onset of aggressive pruritus.15 CAFR and
AD are best ruled out with a strict elimination food trial to rule out CAFR; once CAFR is ruled out, the diagnosis of AD is made and intradermal and serum allergy testing can be considered for management of AD.14,18
Orthopedic abnormalities (such as arthritis) and arthropathies can also be associated with canine ALD.15 As the ALD lesion generally manifests on a distal limb and over a joint, osteoarthritis is a possible primary condition. Radiographs of an ALD
4 lesion often reveal a secondary periosteal reaction of underlying bones, but joint disease is not induced by the licking.14 If present the joint disease may be the cause of the excessive licking.14
Neuropathies have also been associated with ALD lesions. Hereditary sensory autosomal neuropathy (HSAN) is a condition characterized by insensitivity to pain, sometimes combined with self-mutilation.37 This syndrome occurs in dogs and humans and is associated with a substance P deficiency in the dorsal root ganglia of the spinal cord.15 Severe forms of HSAN have been reported in German short-haired pointers,
English pointers, English springer spaniels, French spaniels and a miniature pinscher.37,38
Through a genome wide association study, Plassais et al. identified a 1.8Mb homozygous locus on canine chromosome 4. High-throughput sequencing of the region identified a point mutation upstream to the Glial cell-Derived Neurotrophic Factor (GDNF) gene in four sporting breed dogs with a severe form of HSAN.37 GDNF is involved in neuronal development and adult neuronal survival.37 Aside from mutations, the excessive licking itself causes erosions in the skin and can expose sensory nerve endings which causes the area to be pruritic, thus perpetuating the desire to lick.15 Severe traumatic injuries such as brachial plexus avulsions can be an underlying cause for licking.16 Peripheral nerve tumors can irritate the nerve and cause hyperaesthesia that causes the dog to lick the limb.39
Other primary causes for ALD include a foreign body reaction or trauma.
Previous wounds, venipuncture or catheter placement can be inciting causes for excessive licking resulting in an ALD lesion.14,15,19,40-42 A possible hypersensitivity reaction to an
5 orthopedic pin was the reported cause of excessive licking in one dog with resolution of the lesion when the pin was removed.41
Hypothyroidism is an endocrinopathy that is another primary cause of ALD lesions in dogs.16,43 Hypothyroidism has been reported to cause behavior changes in dogs including restlessness and nervousness.44 The exact pathomechanism for why hypothyroidism causes dogs to lick and form ALD lesions is unknown.
Parasitic dermatoses should be considered as primary causes of canine ALD.
Sarcoptic mange, Cheyletiella, lice and flea allergy dermatitis are allergic parasitic diseases that may result in the dog licking, with subsequent development of an ALD lesion.15,16,18
Psychogenic causes for ALD are common and should be considered when all other primary causes for the licking have been ruled out.14 Animals predisposed to behavioral self-mutilation are often nervous breeds such as the Doberman pinscher,
German shepherd, Great Dane, Labrador retriever, golden retriever, boxer, Weimaraner and Irish setter dogs.14,15,42,44 In one study of 11 dogs a potential psychogenic cause was identified in 5 dogs.20 Psychogenic causes can be sequelae to an underlying organic cause. Excessive licking may cause production and release of endorphins, making the dog feel better and causing an analgesic effect that decreases the dog’s perception of pain.14,15
It has been estimated that psychogenic and idiopathic ALD combined may comprise as many as 50% of cases.45 In addition, as many as 70% of dogs diagnosed with ALD have concurrent fear and/or anxiety-based conditions such as noise phobia or separation anxiety.45
2.1.1.2 Differential diagnoses:
6 Canine ALD lesions are usually clinically distinct. However, other diseases can mimic an ALD lesion and should be considered when a dog presents with a raised plaque-like lesion on a distal limb. Neoplasia is a differential for ALD. Histiocytomas and mastocytomas may be mistaken for ALD if they occur on the cranial surface of the limb.14 Denerolle et al. reported a case of a 10 year old male Labrador retriever presenting with complaint of a lick granuloma of 6 months duration; biopsy of the lesion revealed a grade II mast cell tumor.41 A diagnosis of lymphoblastic lymphoma was made in a 9 year old male Beauceron via a biopsy of a lesion mistaken for ALD.41 Appropriate diagnostic testing (fine needle aspirate and biopsy) is required to rule out neoplasia.
Fungal infections are another differential for ALD lesions.14,19,40,41 Fungal culture of the lesion is required to confirm the diagnosis.14 In one study, an ALD-like lesion cultured positive for Microsporum gypseum and only resolved once antifungal treatment was instituted.19 Sporothrix schenckii was cultured from an ulcerative lesion on the dorsal right carpus in a male Labrador retriever-cross.41 The lesion began to resolve within one month of treatment for sporotrichosis.41
Parasitic and protozoal dermatoses should also be considered as differentials for canine ALD. Lesions of demodicosis can mimic ALD, as this parasite can cause folliculitis, furunculosis resulting in excessively licking the skin lesion.15,16,18
Leishmaniasis is a serious protozoal infection that is caused by a variety of Leishmania spp.46 Leishmania organisms were seen within macrophages from a biopsy of an ALD- like lesion on the right carpus of a female Beauceron dog who lived outdoors in France.41
The antibody titer was positive and the lesion improved and licking resolved in this dog when treatment with meglumine and allopurinol was started.41
7 2.1.1.3 Predisposing factors
Predisposing factors play a role in the development and continuation of licking behaviors in ALD. These factors can be environment related, such as boarding or hospitalization, moving to a new home, or crating for extended periods of time.14,15,42,44,45
Other social factors can be involved in inducing licking behavior such as addition or loss of pets or human companions, anxiety, stress or inadequate exercise.14,15,40,42,44,45,47
2.1.1.4 Perpetuating factors
Perpetuating factors can be as significant as the primary cause and if left undiagnosed can delay resolution of ALD lesions. The most common perpetuating factor of ALD is secondary bacterial infections, which are often deep tissue infections.14,19,20
Previous studies report deep bacterial infections in 82 to 94% of ALD lesions. Organisms cultured included Staphylococcus (pseud)intermedius (most common isolate),15
Pseudomonas spp., Enterobacter spp., Streptococcus spp., Clostridium, Proteus spp.,
Actinobacillus spp. and E.coli.19,20 Methicillin-resistant staphylococcal organisms have been identified in 26% of deep cultures.19 Comparison of organisms cultured from the surface and tissue of ALD lesions found surface cultures predicting deep tissue isolates in only 8 of 22 (36%) lesions.19 Thus, deep tissue cultures are necessary for identification of the bacterial organisms and selection of antimicrobials to treat ALD lesions.
Osteomyelitis is another perpetuating factor that may exacerbate ALD.14,15
Osteomyelitis is inflammation of the bone marrow and adjacent bone48 and is generally the result of infection. Diagnosis is made based on bony changes radiographically, which include lytic lesions, periosteal new bone and increased bone density.48 Periosteal proliferation of underlying bone and soft tissue swelling are common findings in dogs
8 with ALD and are secondary to the ALD lesion and licking.20,42 Treatment of osteomyelitis includes antibiotic therapy and surgical management in severe cases.48
Keratin foreign bodies may be present in ALD lesions.15 The excessive licking causes thickened lesions that on histopathologic examination contain hyperkeratotic and elongated hair follicles, folliculitis, furunculosis, dilated and even ruptured epitrichial sweat glands and hidradenitis.14,19,42 These changes can incite a foreign body reaction to the free keratin, thus contributing to the perpetual cycle of licking.14
Dermal fibrosis and vertical streaking of collagen fibers are consistent histologic findings in biopsies of ALD lesions.19,42 These changes within the dermis contribute to a large inflammatory response within the ALD lesion and can exacerbate and perpetuate licking.14 Fibrosis also tends to “wall off” infection, necessitating a lengthy course of antibiotic treatment.18
2.1.2 Clinical signs
ALD lesions generally have a stereotypical presentation. Early lesions may be haired and have crusted or eroded plaques.14 Chronic lesions become hard, thickened, alopecic plaques or nodules that are ulcerated with a proliferative scarred surface and a hyperpigmented halo.14,19,40 These plaques form from licking, and are usually found on the lower cranial portion of a leg.14 In the majority of cases, the lesions are single and unilateral,14 but multiple lesions are possible.19,20,42 Carpus or metacarpus are the most commonly affected areas.14,19,40,42; however, the lesions may also be found on the tarsus and metatarsus, and are mainly on the anterior surface, but can also be found on the lateral surface.14
2.1.3 Diagnostic approach
9 The diagnostic approach begins with obtaining a complete and thorough history and performing a dermatologic exam.15 ALD lesions are distinct and a tentative diagnosis is generally made from the clinical presentation.14 Since ALD can mimic other diseases,41 diagnostic tests are recommended for definitive diagnosis.
Basic dermatologic diagnostics such as skin cytology, deep skin scraping and fine needle aspirate are recommended to rule out bacterial infection, demodicosis and neoplasia, respectively.14,15 Common cytologic findings from the surface of the ALD lesion are coccoid and rod bacteria in 84% and 6% of cases, respectively;19 however, rarely are indicative of infection in the deep tissue. Demodicosis has not been reported in dogs with ALD but should always be ruled out.19,20,42 Fine needle aspirates are recommended as early recognition of cutaneous neoplasia may be found.15,41 Cytology of fluid or tissue from ALD lesions have very little cellularity aside from representative inflammatory cells and fibrocytes.15
Routine bloodwork, including a complete blood count (CBC) and chemistry profile, along with urinalysis are recommended.15,16,20 If indicated, a thyroid panel should be performed as well.16. Radiographs of the lesion are recommended to investigate any underlying arthropathies, neoplasia or osteomyelitis.14,15,20,42
The gold standard to diagnose ALD is a biopsy for histopathology. Biopsy of the lesion is recommended for diagnosis of ALD and ruling out other diseases that may mimic ALD lesions.14-16,20,42 Characteristic histopathological abnormalities in ALD lesions include dermal fibrosis and vertical streaking of collagen fibers.20,40,42 Additional changes include epidermal hyperplasia and hyperkeratosis.20 The perivascular inflammatory infiltrate is mixed and consists of variable proportions of neutrophils,
10 lymphocytes, histiocytes, plasma cells and eosinophils.20,42 Apocrine sweat glands are surrounded by plasma cells and sebaceous glands are often hyperplastic.42 Focal areas of mild perifolliculitis are occasionally seen.42 Bacteria are rarely observed in histological sections.15 In a recent study additional histological features not previously described were associated with the epitrichial glands including dilation, hypertrophy, retained and inspissated secretions; perihidradenitis and hidradenitis with occasional glandular rupture.19
Bacterial culture is indicated in the diagnostic work-up for ALD as these lesions are commonly infected.14-16,19 A sterile culture should be obtained using a biopsy punch for a deeper, more representative tissue sample.14-16,19 Shumaker et al. found that only
36% of cases were in complete agreement with respect to species isolated and antibacterial susceptibility pattern when superficial and deep cultures were compared, therefore, deep tissue cultures are indicated for identification of the bacterial organisms and selection of antimicrobials to treat ALD lesions.19 Fungal cultures are also recommended, as dermatophytosis and sporotrichosis have been cultured from ALD-like lesions.19,41
Electrodiagnostic testing, such as needle electromyography (EMG) and motor/sensory nerve conduction velocities can be performed in dogs with ALD.14,15
Typically, the test may demonstrate an absence of nerve conduction across the lesion, but these abnormalities are likely a consequence and not a cause of the lesion.14,15,49
However, in one report, nine of 16 dogs with ALD had EMG abnormalities. The most common finding was abnormal activity in the paraspinal muscles supplied by the same spinal cord segment that provides innervation to the area of the ALD lesion suggesting a
11 nerve root lesion.50 These changes were considered primary and not secondary to the licking behavior.50 Although these neurological abnormalities may not be able to be surgically corrected, conservative management aimed at pain relief should be considered.
This test may also be useful in scenarios when automobile accidents or other trauma have caused peripheral neuropathy though entrapment of the nerve or by direct damage to the afferent sensory pathway.15
Food trials should be considered in animals where there is suspicion for CAFR as an underlying cause for ALD.14-16,18 In dogs diagnosed with atopic dermatitis, intradermal allergy testing (IDT) or serum allergy testing (SAT) should be considered for identification of allergens with the goal of implementing allergen-specific immunotherapy (ASIT).14-16
2.2 Treatment
Determination of any primary causes, as well as predisposing and perpetuating factors are important for successful management of ALD.14,15 Treating the primary cause and, equally important, the perpetuating factors is critical as chronic unresponsive lesions have a poor prognosis for resolution.15
2.2.1 Antibiotics
Oral antibiotics are crucial for treatment of the underlying, and often deep, bacterial infection.15,16,18 The antibiotic choice should be based on culture and susceptibility testing from a deep tissue sample and may require a protracted treatment time (4 weeks up to 4 months).15,16,18 Cephalosporins have been used commonly, but have limited success in chronic cases, especially if used empirically, likely due to methicillin-resistant bacteria.15 In one study, deep tissue bacterial cultures from ALD
12 lesions in 48% of the dogs grew one or more isolates resistant to multiple classes of antimicrobials; 26% were methicillin-resistant staphylococcal species.19 Of all the bacteria isolated, 91% were susceptible to fluoroquinolones and 89% susceptible to trimethoprim sulfamethizole.19 There results imply if empirical therapy is being implemented, administration of a fluoroquinolone or potentiated sulfonamide would be more likely to result in therapeutic success than cephalexin or amoxicillin-clavulanic acid.19
2.2.2 Topicals
Topical anti-inflammatory drugs may be used after initial resolution of the ALD lesion with antibiotic therapy.15 Topical corticosteroids can be helpful for early mild lesions.14 A combination product of fluocinolone in dimethyl sulfoxide (DMSO)
(Synotic®) mixed with flunixin meglumine (Banamine®) showed regression of lesions in
82.3% of dogs when used twice daily for one to seven weeks.42 The topical analgesic capsaicin mixed with bitter apple has been reported to be effective for treatment of ALD lesions as it may help decrease reinforcing sensations.14,15 Topical antimicrobials, such as mupirocin with or without capsaicin, have limited benefit in penetrating deep tissues,14 but can be beneficial when used in acute lesions and adjunctive with oral antibiotics.14,15
The efficacy of topical liquid bandages and deterrents such as Bitter Apple® spray for treatment of ALD lesions is based solely on anecdotal reports.14,18
2.2.3 Treatment of pruritic disease
Treatment of any suspected underlying allergic disease (e.g. CAFR, AD) or pruritic parasitic disease is necessary when treating ALD. If non-seasonal symptoms are present an underlying CAFR should be ruled out with a strict elimination food diet trial.51
13 AD may require multimodal therapy. ASIT is the only treatment of AD that modulates the immune system from over-reactive to tolerant52 and should be considered for management of underlying AD.15 ASIT will not provide an immediate benefit, but may be helpful for prevention of further lesion development.15
2.2.3.1 Glucocorticoids
Glucocorticoids may be helpful in the treatment of ALD, as they have a broad anti-inflammatory effect and act to inhibit pro-inflammatory and pruritogenic cytokines.53
Glucocorticoids also inhibit nerve hypersensitivity secondary to inflammation.53
Glucocorticoids are administered orally, intramuscularly (IM), subcutaneously (SC), intralesionally, intravenously (IV) or topically.54 While glucocorticoids are good anti- inflammatories, they are often best avoided while infection is present, which is the case in most ALD lesions.16 Intralesional injections of glucocorticoids have been used in ALD but should be used after resolution of the deep bacterial infection.14,15 Triamcinolone or methylprednisolone injections may be helpful in lesions smaller than 3cm in diameter but are not useful for large chronic lesions.14
2.2.3.2 Janus-kinase inhibitors
Oclacitinib (Apoquel®) is a medication that preferentially inhibits JAK1- dependent cytokines involved in allergy, inflammation and pruritus.55,56 Oclacitinib also inhibits JAK3 and has little effect on JAK2, which is involved with hematopoiesis.55
Oclacitinib has an inhibitory effect on IL-31, a cytokine which plays a key role in canine pruritus.55 Studies have shown good efficacy with oclacitinib when used in dogs with AD and other allergic skin diseases.57-61 In a recent abstract treatment with oclacitinib lead to a 50% reduction in size of an ALD lesion after 70 days of therapy with complete
14 regression of the lesion after 4 months.62 Oclacitinib may be a useful therapy to help decrease the licking behavior in dogs with ALD.
2.2.3.3 Monoclonal antibody therapy
Lokivetmab (Cytopoint®) is a caninized, anti-canine interleukin-31 (IL-31) monoclonal antibody that binds specifically to circulating IL-31, inhibiting its binding to the IL-31 receptor.63,64 A clinical trial with client-owned dogs reported that a single dose of 2.0 mg/kg administered subcutaneously (SC) to dogs with AD decreased pruritus within 1 day, decreased CADESI-03 scores within seven days and had continued efficacy in most dogs for at least 1 month.63 Adverse events were similar between lokivetmab and placebo groups.64 There were no clinically apparent adverse interactions between lokivetmab and a variety of concomitant medications.64 There are no published studies using lokivetmab for treatment of canine ALD, but it may be an option as lokivetmab is effective for controlling pruritus in atopic dogs.
2.2.4 Anti-anxiety medications
Anti-anxiety medications should be considered in dogs with ALD, as psychogenic causes are associated with this disease.14-16 There may be a correlation between the uncontrollable licking experienced by dogs with ALD and the actions of humans suffering from obsessive-compulsive disorder (OCD); ALD has been described as an animal model for OCD.14,15,21,28,43,65,66
2.2.4.1 Serotonin antagonist and reuptake inhibitor
Trazodone hydrochloride is a member of the phenylperazine class of drugs and is classified as a serotonin antagonist and reuptake inhibitor.23 Its primary pharmacologic mechanism of action is to antagonize serotonin 2A receptors and its secondary
15 mechanism is to inhibit serotonin uptake.23,67 In dogs this drug has been used to treat anxiety disorders, to facilitate postsurgical confinement and calming and to reduce stress- related signs and behaviors in hospitalized dogs.23,67,68 Biochemically, it appears that serotonin is directly linked to the presence of OCD as serotonin neuronal levels are low in patients with OCD. The repetitive motor activities of a patient with OCD results in an increase in serotonin levels, ultimately self-medicating them. The chronic licking of ALD may serve to increase serotonin neuronal activity as well.15 While there are no published studies using trazodone specifically in dogs with ALD, its actions on serotonin make it a possible treatment option for dogs with ALD.
2.2.4.2 Tricyclic antidepressants
Tricyclic antidepressants, such as clomipramine, have been widely evaluated as a treatment for OCD in humans.69 Clomipramine combines the properties of a selective serotonin reuptake inhibitor and a tricyclic antidepressant.70 Several studies have evaluated clomipramine for the treatment of canine ALD.21,65,71 In the 13 week single- blind crossover study by Goldberger et al. nine dogs with ALD were treated with clomipramine and desipramine in an A-B-A design with clomipramine as the first phase, desipramine the second, and clomipramine again as the third phase.71 Desipramine is another tricyclic antidepressant; however, it lacks the anti-obsessional activity of clomipramine but has similar side effects. Six of nine dogs showed an initial response to clomipramine, but not to desipramine, with a significant decrease in licking from baseline.71 Rapoport et al. reported a 43% reduction in licking from baseline in 13 dogs with ALD treated with clomipramine .21 Six of the 13 dogs had a reduction of 50% or more in their licking behaviors.21 Hewson et al. assessed the clinical efficacy of
16 clomipramine in 51 dogs for treatment of canine compulsive disorders (CCD), 12 of which had ALD.65 Owners of dogs with CCD were four times more likely to report an improvement in behavior when receiving clomipramine compared to placebo.65 The most common side effects reported with clomipramine include lethargy, loss of appetite, diarrhea, vomiting, growling, weight gain, and mild liver enzyme elevation.21,65,70,71
2.2.4.3 Selective serotonin reuptake inhibitors (SSRIs)
Fluoxetine hydrochloride is a commonly used SSRI in small animal behavioral medicine.72 In 2007 the FDA approved Reconcile™ for use in dogs for separation anxiety.72 Fluoxetine has been used in the treatment of canine ALD lesions.21,22 Rapoport et al. reported a 39% decrease from baseline of licking in dogs with ALD treated with fluoxetine. 21 Two of six dogs in the fluoxetine trial had complete remission of symptoms, while the other four had a reduction of 50% or more in their licking behaviors.21 Stein et al. enrolled five dogs with a history of ALD in an 8-week open trial with fluoxetine.73 One dog had almost complete cessation of licking, two dogs did not continue the medication and two dogs showed marked improvement after 8 weeks of treatment.73 In a placebo-controlled randomized double-blind trial in 58 dogs with ALD there was a significant improvement in the appearance of the lesion and licking behavior as judged by the owner. In contrast, there were no significant changes in the placebo group.22 The most common side effects reported with fluoxetine are lethargy, loss of appetite, hyperactivity, polyuria and polydipsia.21,73
Citalopram is the most selective of the SSRIs and has been useful in treating
OCD in humans.74 Stein et al. reported six of nine dogs diagnosed with ALD (66.7%) treated with citalopram for 8 weeks had significant improvement in owner and
17 veterinarian-assessed clinical global impression and lesion severity.74 Reported side effects included sedation in three dogs, loss of appetite in two dogs and constipation in one dog.74
2.2.4.4 Narcotic antagonists
Naltrexone is a narcotic antagonist that antagonizes the effects of endogenous opioids by binding to endogenous opioid receptors.20 The exact mechanism by which these drugs control stereotypic behavior is unclear. However, it is theorized that by reducing the analgesic state, the narcotic antagonists increase the perception of pain and decrease the endorphin-mediated reward or reinforcement of the self-injurious behavior.20
White et al. found that seven of nine dogs with ALD had improvement of their ALD lesion when treated with naltrexone for 1 month.20 All seven dogs relapsed after naltrexone treatment was stopped.20
Nalmefene is an antagonist at the mu- and delta-opioid receptor and a partial agonist at the kappa receptor.75 It has been studied in humans mainly for use in substance abuse disorders, especially alcoholism.75 Dodman et al. used nalmefene or naltrexone in
11 dogs that had self-licking, self-chewing, and scratching behaviors, five of which had
ALD. The five dogs with ALD were each given a SC injection of nalfemene and then monitored via video camera for at least 90 minutes to observe any licking behavior.76
Nalmefene was completely effective in preventing self-licking or chewing for 75 minutes in two dogs and two dogs had slight but measureable licking.76 One dog with ALD did not have significant reduction in licking or chewing.76
2.2.5 Laser therapy
18 Laser therapy has demonstrated some success, particularly in treatment of small and early ALD lesions.15,16 Carbon dioxide laser emits infrared light at 10,600nm and vaporizes intracellular water and ablates the treated cells without damaging tissue.15
There is also an advantage of sterilizing the lesion as the tissue is vaporized and less postoperative pain associated with sealing of nerve endings.14
Low-level laser therapy (LLLT) is an alternative therapy used to treat a multitude of conditions that require stimulation of healing and relief of inflammation, pruritus and pain.1 It is a form of phototherapy that uses either coherent light sources (lasers) or non- coherent light sources consisting of filtered lamps or light-emitting diodes (LEDs).1
While the mechanism of action of LLLT is poorly understood, it is thought to be through the absorption of visible red light (620-690 nm) and near-infrared light (720-1260 nm) by mitochondrial chromophores, particularly cytochrome c oxidase, which is located within the mitochondria.1 This leads to production of adenosine triphosphate (ATP),1 which alters factors associated with cell proliferation, survival, tissue repair, and regeneration.2
Blue LED, in the range of 405 to 470 nm wavelength, is bactericidal77 and has been shown to kill methicillin-resistant Staphylococcus aureus in vitro.7-9,78 Red LEDs (630-
700-nm) are used to target dermal structures and have been used for treatment of wound healing and photodamage.3 As ALD lesions are inflammatory, painful or pruritic, and commonly secondarily infected,19 the use of LLLT and both red and blue LED light may be a useful treatment option. LLLT treatment protocols are variable and established by the company, but most require a series of visits over a number of weeks. There are currently no published studies using LLLT or LED in dogs with acral lick granulomas.
2.2.6 Radiation therapy
19 Radiation therapy is another treatment option for ALD and has shown success in treating small lesions, but large chronic lesions are much less likely to respond favorably.14,15 The X-ray irradiation destroys nerve endings in the affected area and breaks the cycle of persistent licking.79 Owen et al. performed irradiation using 8 MeV x- rays from a linear accelerator on 13 dogs with ALD.79 Dogs received two to four doses at weekly intervals. Responses were considered good in 9 of 11 dogs and healing was complete by 2 to 3 months after radiotherapy.79 Rivers et al. used radiation therapy in 17 dogs diagnosed with ALD; 12 were treated with orthovoltage teletherapy and five were treated with colbalt-60 teletherapy given on a modified alternate day schedule (Monday,
Wednesday, Friday).25 Clinical response to radiation therapy was noted in 10 dogs (59%), six (50%) treated with orthovoltage teletherapy and 4 (80%) treated with colbalt-60 teletherapy. Response without recurrence was noted in six (three orthovoltage teletherapy, three colbalt-60 teletherapy) cases (35%).25 Cost, limited availability and the need for multiple treatments make this option impractical in most cases, but may be an option if the ALD lesion is refractory to all other treatment therapies.15,25
2.2.7 Surgery
Surgical excision may be an option for some cases if the lesion is small enough to allow repair without undue skin tension.14 But surgical intervention often has postoperative complications and incomplete resolution of the problem, resulting in a wound that is very difficult to manage.14,15 Reconstructive surgery using a phalangeal fillet technique of a nonhealing lick granuloma (previously excised with subsequent dehiscence) has been described.24 It is important to note that surgical excision of the
20 lesion does not prevent recurrence of the ALD lesion and the primary disease must be treated.15
Cryosurgery may be used as a last resort option for lesions that are so large they cannot be removed surgically or a graft is not possible.14 Cryosurgery can be used alone or in combination with surgical excision, radiation, curettage, chemotherapy and cytotoxins.26 Freezing destroys nerve ending, thereby blocking the itch/lick cycle and in order to be effective the procedure must be repeated two or three times.14 Liquid nitrogen and nitrous oxide are the two most common cryogens in veterinary dermatologic cryosurgery.26 Nitrous oxide is useful only for small, superficial lesions while liquid nitrogen is more potent and capable of penetrating very large lesions.26 Anecdotally cryosurgery has had limited success with ALD lesions with less than 50% of the lesions healing following treatment.26
2.2.8 Electronic stimulation
Behavior modification techniques, such as electronic shock collars, have been used to treat animal behavior problems.28 The punishment must be delivered consistently, be sharp and intense, and coincide with or immediately follow the behavior in question.28
Eckstein et al. studied electronic stimulation in 10 dogs with ALD lesions.28 Of these 10 cases, five were dropped due to unwillingness or inability of the owner to follow study instructions. Of the five remaining cases, four (80%) had resolution of the problem; the range of shocks delivered for resolution was seven to 21.28 Two dogs did not relapse during the follow-up period; two dogs relapsed but the problem resolved again with additional training.28
2.2.9 Orgotein
21 Orgotein is a naturally occurring protein that contains copper and zinc and has anti-inflammatory effects.80 Ten dogs with ALD were treated with two weekly 5mg intralesional injections of orgotein. Three dogs received two more weekly 5mg injections, administered intramuscularly. Lesions healed in 9 dogs and in all dogs the licking was considerably reduced after the first injection with orgotein.80
2.2.10 Acupuncture
Acupuncture has been used for the control and management of pain, degenerative disease and other chronic ailments that have not responded to conventional methods.27
One study used acupuncture for the treatment of ALD in four dogs.27 Two dogs had a poor response (10-25%) or no response (0-10%) to treatment, while two had a good (50-
75%) or excellent response (75-100%).27 Acupuncture therapy requires a trained veterinary professional for administration and multiple visits.
2.3 Prognosis
A thorough search for underlying disease, secondary infection and psychogenic components are essential for resolution of ALD lesions.14 Treatment of any predisposing and perpetuating factors are also critical for management.15 In general the prognosis is guarded, but combination therapy and behavior modification with good client compliance is essential for a successful outcome.14
2.4: Photostimulation
Photostimulation is the use of light to activate biological cells or tissues. Therapy with photostimulation is called phototherapy, light therapy, or photomodulation.
2.4.1 Low-level light therapy
2.4.1.1 Classification
22 Low-level light therapy, a form of phototherapy, uses either coherent light sources
(lasers “light amplification by stimulated emission of radiation”81) or non-coherent light sources consisting of filtered lamps or light-emitting diodes (LEDs).1 It was originally believed that this form of phototherapy required the use of laser light but recently LEDs have been used as a cheaper alternative – hence the term in the literature that is most commonly used is low-level laser therapy (LLLT).81 LLLT involves exposing cells or tissue to low levels of red and near infrared (NIR) light.81 It is referred to as “low level” because of its use of light at energy densities that are low compared to other forms of laser therapy that are used for ablation, cutting, and thermally coagulating tissue.81
LLLT devices are categorized into six different classifications (Class I, IA, II,
IIIA, IIIB, IV) based on the increasing potential for damage such as eye injuries and thermal injuries.82 Class I lasers have minimal to no side effects reported, unlike Class IV laser which require eye protection and advanced training to avoid burns.82
2.4.1.2 Mechanism of action
The mechanism associated with the cellular photobiostimulation by LLLT is not yet fully understood.1,81 The basic mechanism is thought to be through absorption of red and NIR light by mitochondrial chromophores, particularly cytochrome c oxidase.1,81
Cytochrome c oxidase is contained in the respiratory chain located within the mitochondria.1 The hypothesis is that the absorption of light energy may cause photodissociation of inhibitory nitric oxide leading to enhancement of enzyme activity and adenosine triphosphate (ATP) production.1,81 As a result, LLLT alters the cellular redox state which induces the activation of numerous intracellular signaling pathways,
23 and alters the affinity of transcription factors involved in cell proliferation, survival, tissue repair and regeneration.1,81
2.4.2 Uses of LLLT
2.4.2.1 Human medicine
LLLT has been widely used as a non-invasive therapy in human medicine.2
Several studies have evaluated the use of LLLT to stimulate hair regrowth both in animal and human models.2,83-86 A hand-held LLLT device (Hairmax LaserComb® ) was used to evaluate to effects of LLLT on hair regrowth in a mouse model of alopecia areata.84 The mice in the laser treated group (655nm) had hair regrowth after 6 weeks of three-times a week treatment, compared to no hair regrowth in the sham-treated group.84 This same device was used in another study to evaluate hair regrowth in chemotherapy-induced alopecia (CIA) in rats.85 Rats receiving LLLT (655nm) once daily for 10 days had hair regrowth five days earlier than sham-treated rats.85 Jimenez et al. used the Hairmax
LaserComb® device in a study on human subjects with male and female pattern hair loss.83 Subjects were treated three times weekly for 26 weeks with LLLT (635 or 655nm) or sham laser.83 A significant increase in terminal hair density was seen in those treated with laser compared to sham-treated patients.83 While current studies support the use of
LLLT for hair regrowth, more studies are needed to optimize treatment parameters.2
LLLT has been used for the management of pain in patients with musculoskeletal and joint disorders.87 A systematic review of 11 trials using LLLT for joint disease in the knee, temporomandibular or zygapophyseal joints88 concluded there was a mean weighted difference in change of pain score in favor of the LLLT groups.88 A review of over 4000 studies concluded that in the majority of laboratory and clinical studies, LLLT
24 has a positive effect on acute and chronic musculoskeletal pain.89 A recent systematic review and meta-analysis concluded that LLLT is an effective treatment modality to achieve pain relief in adult patients with musculoskeletal disorders.87 In addition, the beneficial effects of LLLT were unaffected by the anatomical site of the lesion and World
Association of Laser Therapy (WALT) dosage recommendations are needed for the best pain relieving results.87 In a systematic review of the use of LLLT in patients suffering from temporomandibular disorders, seven of 13 studies reported a significant improvement in pain in the LLLT group, while in 6 studies there was no significant improvement between control and LLLT groups.90
LLLT has also been considered as a treatment in patients suffering from strokes and traumatic brain injuries (TBI).91 There was significantly greater improvement (using the NIH Stroke Severity Scale) in the treatment group, but not in the sham-group when
LLLT was used in a clinical trial with stroke patients.91 It was also discovered that the severity of the stroke may also affect the outcome when using LLLT.91
LLLT has been used in human medicine as treatment for neuropathic pain.92 A systematic review of the literature was performed to evaluated the use of LLLT to control neuropathic pain and with a goal of establishing a therapeutic window for the use of
LLLT.92 These studies revealed better results for LLLT above 70 mW, but overall LLLT has positive effects on the analgesia control for neuropathic pain.92 Rats with a standardized crush injury to the right sciatic nerve were treated daily with 6 J of LLLT
(820nm) for 28 days.93 At 21 days post-injury the laser-treated rats had improved nerve function, but there was no difference in evoked compound action potentials (nerve regeneration) between the laser and sham-treated groups.93 In six of 10 studies performed
25 in humans with peripheral neuropathies, LLLT led to a reduction in sensory impairment and improvement of the physiological function of the sensory nerves.94 The lasers used had wavelengths that varied from 660-860nm. Further studies are needed to recommend a specific protocol.94
LLLT has been evaluated in numerous wound healing studies in mice and rat in vivo models as well as in vitro studies on human keratinocyte lines.95-100 LLLT affects cellular metabolic processes and promotes effects such an analgesia and healing while providing the body with a better inflammatory response.98 Healing was maximal in mice treated with 810-nm diode laser, evidenced by significant wound reduction, enhanced collagen accumulation and complete re-epithelialization of the wound.96 In a diabetic wound healing mouse model, histopathological analysis of the wounds revealed collagen fibers were more organized in LLLT irradiated control and irradiated diabetic mice when compared to non-irradiated groups.97 In a study of wound healing in rats treated with
LLLT, an increased migration of cells were found and the wounds closed faster.95 A qualitative review concluded that LLLT applied to skin wounds was able to promote physiological effects such as resolution of inflammation, neoangiogenesis, epithelial and fibroblasts proliferation, collagen synthesis and deposition, revascularization and wound contraction.99 The use of LLLT has also been evaluated in humans in vivo for healing of pressure sores.101 A systematic review found significant improvement in healing with the use of LLLT at 658-nm wavelength but not at 808, 904 or 940 nm.101 Dose is important as deleterious effects were associated with doses above 10 J/cm2.99 Similar results were found with skin flap survival in rats, where a higher dose (100 J/cm2) increased the necrotic area when compared to sham-treated skin flaps.100
26 LLLT has also been used in human medicine for various dermatologic conditions.1,30 LLLT in the red to near infrared range alone or in combination with other treatments, mainly blue light, has been effective in treatment of acne vulgaris.1 It has also been suggested as an alternative treatment for accelerated healing and reducing symptoms associated with herpes virus lesions.1 LLLT used in human patients with vitiligo showed marked repigmentation after 6-8 months of treatment.1 LLLT at wavelengths of 830 and 630-nm has been used in patients to help resolve plaque psoriasis lesions resistant to conventional therapy.1 In patients suffering from AD there was a 62% decrease in skin symptom scores and 79% decrease in itch score of more than 1 point, on a 0 to 3 scale, after using LLLT.30
Recently, it has become apparent that LLLT can be effective if delivered to normal cells or tissue before the actual insult or trauma, in a pre-conditioning mode.102
Pre-conditioning with LLLT can protect skeletal muscle against exercise-induced damage and help accelerate recovery and increase athletic performance.102
2.4.2.2 Veterinary medicine
LLLT has been used in veterinary medicine, mainly in dogs and horses, for treatment of osteoarthritis, post-operative pain, wound healing and dermatologic conditions such as pruritus and alopecia.31,103-107 When using LLLT in animals, organic debris, pigment and hair, may hinder depth of light penetration. In a previous study, equine tendons that were clipped and cleaned with alcohol had a greater transmission of light than those that were unprepared.107 While this study was performed in horses, it is likely that hair and organic debris would similarly affect light transmission in companion animals and should be considered when utilizing LLLT.
27 Combining LLLT with systemic pain medications along with weight management and nutritional joint support has been found to be an effective, non-invasive way to manage chronic pain in animals.29 LLLT has been shown to decrease arthritis pain, decrease muscle spasm and improve circulation.29 LLLT used in dogs daily for 5 days post-hemilaminectomy surgery significantly improved neurological dysfunction and time to ambulation (median 3.5 days) compared to the control group (14 days).103
LLLT does not appear to be beneficial for stimulating wound healing in dogs.
When wounds were surgically created in beagle dogs, there was no difference between
LLLT-treated (1.125 J/cm2) and control wounds for all healing parameters, including histology.104 Control wounds on dogs in a previous study that did not receive laser therapy had significantly greater contraction and epithelialization compared to laser- treated wounds (treatment group) and untreated distant wounds (control group) indicating no beneficial effects of LLLT on the healing of acute wounds in healthy dogs. These results also suggest a possible systemic adverse effect of the laser on healing on both the laser treated wound and the untreated control wound distant to the treated wound.104
LLLT has been used as a preoperative treatment in order to induce a protective response, decrease inflammation, and reduce damage from surgery.106 Preoperative LLLT was used in a study of dogs undergoing tibial plateau leveling osteotomy (TPLO) surgery.106 When given a single preoperative LLLT treatment there was a significant improvement in weight bearing eight weeks post-operatively in the laser-treated dogs compared to the sham-treated dogs.106 Although not significant, a greater proportion of
LLLT treated dogs had healed at the eight-week time point than dogs in the sham group,
28 even though dogs were older in the LLLT group.106 It would be expected that older dogs would have had slower healing overall, which was not the case.106
As in humans, LLLT has also been used in veterinary medicine for treatment of dermatologic conditions such as pedal pruritus and alopecia.31,105 The use of LLLT was evaluated in a pilot study of seven dogs affected with canine non-inflammatory alopecia
(CNA); two dogs each had postclipping alopecia and recurrent flank alopecia, and three dogs had pattern alopecia.105 Each dog was treated twice weekly with the laser device containing a cluster probe of three wavelengths (470, 685, and 830 nm) for up to two months. At the end of the study coat quality was greatly improved in six of seven dogs and improved in one of seven.105 Histological and morphometric analysis were performed on a post-treatment biopsy and control site in a single dog which revealed an increase in the percentage of hair follicles per unit area in the laser-treated versus control area.105
LLLT has been evaluated as a local treatment of pedal pruritus in dogs diagnosed with AD.31 Each dog received three laser treatments (dual wavelength of 810nm (20%) and 980nm (80%) and a 650 nm wavelength aiming beam) per week for weeks one and two, then two laser treatments for weeks three and four on one paw. Each dog served as their own placebo control, receiving just the aiming beam (650 nm wavelength) on the control paw. There was no significant difference in pruritus scores between LLLT and placebo treatments, although there was a significant difference in pruritus scores from baseline at weeks 2, 4 and 5, suggesting a possible systemic benefit.31
2.4.3 Light emitting diodes
2.4.3.1 Definition
29 Light emitting diodes, or LEDs, produce a narrow spectrum of light in an incoherent manner, where the light is randomly spread out once emitted from a light source.3 LEDs have different depth of penetration based on their wavelength and can affect cellular metabolism by triggering intracellular photobiochemical reactions.3
Observed effects include increased ATP, modulation of reactive oxygen species, the induction of transcription factors, alteration of collagen synthesis, stimulation of angiogenesis, and increased blood flow.3
2.4.3.2 Classification
LEDs are classified by wavelength of light. Red LEDs (630-700-nm) have been shown to activate fibroblast growth factor, increase type 1 pro-collagen, increase matrix metallo-proteinase -9 (MMP-9) and decrease MMP-1.3 They have the deepest tissue penetration of the visible wavelengths and are used to target dermal structures, such as adnexa and fibroblasts.3 Red LEDs have been studied for use in wound healing, photodamage, the treatment of nonmelanoma skin cancers, precancers, warts; and prevention of oral mucositis in cancer patients.3 Red LEDs can also be used for treatment of actinic keratosis and follicular inflammatory disorders such as acne vulgaris.3
Yellow LED (570-590-nm) alters ATP production, gene expression and fibroblast activity and penetrates to the level of the papillary dermis.3 Its main applications are photoaging and as an adjuvant to laser therapy.3 In a study of 900 patients with photoaged skin, those receiving yellow LED alone self-reported a softening of skin and reduction in fine lines.3,108 However, results may be complicated by placebo effect.3 Yellow LED has also been reported to reduce erythema and pain and speed healing after treatment with fractionated laser therapy for treatment of photodamaged or scarred skin109 or with
30 intense pulsed light.3 Yellow LED may help reduce the incidence and degree of radiation- induced skin reactions as well as the incidence of treatment interruption of radiation therapy because of skin reaction.3,110 However, in contrast, a similar study found no significant differences in radiation-induced skin reactions using yellow LED in patients with radiation dermatitis.3,111 Further studies are needed to determine if yellow LED is beneficial for radiation dermatitis in humans.
Infrared (IR) light (800-1200-nm) is believed to stimulate circulation by inducing the release of guanylate cyclase and nitrous oxide (NO), which then promotes vasodilation and growth factor production, and angiogenesis.3 IR LEDs can penetrate to the level of the adnexa and reticular dermis and are frequently used in combination with other light devices.3 IR LED was shown to decrease the role of fibroblast proliferation in normal human skin, indicating IR LED could be a promising treatment for wound healing and scars.3,112 As such, IR LED has been used to treat several patients including those with diabetic non-healing wounds, full-thickness pressure wounds, and a bed-bound patient with MRSA furuncles.3,113 IR LED has been shown to attenuate the formation of hypertrophic scars or keloids.1 Combination therapy using IR A plus visible light treatment has been effective in treating patients with cutaneous scleroderma, with seven out of 10 patients showing marked improvement in one study, which could prove to be beneficial in treatment of dysmorphism, contractures and restricted movement.3,114
Blue LED (400-470-nm) has both anti-inflammatory and antibacterial properties and penetrates to the level of the epidermis, so is best used for superficial conditions.3
Irradiation with blue LED leads to photoexcitation of bacterial porphyrins, singlet oxygen production and eventually bacterial destruction.4 Blue LED has been used successfully in
31 treating Propionibacterium acnes in patients suffering from acne vulgaris.3-5,115,116 P. acnes produces porphyrins which absorb blue light.4 Fungal organisms, such as Candida albicans, also contain endogenous porphyrins and are susceptible to blue LED.117 A single exposure of 415-nm blue light significantly reduced the fungal burden of C. albicans in infected mouse burns.117 Blue LED was also effective for inactivation of
Pseudomonas aeruginosa both in vitro and in a mouse model.6,118,119 Wang et al. found that 415-nm blue LED inactivated both P. aeruginosa and Acinetobacter baumannii biofilms both in vitro and in infected mouse burn wounds;120 both A. baumannii and P. aeruginosa contain endogenous porphyrins.120 At doses of 165 or 220 J/cm2, 470-nm blue
LED light completely inactivated Salmonella typhimuirum and Salmonella heidelberg.7
The effects of blue LED on Staphylococcus, specifically Staphylococcus aureus, have been studied extensively.6-9,78,119,121-125 Both methicillin-susceptible and methicillin- resistant S. aureus have been shown to contain porphyrins.124 Using a 405-nm blue light combined with 880-nm IR light, there was a significant reduction in bacterial colonies of
S. aureus at all doses, with the highest decrease (72%) at the highest dose tested (20
J/cm2).119 405-nm blue light produced a dose-dependent bactericidal effect on S. aureus, with up to 90% kill rate; the highest kill rate was at the highest dose of 15 J/cm2.6
However, at the same dose (15 J/cm2) 470-nm blue LED only killed 62% of S. aureus isolates.6
Methicillin-resistant strains of S. aureus (MRSA) are a serious cause of disease in people and treatment is difficult due to high-level antimicrobial resistant isolates.123
Using a dose of 60 J/cm2, 405-nm blue LED killed 93.5% of MRSA in vitro.123 Similar results were found in another study comparing the antimicrobial effect of 405-nm blue
32 LED and blue 405-nm laser on MRSA in vitro.78 Regardless of device, irradiation at each dose (40, 54, 81, or 121 J/cm2) resulted in significant bacterial growth suppression compared to non-irradiated controls.78 The antimicrobial effect of both light sources,
LED and laser, were not significantly different in 35 of 36 trials.78 MRSA growth suppression with either light source increased with repeated irradiation, particularly at the
15- or 30-minute treatment time interval.78 Enwemeka et al. found as much as 90.4% reduction in colonies of MRSA when using 470-nm blue light in vitro at 55 J/cm2.9
Another study reported that 100% of MRSA colonies were suppressed with a single exposure to 55 or 60 J/cm2 of 470-nm or double treatment with 50, 55, or 60 J/cm2 of
405-nm blue light.8 Denser bacterial cultures required higher doses to achieve 100% suppression.8 470-nm blue light completely (100%) inhibited growth of MRSA when used at a dose of 110 and 220 J/cm2.7 460-nm blue light was able to eradicate MRSA in both planktonic and biofilm states in a dose-like fashion in vitro.126 In a mouse model of skin abrasions infected with MRSA, 415-nm blue light rapidly reduced the bacterial burden in both acute and established MRSA infections.122
Blue light LED has been combined with sliver nanoparticles and antimicrobial agents as a new strategy to treat serious MRSA infections.127 The antimicrobial activity of silver nanoparticles along with 460-nm blue light therapy was enhanced when both agents were applied to MRSA in vitro, compared to each agent alone.127 The bactericidal activities were highest when silver nanoparticles and blue light were combined with azithromycin or clarithromycin.127
2.5 Photodynamic therapy
33 Photodynamic therapy (PDT) is a treatment that involves photosensitization of a target using a topical or systemic agent that can be activated by light in the presence of oxygen and produce a cytotoxic reaction.128 PDT can be performed using 5- aminolevulinic acid (5-ALA) or its methylester aminolevulinic acid (MAL),129 a natural amino acid that is the precursor of a strong photosensitizer, protoporphyrin IX within cells.128 5-ALA can be administered locally, systemically (intravenous and intraperitoneal) and orally. 5-ALA-PDT combined with a 410-nm blue LED had an antibacterial effect on MRSA in vitro with a 5 log10-unit decrease in organisms at 50
J/cm2.128 In the same study, in a mouse model of MRSA-infected wounds, the use of 5-
ALA-PDT with a 410-nm LED accelerated wound healing and decreased bacterial counts on the wound surface.128
The use of PDT has been evaluated in patients with surgical scars.129 PDT with either topical ALA or MAL combined with red LED (635-nm) statistically improved scar appearance after two to three sessions.129
PDT with MAL and red light have been used to treat facial photodamage.130 A double-blind randomized placebo-controlled trial found that MAL with red light had significantly greater treatment success than placebo with red light, with improvements in variables such as fine lines, coarse lines, mottled pigmentation, roughness, erythema, sallowness and sebaceous hyperplasia.130
34
Chapter 3
In vitro bactericidal activity of blue light (465-nm) phototherapy on methicillin-
susceptible and methicillin-resistant Staphylococcus pseudintermedius
3.1 Abstract
Background: Staphylococcus pseudintermedius is the most common cause of bacterial skin infections in dogs. Methicillin-resistant infections have become more common and are challenging to treat. Blue light phototherapy may be an option for treating these infections.
Hypothesis/Objectives: The objective of this study was to measure the in vitro bactericidal activity of 465-nm blue light on methicillin-susceptible Staphylococcus pseudintermedius (MSSP) and methicillin-resistant Staphylococcus pseudintermedius
(MRSP). We hypothesized that irradiation with blue light would kill MSSP and MRSP in a dose-dependent fashion in vitro as previously reported for methicillin-resistant
Staphylococcus aureus (MRSA).
Methods: In six replicate experiments, each strain (MSSP: n=1), (MRSP ST-71
[KM1381]: n=1) and (MRSA [BAA-1680]: n=1) were cultivated on semisolid media, irradiated using a 465-nm blue light phototherapeutic device at the following cumulative doses: 56.25, 112.5, and 225 J/cm2 and incubated overnight at 35oC. Controls were not irradiated. Colony counts (CC) were manually performed. Descriptive statistics were performed and treatment effects assessed using the Mann-Whitney-Wilcoxon rank-sum
35 test. Bonferroni-corrected rank sum tests were performed for post-hoc analysis when significant differences were identified.
Results: There was a significant decrease in CC with blue light irradiation at all doses for
MRSA (P=0.0006) but not for MSSP (P=0.131) or MRSP (P=0.589).
Conclusions: Blue light phototherapy significantly reduced CC of MRSA, but not of
MSSP or MRSP. The mechanism for the relative photosensitivity of the MRSA isolate is unknown, but is hypothesized to be due to an increased concentration of porphyrin in S. aureus relative to S. pseudintermedius, which would modulate blue light absorption.
3.2 Introduction:
Staphylococcus pseudintermedius is a common commensal and opportunistic pathogen of the skin of dogs and is the most common cause of bacterial skin infections.10
In recent years, methicillin-resistant S. pseudintermedius (MRSP) infections have become much more common.11 The prevalence of MRSP in clinical samples from dogs with canine pyoderma ranges from 15.6% to 38.2%.131,132 With the emergence of MRSP the number of oral antimicrobial drugs to which bacterial isolates are susceptible is limited often leading to pharmacotherapeutic choices that have profound side effects or are reserved for human infections.12 Topical antimicrobial therapy has been shown to be effective for treatment of some MRSP infections,13 but these treatments can be time consuming and inconvenient for owners. There is a need to develop new therapies to treat these infections that are both effective and have minimal side effects.
Photostimulation is the use of light to activate biological cells or tissues. Therapy with photostimulation is called phototherapy, light therapy, or photomodulation.
Photostimulation can be performed using light emitting diodes (LEDs). LEDs produce a
36 narrow spectrum of light in an incoherent manner, where the light is randomly spread out once emitted from the light source.3 LEDs have different depth of penetration based on their wavelength and can affect cellular metabolism by triggering intracellular photobiochemical reactions.3 Wavelengths available in commercial LED units include ultraviolet (100 to 400-nm),133,134 blue (400 to 470-nm)3, yellow (570 to 590-nm)3, red
(630 to 700-nm)3, and infrared (800 to 1200-nm)3. The deepest target of LED light penetration varies. In humans blue light targets the epidermis (less than 1 mm), yellow light the papillary dermis (0.5 to 2 mm), red light the adnexa (2 to 3 mm) and infrared light (5 to 10 mm) both the adnexa and reticular dermis.3 These depth penetrations would be expected to be similar in animal integument.
Blue light phototherapy has recently been shown to be a treatment option for bacterial infections. While the exact mechanism is unknown, blue light is thought to excite intracellular porphyrins and produce cytotoxic reactive oxygen species.5 These reactive oxygen species have an antimicrobial effect on the bacteria, but are not detrimental to the host cells.5
Blue light phototherapy has been shown to be bactericidal against Staphylococcus species in vitro.6-9 A dose-dependent bactericidal effect was observed on Staphylococcus aureus, with the greatest reduction of bacteria (62%) at the highest dose of 15 J/cm2 using
470-nm blue light.6 Optimal doses of 470-nm blue light phototherapy have been reported to be between 55 to 220 J/cm2 resulting in 90.4 to 100% reduction of MRSA, respectively.6-9 The bacterial densities of MRSA in these studies ranged from 3x106
CFU/mL up to 7x106 CFU/mL.7-9 Denser bacterial cultures, (7x106 CFU/mL) reflecting increasing bacterial loads, required higher doses.8
37 Since blue light phototherapy has been shown to be effective in killing bacteria associated with human Staphylococcal infections, it may be a useful adjunctive or sole therapy option for Staphylococcal infections in dogs, specifically methicillin-resistant infections. As there are no published studies evaluating the bactericidal effect of blue light phototherapy on MRSP or methicillin-susceptible Staphylococcus pseudintermedius
(MSSP), the objective of this study was to determine the in vitro bactericidal activity of
465-nm blue light on MSSP and MRSP. We hypothesized that irradiation with 465-nm blue light would suppress growth of MSSP and MRSP in a dose-dependent manner, as previously reported for MRSA.7,9
3.3 Materials and methods:
3.3.1 Bacterial isolates and culture:
A sequence-typed strain of MRSP ST-71 (KM1381), sequence-typed strain of
USA300 MRSA (BAA-1680) and an untyped clinical MSSP isolate were selected for use as the test isolates. The MRSP ST-71 (KM1381) isolate was obtained from the University of Tennessee College of Veterinary Medicine bacteriology laboratory in Knoxville,
TN.135,136 The USA300 reference strain of MRSA was obtained from American Type
Culture Collection (ATCC® BAA-1680).137 The clinical MSSP isolate was speciated using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry.138 A standardized inoculum of 15µL of approximately 104 CFU/mL of each isolate was spread-plated onto 35mm petri dishes containing tryptic soy agar (TSA). This plate size was chosen to attain uniform irradiation as the blue light was emitted from an opening that was approximately 25mm in diameter. Briefly, a 0.5 McFarland standard suspension (approximately 108 CFU/mL concentration) was made for each isolate in
38 sterile water. Sterile ten-fold serial dilutions were performed by placing 40µL of the bacterial suspension in 360µL of phosphate-buffered saline (PBS) to reach a final dilution of approximately 104 CFU/mL. Dilutions were obtained to achieve a range of 10 to 100 colony forming units (CFU) on the 35mm TSA plates. Plates for the treatment groups
(MSSP, MRSP and MRSA isolates) were inoculated with 15µL of the 104 CFU/mL dilution and spread onto the 35mm TSA plates. Plates for controls for each isolate were spread in the same manner. After inoculation the treatment group plates were irradiated with blue light (see below) and incubated overnight at 350C in ambient air. Plates for the controls were not irradiated prior to incubation. Incubation conditions (time and temperature) were identical for control and treated plates.
3.3.2 Blue light therapy:
A 465-nm blue light therapeutic device (MR4 ACTIVet PRO™ device, Multi
Radiance Medical®; Solon, OH, USA) was used for all irradiations (Table 1). The device was clamped approximately 6mm above the TSA plates for even dispersion of light across the inoculated area (Figure 1). Treatment groups (MSSP, MRSP and MRSA) were irradiated once with the following doses: 56.25 J/cm2 (15 minute exposure), 112.5 J/cm2
(30 minute exposure), and 225 J/cm2 (60 minute exposure). The controls were not irradiated. The three doses were chosen based on a previous study in which irradiation of
USA300 MRSA (ATCC® BAA-1680) with 470-nm blue light at 55, 110, and 220 J/cm2 produced 69-92%, 80-100% and 100% suppression of bacterial growth, respectively.7
Each dose was repeated in sextuplicate for each isolate. Colony counts (the total numbers of colonies on each individual plate; CC) were manually performed.
39 3.3.3 Statistical Analysis:
Descriptive statistics were generated for CC for control and treatment groups at each dose and the data were tested for distribution and normality by visual inspection and with the D’Agostino – Pearson test (MedCalc® for Windows, version 15.0; Ostend,
Belgium). As data were not normally distributed the results are displayed as medians, interquartile ranges and ranges (MedCalc® for Windows, version 15.0; Ostend, Belgium) and percent reduction for CC. The control and treatment groups were compared within each bacterial isolate using a Mann-Whitney-Wilcoxon rank sum test (SPSS IBM version
24; Armonk, NY, USA). When a significant difference was identified, Bonferroni- corrected rank sum tests (SPSS IBM version 24; Armonk, NY, USA) were performed at each dose for post-hoc analysis. Statistical significance was set at P<0.05.
3.4 Results:
The median CC and percent reduction of the treatment groups (MSSP, MRSP,
MRSA) after irradiation with 465-nm blue light at 56.25, 112.5 and 225 J/cm2 are shown in Table 2. The percent reduction of CC was greatest for MRSA at all doses, reaching
100% at the two highest doses. In contrast, the percent reduction of CC for MSSP and
MRSP was minimal with the maximum reduction found at the highest dose (225 J/cm2) of 11.7% for MSSP and 21.2% for MRSP for CC.
The effect of dose on CC for the treatment groups is shown in Figures 2a-c with representative plates depicted in Figures 3a-l. A significant reduction in median MRSA
CC after treatment (P<0.0005, Table 3) at all doses (P=0.006, Figure 1) was present.
There was no significant reduction in median CC after irradiation for MSSP (P=0.131) or in median CC for MRSP (P=0.589) (Table 3).
40
3.5 Discussion:
Blue light 465-nm phototherapy significantly reduced CC for MRSA, but not for
MSSP or MRSP. An earlier study using 470-nm blue light phototherapy reported a significant dose dependent reduction in MRSA colony counts with 90.4% of the colonies killed at a dose of 55 J/cm2.9 However, 100% reduction was not obtained, even at the highest dose tested (60 J/cm2).9 Our study had similar results for MRSA with a 93.3%
CC reduction at 56.25 J/cm2. In two recent studies, 470-nm blue light suppressed MRSA at 55 J/cm2; however, to achieve 100% bacterial suppression irradiation had to be performed twice for standard or less dense cultures. Denser cultures, when irradiated once, required the highest dose of 220 J/cm2 to achieve 100% kill.7,8 Similarly, in our study, the two highest doses, 112.5 and 225 J/cm2 were required to achieve 100% reduction in CC for MRSA. While Staphylococcus pseudintermedius is the most common organism isolated in dogs with bacterial skin infections,10 MRSA infections are an emerging problem in veterinary medicine.11,139,140 As handheld LED blue light phototherapic devices are commercially available, blue light phototherapy may be an effective option for treating MRSA. Based on the results of our study, to achieve 100% kill, treatment times would need to be at minimum 30 minutes, so this therapy would be best suited for localized rather than generalized infections. The effect of factors such as hair and organic debris need to be considered for use in vivo. Clipping the hair and cleaning the skin over the area to be irradiated with alcohol has been shown to increase the depth of penetration of the light when a low level laser therapy probe was used on the flexor tendon in the horse.107 In a mouse model of MRSA skin abrasion infections, 415-
41 nm blue light delivered at 108 J/cm2 rapidly reduced the bacterial burden, suggesting the use of blue light may be an option for treatment of MRSA skin infections.122
Interestingly, the percent CC reduction for MSSP and MRSP were minimal, with the greatest reduction of 11.7% and 20.5% respectively at the highest dose of 225 J/cm2.
These results were not expected, as blue light phototherapy had been effective in vitro against MRSA in previous studies7-9 and in ours. While the mechanism of action of blue light is not fully understood, it is believed to excite intracellular porphyrins, thus generating the production of cytotoxic reactive oxygen species that kill the bacteria.5
Since porphyrins are a key factor in absorption of blue light, it is possible that differences in the amount of endogenous porphyrins between bacterial strains would result in differences in blue light absorption and killing of the bacteria. Lipovsky et al. compared the difference in absorption of visible light (400-800-nm) of two different strains of
Staphylococcus aureus, one methicillin-susceptible strain (MSSA) and one resistant strain (MRSA).124 MSSA strain had a maximum reduction in bacterial viability of 99.8% compared to 55.5% for MRSA. Porphyrins were extracted and the relative fluorescence measured using a spectrometer. The MSSA had a higher porphyrin concentration and cytotoxic oxygen radical production compared to MRSA which likely accounted for more absorption of light and a greater reduction of the bacteria.124 The relative percent production of porphyrin has also been measured in Staphylococcus aureus using high performance liquid chromatography (HPLC).141 To the author’s knowledge there are no studies of endogenous porphyrin concentration measurement in MSSP or MRSP.
Photodynamic therapy (PDT) is a treatment option that involves photosensitization of a target using a topical or systemic agent that is activated by light in
42 the presence of oxygen and produce a cytotoxic reaction.128 PDT can be performed using
5-aminolevulinic acid (5-ALA), a natural amino acid that is the precursor of a strong photosensitizer, protoporphyrin IX within cells.128 5-ALA can be administered locally, systemically (intravenous and intraperitoneal) and orally. 5-ALA-PDT combined with a
410-nm LED had an antibacterial effect on MRSA in vitro with a 5 log10-unit decrease in organisms at 50 J/cm2. In the same study, in a mouse model of MRSA-infected wounds, the use of 5-ALA-PDT with a 410-nm LED accelerated wound healing and decreased bacterial counts on the wound surface.128 5-ALA-PDT may be an option both in vitro and in vivo for MRSP and MSSP to help increase bacterial kill.
In conclusion, blue light phototherapy significantly reduced CC of MRSA, but not of MSSP or MRSP. The mechanism for the relative photosensitivity of the MRSA isolate is unknown, but it hypothesized to be due to an increased concentration of porphyrin in S. aureus relative to S. pseudintermedius, which would modulate blue light absorption.
Future studies are needed to measure the concentration of porphyrins and assess porphyrin relevance in blue light absorption in MSSP and MRSP as well as assess the use of PDT combined with blue light for MSSP and MRSP infections.
43 Table 1. Properties of the Blue Light Phototherapeutic Device (MR4 ACTIVet PRO™,
Multi Radiance Medical®; Solon, OH, USA)
Number of blue LEDs 3 Blue Wavelength of blue LEDs (nm) 465 (±10) Mode Continuous Average optical output (mW) - each 83.33 Power density (mW/cm2) - each 111.11 Energy density (J/cm2) - each 100, 200, 400 Dose (J) - each 75, 150, 300 Spot size of blue LED (cm2) - each 0.75
Magnetic Field (mT) 45
Irradiation time (sec) 900, 1800, 3600 Total dose (J) 225, 450, 900 Aperture of device (cm2) 4 Energy density at aperture (J/cm2) 56.25, 112.50, 225.00 Power density at aperture (mW/cm2) 62.50
Dosages and irradiation times used in the study are bolded.
44 Table 2: Median Colony Counts and Percent Reduction for Control Group and Treatment
Groups (Methicillin-Susceptible Staphylococcus pseudintermedius, Methicillin-Resistant
Staphylococcus pseudintermedius [KM1381] and Methicillin-Resistant Staphylococcus aureus [BAA-1680]) after Irradiation with 465-nm Blue Light
MSSP MRSP (KM1381) MRSA (BAA-1680) Blue Light Dose CC CC CC CG TG % Red CG TG % Red CG TG % Red 56.25 J/cm2 29 27 6.9 23 25 -8.7 67 4.5 93.3 112.5 J/cm2 31 28 9.7 29.5 29.5 0 72.5 0 100 225 J/cm2 38.5 34 11.7 26 20.5 21.2 90.5 0 100
MSSP = methicillin-susceptible Staphylococcus pseudintermedius, MRSP = methicillin- resistant Staphylococcus pseudintermedius, MRSA = methicillin-resistant Staphylococcus aureus, CC=median colony counts, CG= control group (not irradiated), TG= treatment group, % Red=percent reduction
45 Table 3: Median, Interquartile Range and Range of Colony Counts for Control Groups and Treatment Groups (Methicillin-Susceptible Staphylococcus pseudintermedius,
Methicillin-Resistant Staphylococcus pseudintermedius [KM1381] and Methicillin-
Resistant Staphylococcus aureus [BAA-1680]) after Irradiation with 465-nm Blue Light
Bacterium Group Median (IQR) Range P Value MSSP CC CG 31.5 (27.8, 38.3) 24-51 0.131 TG 28.5 (25.5, 33.5) 20-41 MRSP (KM 1381) CC CG 24 (16.8, 42.0) 9-47 0.589 TG 24 (15.8, 35.3) 13-42 MRSA (BAA-1680) CC CG 75.5 (66.5, 88.5) 57-116 <0.0005 TG 0.5 (0, 2.5) 0-7
The control and treatment groups (all doses combined) were compared within each bacterial isolate using the Mann-Whitney-Wilcoxon rank sum test (SPSS IBM version
24; Armonk, NY, USA). Statistical significance was set at P<0.05.
MSSP = methicillin-susceptible Staphylococcus pseudintermedius, MRSP = methicillin- resistant Staphylococcus pseudintermedius, MRSA = methicillin-resistant Staphylococcus aureus, CC=colony counts (number of colonies), CG= control group (not irradiated),
TG= treatment group, IQR = interquartile range
46
Figure 1. The 465-nm blue light therapeutic device (MR4 ACTIVet PRO™ device, Multi
Radiance Medical®; Solon, OH, USA) irradiating a 35mm petri dish containing tryptic soy agar (TSA) plated with a bacterial isolate.
47 a. MSSP
b. MRSP (KM1381)
c. MRSA (BAA-1680)
Figure 2: a. MSSP, b. MRSP (KM1381), c. MRSA (BAA-1680)
Figure 2 Legend: Box plots showing the bacterial colony counts at each dose (56.25,
112.5, 225 J/cm2) for the control (not irradiated) and treatment groups (irradiated with
Continued
48 Figure 2 continued
465-nm blue light) for MSSP (a), MRSP (KM1381) (b), and MRSA (BAA-1680) (c).
Black circles and open triangles represent the colony counts of each individual plate
(gray squares are outliers). The control and treatment groups were compared within each bacterial isolate using a Mann-Whitney-Wilcoxon rank sum test (SPSS IBM version 24;
Armonk, NY, USA). When a significant difference was identified, Bonferroni-corrected rank sum tests (SPSS IBM version 24; Armonk, NY, USA) were performed at each dose for post-hoc analysis. Statistical significance was set at P<0.05. There was a significant difference in CC for MRSA between treatment and control groups at each dose
(P=0.006). MSSP = methicillin-susceptible Staphylococcus pseudintermedius, MRSP = methicillin-resistant Staphylococcus pseudintermedius, MRSA = methicillin-resistant
Staphylococcus aureus.
49
Figure 3 Legend: Representative culture plates of MSSP (a-d), MRSP (KM1381) (e-h) and MRSA (BAA-1680) (i-l) irradiated with 56.25, 112.5, and 225 J/cm2 465-nm blue light. Control plates (a,e,i) were not irradiated. MSSP = methicillin-susceptible
Staphylococcus pseudintermedius, MRSP = methicillin-resistant Staphylococcus pseudintermedius, MRSA = methicillin-resistant Staphylococcus aureus
50
Chapter 4
Low-level laser therapy as an adjunctive treatment for canine acral lick dermatitis:
A randomized, double-blinded, sham-controlled study
4.1 Abstract:
Background: Canine acral lick dermatitis (ALD) is a frustrating, chronic disease that can have numerous primary causes including pruritic and painful diseases. Conventional therapy generally consists of systemic antibiotics and anti-anxiety medications. Low- level laser therapy (LLLT) is an alternative therapy that has been used to treat conditions associated with inflammation and pain.
Hypothesis/Objectives: The objective of this study was to determine whether the use of
LLLT combined with conventional therapy would result in a significant decrease in the licking of the ALD lesion than conventional therapy alone. We hypothesized that the combination of LLLT and conventional therapy would result in a >50% reduction in licking visual analog score (LVAS) than conventional therapy alone.
Methods: Dogs were randomly assigned to two groups. The treatment group received
LLLT with blue and red light-emitting diodes (LEDs), while control group had sham therapy (laser off). Treatments were three times weekly for two weeks, then twice weekly for two weeks for a total of 10 visits. All dogs received systemic antibiotics and trazodone. Descriptive statistics were performed (mean, standard deviation).
51 Results: There was an overall decrease in LVAS in both control and treated dogs.
Treatment dogs had a 20% greater decrease in LVAS compared to control dogs.
Conclusions: The use of LLLT as a non-invasive treatment resulted in an additional decrease in licking behavior in dogs with ALD. LLLT should be considered as an adjunctive treatment in the management of dogs with ALD.
4.2 Introduction:
Canine acral lick dermatitis (ALD) is a disease that manifests as excessive, compulsive licking at a focal area on a distal limb resulting in a proliferative, ulcerative and alopecic lesion that has secondary deep infection.14,15 There are numerous primary causes of ALD including allergic disease, hypothyroidism16, orthopedic abnormalities, neuropathies, trauma and psychogenic.17 Some of these causes are considered pruritic
(e.g. allergic disease), which can manifest as licking; however, some are not (e.g. osteoarthritis) and the licking behavior in those diseases may be due to pain. While pain and itch are two distinct sensations, there is crossover between the two with similar neurologic mechanisms and receptors.142,143 It is believed that the licking in ALD is due to both pain and pruritus.18 Chronic licking may result in endorphin release furthering the desire to lick, impeding resolution of the lesion.17 Conventional therapy for ALD includes systemic antibiotics for the deep bacterial infection19 and a systemic behavior-modifying medication (i.e., trazodone, fluoxetine, clomipramine).17,20-23 The deep infection can perpetuate licking and requires a minimum of 4 weeks or more of antibiotic treatment18 with initial improvement of the lesion seen within 4 weeks of starting therapy.144 Non- conventional treatments include surgery,24 radiation,25 acupuncture,27 cryosurgery26 and
52 electronic stimulation,28 which have variable efficacy and numerous side effects; therefore, new safe and effective treatment modalities are needed.
Low-level laser (light) therapy (LLLT), a form of phototherapy, uses either coherent light sources (lasers) or non-coherent light sources consisting of filtered lamps or light- emitting diodes (LEDs) or a combinations of both.1 LLLT is an alternative therapy used to treat a multitude of conditions that require stimulation of healing and relief of inflammation, pruritus and pain.1,29,103,104,106 While the mechanism of action of LLLT is poorly understood, it is thought to be through the absorption of visible red light (620-690 nm) and near-infrared light (720-1260 nm) by mitochondrial chromophores, particularly cytochrome c oxidase, which are located within the mitochondria.1 This leads to production of adenosine triphosphate (ATP),1 which alters factors associated with cell proliferation, survival, tissue repair, and regeneration.2 LLLT devices are categorized into six different classifications (Class I, IA, II, IIIA, IIIB, IV) based on the increasing potential for damage such as eye injuries and thermal injuries.82 Class I lasers have minimal to no side effects reported, unlike Class IV lasers which require eye protection and advanced training to avoid burns.82
Light emitting diodes, or LEDs, produce a narrow spectrum of light in an incoherent manner, where the light is randomly spread out once emitted from a light source.3 LEDs have different depth of penetration based on their wavelength and can affect cellular metabolism by triggering intracellular photobiochemical reactions.3 Blue LED, in the range of 405 to 470-nm wavelength, is bactericidal77 and has been shown to kill methicillin-resistant Staphylococcus aureus both in vitro and in vivo.6-9,78,121-123,126 Red
LEDs have deep tissue penetration and can target dermal structures such as adnexa and 53 fibroblasts.3 Red LEDs have been used for wound healing, to treat photodamaged skin and scars, and for treatment of skin cancers.3,129,130 As ALD lesions are inflammatory,19 painful or pruritic, and commonly secondarily infected,19 the use of both red and blue
LED may be beneficial.
LLLT is a non-invasive, drug-free option that has been evaluated in human medicine for treatment of chronic joint pain,87-90 neuropathic pain,92-94 hair loss,2,83-85 acne,1 psoriasis,1 wound healing,95-100 vitiligo,1 plaque psoriasis,1 and atopic dermatitis.30 In veterinary medicine LLLT has been evaluated for use for treatment of osteoarthritis,29 post-hemilaminectomy surgery,103 wound healing,104 tibial plateau levelling osteotomy
(TPLO) surgery,106 pedal pruritus,31 and non-inflammatory alopecia.105 Certain factors should be considered when using LLLT in animals, such as organic debris and hair, which can affect the depth and penetration of the light.107 Only anecdotal reports exist for use of LLLT for ALD; thus randomized, controlled double-blind studies to evaluate the efficacy of LLLT for the treatment of ALD lesions are needed. As the ALD lesion is direct result of excessive licking from pain and/or pruritus and often infected, LLLT
(delivered by laser and blue and red LED) may be an effective therapy for ALD, regardless of the underlying condition.29-31 The objective of this study was to determine whether the use of LLLT combined with conventional therapy would result in a significant decrease in the licking of the ALD lesion by the dog compared to conventional therapy alone. Our hypothesis was that the combination of LLLT and conventional therapy for canine ALD would result in a >50% reduction in licking compared to conventional therapy alone.
4.3 Materials and Methods: 54 4.3.1 Animals:
The study was approved by the Institutional Animal Care and Use Committee.
Dogs of any gender, breed, and age with a lesion consistent with ALD (firm, plaque-like lesion) were enrolled in the study. The lesion had to have been present for at least three weeks. If an individual dog had more than one ALD lesion, the lesion present the longest was the one evaluated in the study. Dogs had to be on topical or oral flea preventive for the previous two months prior to enrollment or if not on flea preventive were not allowed to commence with preventive until study completion. Dogs had to be on the same diet for the previous two months prior to enrollment. Dogs were allowed to be on the following long-term medications: allergen-specific immunotherapy if used for greater than one year; oral or injectable glucocorticoids, fatty acid supplements, oclacitinib or cyclosporine if used for greater than four months; oral anti-anxiety medications
(clomipramine, trazodone, fluoxetine) if used for greater than four months; oral antihistamines if used for greater than one month. Antimicrobial topical shampoos, sprays, mousses and lotions were permitted if used for greater than one month and not applied directly on the lesion. Dogs were excluded if they had received oral antibiotics within the previous seven days but were subsequently enrolled after a seven-day washout period.
Upon enrollment into the study, all dogs received a physical, neurologic, orthopedic and dermatologic examination. Dogs with abnormal physical examination findings precluding sedation were excluded.
Surface cytology to identify infectious organisms (bacteria, yeast) and inflammatory cells was performed by obtaining impression smears from the ALD 55 lesion.19 Samples were heat-fixed, stained with Diff-Quick® (Baxter Healthcare Co.,
McGraw Park, IL, USA) and evaluated microscopically at 10x (scanning power) and
100x (oil immersion) magnification.19 Numbers of organisms and inflammatory cells were counted from 10 oil immersion fields (OIF) and recorded as rare (one organism every 4 to 5 OIF), occasional (two to three organisms every 4 to 5 OIF), 1+ (10 to 20 organisms/OIF), 2+ (20 to 30 organisms/OIF), 3+ (30 to 40 organisms/OIF), 4+ (greater than 40 organisms/OIF).
Deep skin scrapings were obtained to look for Demodex spp. in the acral lick lesions. Scrapings were performed from the edge of the lesion using a scalpel blade and mineral oil. The material was placed onto a microscope slide with a cover slip and evaluated microscopically at 10x (scanning power).19
4.3.2 Study design:
This study was a randomized, double-blinded, sham-controlled design, with an investigator and owners blinded to treatment. The dogs were randomized into one of two groups (treatment [laser] or control [sham]) using the random function in Microsoft
Excel. All owners completed a Licking Visual Analog Scale (LVAS) (Figure 4) at the initial visit to determine baseline licking behavior. In order to obtain a numeric value for the investigators LVAS, a scale was applied over the owners LVAS response (Figure 5).
LVAS form was completed by the owner at each visit until study end and the scores were recorded by the unblinded investigator. The ALD lesion was measured (area and thickness) by one blinded investigator. The surface area of the lesion was measured by placing plastic kitchen wrap (Press ‘n Seal®; The Glad Products Company, Oakland, CA) on top of the lesion and tracing the edges of the lesion using a felt-tip marker.15 The 56 thickness was measured in millimeters using a ruler. The unblinded investigator calculated the surface area in cm2 of the lesion using an image analysis software program
(Image J®, v1.50i, National Institutes of Health, USA) by outlining the border of the lesion (referred to as overall lesion size) and individual ulcers contained within the boundary of the lesion. The sum of the surface areas for each of the individual ulcers within the boundary of the lesion was then added together to obtain the total calculated surface area of all ulcers in the ALD lesion (referred to as composite ulcer size).
The dogs were sedated for the following procedures using dexmedetomidine (5 to
7 mcg/kg) IV and butorphanol (0.2 mg/kg) IV. Two-view radiographs of the affected limb with the ALD lesion were performed for subjective evaluation of underlying orthopedic disease by a radiologist. Dogs with orthopedic conditions were not excluded from the study, as LLLT has been shown to be an effective treatment for osteoarthritis in dogs.29 Hair was collected across the entire lesion using the Mackenzie toothbrush technique.145 Two 6-mm skin punch biopsies were obtained from a non-ulcerated area of the ALD lesion. One biopsy sample was submitted for histopathological confirmation of
ALD and the other was submitted for a bacterial and dermatophyte tissue culture. The hairs from the toothbrush sample and tissue sample were plated together for the dermatophyte culture. The site for bacterial and dermatophyte culture collection was clipped and prepped with chlorhexidine scrub and sterile saline to remove surface contaminants to obtain skin punch biopsy samples. The sample was bisected at the level of the epidermis with a scalpel blade; the epidermis was submitted for dermatophyte culture and the other half (dermis) was submitted for bacterial culture both in separate sterile containers. The tissue samples were transported to the Clinical Microbiology 57 Laboratory at The Ohio State University College of Veterinary Medicine. Each tissue was separately macerated in a sterile 1.5mL polypropylene tube using a sterile disposable pestle. For aerobic culture, the macerated tissue was inoculated onto Columbia agar containing 5% sheep blood, MacConkey agar and CNA (colistin, nalidixic acid) agar supplemented with 5% sheep blood (Becton, Dickinson and Company; Franklin Lakes,
NJ, USA) and incubated overnight at 35°C in ambient air. For the dermatophyte culture, the macerated tissue was inoculated onto Sabouraud’s dextrose agar and incubated at room temperature for 30 days. Identification of bacterial isolates was performed via
Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (Bruker; Billerica, MA, USA) and antimicrobial susceptibilities were determined via broth microdilution (Sensititre COMPGP1F, Thermo Fischer Scientific;
Waltham, MA, USA). Staphylococci that exhibited resistance to oxacillin (MIC ≥ 0.5
µg/ml) were considered methicillin resistant based on the CLSI interpretive standard (Vet
01-S3).146
The biopsy sample for histopathology was preserved in 10% formalin and routinely processed, sectioned and stained with hematoxylin and eosin (H&E)19 and subjectively evaluated by a board-certified veterinary pathologist. A 3-0 Maxon simple interrupted cruciate suture was placed at the biopsy sites. Sedation was reversed with atipamezole IM. Dogs diagnosed with dermatophytosis, demodicosis or neoplasia were excluded at the time of diagnosis. All dogs (treatment and control) received conventional therapy consisting of oral antibiotics (based on culture results, cephalexin if no growth) and trazodone (4 to 6 mg/kg PO q12h) for the study duration. Trazodone was started by the owners that evening after recovery from sedation. Once the ALD diagnosis was 58 confirmed and the result of the bacterial culture was obtained, the dogs started oral antibiotic therapy. At each recheck visit, the owners reported any side effects of the medications to the unblinded investigator.
4.3.3 Laser treatment:
Laser treatment (or sham) commenced upon recovery of sedation and subsequently occurred on three non-consecutive days per week for the first two weeks, then two non-consecutive days per week for the final two weeks for a total of 10 treatments. Laser treatment was conducted with a cordless super pulsed Class I laser device (MR4 ACTIVet PRO™ device, Multi Radiance Medical®; Solon, OH, USA) on
Setting 1 (50 Hz) in conjunction with blue LED (470 nm) and red LED (640 nm) for two minutes on the periphery of the lesion at an approximate rate of 1cm/s. The total average power was 130mW with a dose of 15.6 J and fluence of 3.93J/cm2, aperture 4 cm2, laser spot 0.4422cm2 and LED spot 0.9cm2. The laser treatment protocol and settings were from the manufacturer’s recommendations.147 Control dogs received sham treatment where the laser was off with no light. Prior to laser treatment (or sham) of the lesion all dogs received an unwind protocol to acclimate animals to treatment, as recommended by the manufacturer.147 This involved using the device on Setting 1 (50 Hz) with red and blue light off and scanning slowly from the base of the skull to tail, up and down the spine for 5 minutes.147 The device was turned off for the unwind protocol in the sham group. Laser treatment was performed by the unblinded investigator. At each recheck before treatment, the lesion under investigation was measured by the blinded investigator and documented with digital images by the unblinded investigator.
59 Data Analysis:
Descriptive statistics (mean, standard deviation [SD]) were used to summarize the data for the treatment (laser) group compared to the control (sham laser) for lesion size, composite ulcer size, LVAS and skin thickness using Microsoft Excel (version 14.7.2).
4.4 Results:
4.4.1 Signalment and history:
Nine dogs were evaluated, met the inclusion criteria and were enrolled in the study; five dogs were in the control group (CG) and four dogs were in the treatment group (TG). The mean age of onset was estimated to be approximately 5 years old (range
1 to 9 years) while the mean age at enrollment was 6.9 years (range 2 to 11 years). Three spayed females and six neutered male dogs were enrolled. There were two German shepherd dogs, two great danes, two mixed breed dogs (one German shepherd mix and one Labrador retriever mix) and one each of the following breeds: old English sheepdog,
Doberman pinscher and English springer spaniel.
Five of nine dogs (56%) had a single ALD lesion; two dogs had three lesions in the same location and two dogs had two lesions in different locations. Seven of nine dogs
(78%) had ALD lesions on the front limbs while two dogs (22%) had ALD lesions on the hindlimbs. The most common location of included ALD lesions was the right carpus
(n=3), followed by left dorsal metatarsus (2), left carpus (1), right front mid- antebrachium (1), left front distal antebrachium (1), and right front paw medial metacarpal bone II (1) (Table 4).
Dog 6 was reported to have had a left front distal limb fracture at approximately 7 months of age that was repaired surgically using screws. When he began licking this 60 distal limb at 5 years of age, surgery was performed to remove the screws. This did not resolve the ALD lesion. No other dogs had a history of trauma or fractures.
The length of time the included ALD lesions were present ranged from three months to over two years. Four of nine dogs had non-seasonal ALD lesions, in two dogs, the ALD lesions were seasonal (one in summer, one in spring and summer) and in the remaining 3 dogs the lesions had been present less than one year. Eight of nine dogs
(89%) had no other dermatologic signs, while one dog also had pedal pruritus. Four of nine dogs (44%) were on concurrent medications other than flea and heartworm preventives (Table 4).
4.4.2 Diagnostic tests:
4.4.2.1 Deep skin scrapings and surface cytology:
All ALD lesions were negative for demodicosis. The findings on skin cytology from eight ALD lesions included bacteria [cocci (n=8)]; nuclear streaming (5), and inflammation [suppurative (5), pyogranulomatous (1), eosinophilic (1)]. Skin cytology from one ALD lesion was negative for inflammatory cells and organisms.
4.4.2.2 Deep bacterial tissue culture and dermatophyte tissue/hair culture:
Eight of nine (89%) ALD lesions were positive for bacterial growth. All eight were positive for Staphylococcus pseudintermedius (two were methicillin-resistant), and one lesion each also grew Streptococcus canis and Streptococcus dysgalactiae.
Antimicrobial susceptibilities for the Staphylococcus pseudintermedius isolates are listed in Table 5. All isolates (100%) whether methicillin-susceptible Staphylococcus pseudintermedius (MSSP) or methicillin-resistant Staphylococcus pseudintermedius
(MRSP) were susceptible to amikacin and gentamicin. As anticipated, none of the 61 isolates were susceptible to ampicillin or penicillin. All the MSSP isolates were also susceptible to the remaining antimicrobial agents: amoxicillin/clavulanic acid, cefazolin, cefpodoxime, chloramphenicol, clindamycin, doxycycline, erythromycin, enrofloxacin, marbofloxacin, oxacillin and trimethoprim/sulfamethoxazole. For the MRSP isolates, in addition to being susceptible to amikacin and gentamicin, one was also susceptible to chloramphenicol, clindamycin, doxycycline, enrofloxacin, erythromycin, and marbofloxacin while the second MRSP isolate was multi-drug resistant as it was only susceptible to amikacin and gentamicin.148 Based on the bacterial susceptibility results, six of eight dogs were prescribed cephalexin (Rilexine®; Virbac Animal Health, Inc.,
Forth Worth, TX), one dog clindamycin and one dog parenteral (subcutaneous) amikacin.
The one dog with a negative culture from the ALD lesion was prescribed cephalexin.
Dermatophyte cultures were negative for all ALD lesions.
4.4.2.3. Histopathology:
The histopathological findings are summarized in Table 6. Epidermal ulceration was identified in four of nine cases (44.4%). There was epidermal hyperkeratosis in seven of nine cases (78%), which was orthokeratotic in six and parakeratotic in one. The most common follicular changes were follicular hyperplasia (n=9; 100%), infundibular hyperkeratosis (8; 89%), follicular elongation (8; 89%), folliculitis (5; 56%) and follicular dilatation (6; 67%). Abnormalities of the adnexa mainly affected both the sebaceous and epitrichial glands. Fibrosis was superficial in six of nine cases and deep in four cases. A dermal inflammatory infiltrate was present in all nine cases, one with eosinophilic inflammation. Pigmentary incontinence was observed in one case. Cocci
62 were identified within the superficial keratin in three cases, and additionally within follicles in two. No fungal elements were identified histopathologically on H&E.
4.4.2.4 Radiography:
Radiographic examination of the tissues underlying the ALD lesion revealed focal soft tissue thickening in all dogs (9/9). Other radiographic findings included osteophytes
(n=3), periosteal proliferation (n=2) and enthesophytes (n=4). Dog 6 had linear lucencies within the mid to distal radius and a focal angular mineral proliferation along the caudal aspect of the mid ulnar diaphysis. There were also multiple small metallic hemoclips within the SC tissues dorsal to the radius.
4.4.3 Side effects:
Two of nine dogs had reported side effects due to trazodone and/or antibiotics.
Two dogs were excessively sedate on trazodone, (Dog 1 and 7) which resolved with a decrease in dose. Dog 1 also had loose stool on cephalexin that resolved without treatment.
4.4.4 LVAS:
The LVAS for all dogs for all visits are listed in Table 7. The mean LVAS ± SD at the start and end of the study for the dogs in the control group was 6.9 ±1.0 and 4.3 ±
2.2, respectively and for the dogs in the treatment group was 5.5 ± 0.3 and 2.3 ± 1.9, respectively which equated to a 38% decrease in LVAS in the control group and a 57% decrease in LVAS in the treatment group (Figure 6).
4.4.5 Overall Lesion size:
The overall size of the included ALD lesion for all dogs at all visits are listed in
Table 8. The mean lesion sizes ± SD at the start and end of the study for dogs in the 63 control group were 10.9 cm2 ± 16.3 and 10 cm2 ± 16.9, respectively. The mean lesion sizes for the dogs in the treatment group at the start and the end of the study were 17.6 cm2 ± 18 and 17.4 cm2 ± 26.5, respectively. This equated to an 8.3% decrease in overall lesion size in the control group and 1% decrease in overall lesion size in the treatment group (Figures 7 and 8). After visual inspection of the raw data in Table 8, lesion size decreased over time in all dogs in both groups with the exception of Dog 5. This dog’s lesion size decreased from visit 1 to 5, but then increased from visit 6 to the end of the study. When the data from dog 5 was excluded from the treatment group the mean lesion size ± SD at the start and end of the study for the remaining 3 dogs in the treatment group was 8.7 cm2 ± 4.6 and 4.1cm2 ± 1.6, respectively which equated to a 52.6% decrease in overall lesion size in the treatment group (Figure 9).
4.4.6 Lesion thickness:
The mean lesion thickness ± SD at the start and end of the study for dogs in the control group was 7.6mm ± 3mm and 6.4mm ± 2.9mm, respectively and for the treatment group was 8.5mm ± 4.2 and 7mm ± 4.1mm, respectively. This equated to a 15.8% decrease in thickness in the control group and 17.6% decrease in the treatment group.
4.4.7 Composite ulcer size:
The composite ulcer size of the ALD lesions for all dogs at all visits are listed in
Table 9. The mean composite ulcer size ± SD of the lesions at the start and end of the study for dogs in the control group was 8.8 cm2 ± 14.4 and 8.1cm2 ± 14.5, respectively and for the dogs in the treatment group was 11.5 cm2 ± 17.7 and 13.2 cm2 ± SD 23.9, respectively which equated to a 7.4% decrease in lesion size in the control group and
14% increase in lesion size in the treatment group (Figure 10). After visual inspection of 64 the raw data in Table 9, composite ulcer size decreased over time in all dogs in both groups with the exception of Dog 5. This dog’s lesion size decreased from visit 1 to 5, but then increased from visit 6 to the end of the study. When the data from Dog 5 was excluded from the treatment group, the mean composite ulcer size for the remaining three dogs in the treatment group at the start and end of the study was 2.8 cm2 ± SD 2.9 and 1.2 cm2 ± SD 1.3, respectively which equated to a 55.4% decrease in overall lesion size in the treatment group (Figure 11).
4.4 Discussion:
We hypothesized that the combination of LLLT, systemic antibiotics and trazodone (treatment group) would result in greater than 50% reduction in licking of
ALD lesions than systemic antibiotics and trazodone (control group) alone. There was a decrease in LVAS in both the treatment group and the control group; however, the decrease was only 20% greater in those dogs in the treatment group compared to the control dogs. At most subsequent visits, regardless of group, there was a steady decrease in mean LVAS score from the first to the last visit (Figure 6). Therefore, treatment of deep infection and controlling any psychogenic components (whether primary or learned behavior) is beneficial to decrease licking of the ALD lesion. The adjunctive use of
LLLT, as a non-invasive, drug-free treatment, resulted in an additional decrease in the licking behavior.
In this study, both overall lesion size and individual ulcers were measured to document clinical improvement as the use of LLLT with red and blue LED are reported to stimulate healing, relieve inflammation, infection, pruritus and pain; and improve wound healing.1,3 Overall lesion size decreased in both groups; however, there was a 65 greater decrease in size in the control group compared to the treatment group.
Unexpectedly, while composite ulcer size decreased in the control group it increased in the treatment group. After closer inspection of the raw data for both overall lesion size and composite ulcer size, the values for dog 5 increased after visits 5,so the data were excluded. Lesion and composite ulcer size increased from visit 6 to visit 10 in dog 5, which may be due to a decrease in treatment frequency from three times weekly to twice weekly after visit 6. Excluding the data from dog 5 resulted in the treatment group having a 52.6% decrease in overall lesion size compared to an 8.3% decrease in the control group and a 55.4% decrease in composite ulcer size compared to a 7.4% decrease in the control group. Excluding this dog, the use of LLLT in combination with antibiotics and trazodone (conventional therapy) resulted in greater than 50% decrease in overall lesion size and composite ulcer size compared conventional therapy alone. Lesion thickness has not been reported in previous studies. In this study, the decrease in thickness was only about 2% more in the treatment group than control group.
The mean age of onset in this study was approximately 5 years old, which was similar to previous reports.14,19 One study of 63 dogs with ALD identified a bimodal distribution of age of onset of licking, with peaks at 1 and 6 years of age.22 Our study had too small a sample size to report any bimodal distributions. More male neutered dogs than female spayed dogs enrolled, which is in agreement with two previous studies.20,42
Both these studies had a low number of dogs enrolled, as did we in our study. However, another study with 31 dogs found a trend for female dogs to be affected with ALD, although this finding was not significant.19
66 All the dogs were large breeds or large mixed breeds, in agreement with previous reports.14,15,19,20,40,42,79,80 German shepherd dogs (GSD) (including a GSD mix) and great danes represented the majority of dogs in our study and are commonly reported breeds with ALD.19,20,40,42,79,80 Doberman pinschers have been reported to be predisposed to
ALD lesions,19,20,40,80 ; however, only one was enrolled in our study.
The majority of dogs had a single ALD lesion (56%) located on the forelimb
(78%), which is in accordance with previous studies.14,19,20,42 Interestingly, only one dog in this study had other skin issues (pedal pruritus) in addition to the lick granuloma.
Previous studies do not report concurrent skin disease in the dogs with ALD lesions.19,20,42,79,80 It is possible that in general dogs with ALD do not have other skin disease aside from the lick granuloma itself; however it may be that this information was not obtained in the history or reported in the studies.19,20,42,79,80
A previous study found surface cultures predicting deep tissue isolates in only eight of 22 (36%) ALD lesions.19 Thus, deep tissue cultures are necessary for identification of the bacterial organisms and selection of antimicrobials to treat ALD lesions. The most common bacterial organism obtained from a deep tissue biopsy from the ALD lesions in our study was S. pseudintermedius. Two (25%) of these staphylococcal organisms were methicillin-resistant. This is in agreement with a previous study where the most common bacterial isolate from deep cultures was S.
(pseud)intermedius; 26% were methicillin-resistant.19 Additional organisms that have been cultured from ALD lesions include Staphylococcus aureus, Staphylococcus xylosus,
Staphylococcus epidermidis, Pseudomonas spp., Proteus mirabilis, Enterobacter spp,
Acinetobacter baumanii, Streptococcus spp. and E.coli.19,20 In our study, the only other 67 bacterial organisms isolated were Streptococcus canis and Streptococcus dysgalactiae; both in conjunction with S. pseudintermedius. No gram-negative organisms were isolated. One of nine ALD lesions (11%) had no bacterial growth on tissue culture, which is similar to other studies in which 3 to 17% of deep tissue cultures from ALD lesions were negative for bacterial organisms.19,20
In our study, while 25% of the S. pseudintermedius infections were methicillin- resistant the majority of the staphylococcal isolates were susceptible to the most common antibiotics used in veterinary medicine. Seventy-five percent or greater were susceptible to routine drug classes used to treat staphylococcal infections including amoxicillin- clavulanic acid, cephalosporins, clindamycin, doxycycline, fluoroquinolones and trimethoprim/sulfamethoxazole. In a previous study, 39% of deep tissue Staphylococcal isolates were resistant to multiple antibiotic drug classes and 92.3% of deep
Staphylococcal isolates were susceptible to enrofloxacin.19 Our results paralleled these findings with 88% of samples susceptible to enrofloxacin.
Histopathological features previously described for ALD include acanthosis with occasional serocellular crusts; ulceration; orthokeratotic to parakeratotic hyperkeratosis; superficial and deep dermal fibrosis with vertical streaking; thickened and elongated follicles; perivascular, perifollicular or diffuse dermal infiltrate of lymphocytes, neutrophils, macrophages and plasma cells; plasmacytic perihidradenitis; folliculitis and furunculosis; epitrichial gland hypertrophy, dilatation and retained secretions, hidradenitis and glandular rupture; and sebaceous gland hyperplasia.19,20,40,42,149 Follicular elongation, plasmacytic periadnexal inflammation and vertical streaking are considered distinctive for ALD.40 Our results are in agreement, however additional features were 68 identified that have not been previously reported such as pigmentary incontinence and eosinophilic dermatitis. The presence of cocci were also noted. Cocci were reported in histopathological samples in 16% of samples in a previous study.19 Pigmentary incontinence is often regarded as a sign of immune-mediated damage to the epidermal melanin unit, but may also be associated with physical injury or trauma to the epidermis.150,151 It is likely that the trauma and inflammation from licking the ALD lesion was responsible for pigmentary incontinence.40,151
Common radiographic findings in dogs with ALD include soft tissue swelling and periosteal reactions.14,20,42 Similarly, all dogs in the present study had soft tissue swelling with the majority having periosteal proliferation, enthesophytes and osteophytes. In addition, dog 6 had numerous lucencies and hemoclips present on radiographs due to a history of previous fracture repair when the dog was a puppy. This history of trauma was possibly the initiating cause for this dog’s ALD lesion.
Anti-anxiety medications such as clomipramine,21,65,71 fluoxetine,21,22,73 citalopram,74 naltrexone,20 and nalfemene76 have been evaluated as sole treatment of canine ALD. Fluoxetine has been shown to decrease licking behavior by up to 50% of baseline as well as significantly improve lesion appearance.21,22,73 Significant improvement in owner and veterinarian-assessed clinical global impression and lesion severity of ALD was reported in dogs treated with citalopram.74 Clomipramine was reported to reduce licking by 43% and dogs receiving clomipramine were four times more likely to be categorized as improved by owners when compared to the placebo group.21,65 Treatment with naltrexone improved ALD lesions in seven of nine dogs which all relapsed when naltrexone treatment stopped.20 Nalmefene was shown to be effective 69 in preventing or reducing self-licking or chewing when administered subcutaneously in four of five dogs.76 To the author’s knowledge, this is the first report of trazodone being used as an adjunctive treatment for canine ALD. Trazodone is a serotonin antagonist and reuptake inhibitor that has been used previously to treat behavioral disorders and reduce stress-related signs and behaviors in dogs.23 Trazodone has minimal side effects, being sedation, and has been reported to take effect within 31 to 45 minutes.23 Other commonly used anti-anxiety medications, like fluoxetine and clomipramine can take four to six weeks for full efficacy.152 The chronic licking of ALD may serve to increase serotonin neuronal activity,14 thus making trazodone a useful treatment option. In our study, when trazodone was combined with an oral antibiotic, the licking decreased, and overall lesion size and composite ulcer size decreased.
The main limitation of this study was the small sample size. Nine dogs were enrolled, which only allowed for four dogs to be in the treatment group and five in the control group. The small sample size prevented performing statistical analysis. The small number of dogs in each group may be the reason for the minimal changes in LVAS, lesion size and ulcer size. The other limitation is the selection of dose of the laser.
Defining the dose for LLLT is either anecdotal or based on manufacturers recommendations.153 Furthermore, the lack of reporting by authors of critical parameters of laser devices makes the assessment and choice of appropriate dose difficult.153 While there are previous studies using LLLT devices in veterinary medicine,29,31,103-106 the different classes of devices used make comparison of dose difficult. There are no previous references for dosing of LLLT for ALD in dogs. Future studies should evaluate different LLLT dosing protocols for ALD. 70 It is important to treat the deep infection in the ALD lesion with systemic antibiotics and the psychogenic component with trazodone; the addition of LLLT can further decrease the dog’s licking of the lesion and decrease the size of the lesion. The most commonly isolated bacterial organism from these lesions is S. pseudintermedius. As there is a one in four chance that the infection will be methicillin-resistant, and if a deep tissue culture is not possible, enrofloxacin would be an appropriate option.
71 Table 4. Historical information for all dogs with acral lick dermatitis
Dog Group Number Location of Length of time Concurrent Concurrent of ALD ALD ALD lesion skin/ear medications lesions lesion(s) present disease 1 TG 1 Left dorsal Seasonal in None Glucosamine, metatarsus summer for 2+ fipronil/(S)- years methoprene
2 TG 3 Right carpus Non-seasonal for Yes, pedal Oclacitinib, 2+ years pruritus selamectin
3 CG 1 Right carpus 3 months None Trazodone prn for anxiety, imidacloprid/ pyriproxyfen 4 CG 1 Left carpus 6 months None Fipronil/(S)- methoprene 5 TG 1 Right front Non-seasonal 1.5 None None mid to 2 years antebrachium 6 CG 3 Left front Non-seasonal for None None distal 1+ years antebrachium 7 TG 1 Right front Non-seasonal for None Amantadine, paw 1 year gabapentin, traumeel, imidacloprid/ permethrin/ pyriproxyfen/ 8 CG 2 Left dorsal 9 months None Spinosad/ metatarsus; milbemycin oxime right distal dorsal metatarsus 9 CG 2 Right Seasonal (spring, None Selamectin carpus; left summer) for 2+ carpus years CG=control group, TG=treatment group, ALD= acral lick dermatitis, ALD lesion included in the study are bolded and underlined when multiple ALD lesions were present in different locations.
72 Table 5: Antimicrobial susceptibility test results of Staphylococcal isolates from deep tissue ALD lesions in eight dogs
Number of susceptible (%) Staphylococus pseudintermedius Antibiotic isolates Amikacin 8 (100%) Amoxicillin/clavulanic acid 6 (75%) Ampicillin 0 (0%) Cefazolin 6 (75%) Cefpodoxime 6 (75%) Chloramphenicol 7 (88%) Clindamycin 7 (88%) Doxycycline 7 (88%) Enrofloxacin 7 (88%) Erythromycin 7 (88%) Gentamicin 8 (100%) Marbofloxacin 7 (88%) Oxacillin 6 (75%) Penicillin 0 (0%) Trimethoprim/sulfamethoxazole 6 (75%)
73 Table 6. Summary of histopathologic findings in nine cases of acral lick dermatitis
Totals Epidermis Complete ulceration 4 (44.4%) Regular acanthosis 1 (11%) Irregular acanthosis 7 (78%) Orthokeratotic 6 (67%) Parakeratotic 1 (11%) Hyperkeratosis 7 (78%) Crust 2 (22%) Hypergranulosis 7 (78%) Spongiosis 0 Cocci (keratin) 3 (33%) Follicles Infundibular hyperkeratosis 8 (89%) Follicular hyperplasia 9 (100%) Follicular elongation 8 (89%) Follicular dilatation 6 (67%) Folliculitis 5 (56%) Furunculosis 1 (11%) Cocci 2 (22%) Adnexa Sebaceous gland hyperplasia 6 (67%) Epitrichial gland hypertrophy 9 (100%) Epitrichial gland dilatation 7 (78%) Epitrichial gland inspissated 3 (33%) secretions Epitrichial gland rupture 2 (22%) Hidradenitis 2 (22%) Dermis Superficial fibrosis 6 (67%) Vertical streaking 6 (67%) Deep fibrosis 4 (44%) Pigmentary incontinence 1 (11%) Dermal infiltrate Superficial LPC perivascular 9 (100%) Superficial neutrophilic 7 (78%) perivascular Middermal LPC 9 (100%) Middermal pyogranulomatous 2 (22%) Deep LPC 7 (78%) Continued
74 Table 6: Continued.
Perihidradenitis 9 (100%) Perifolliculitis 4 (44%) Eosinophils 2 (22%)
Histopathological findings not previously reported in acral lick dermatitis lesions are bolded. LPC = lymphoplasmacytic
75 Table 7. Licking visual analog scale (LVAS) of the acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser) groups over 10 visits. V1 is baseline in which treatment or sham was started. V1 through V6 occurred three times weekly for two weeks and V7 to V10 occurred two times weekly for two weeks.
Dog Group V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 3 CG 6.5 5.7 6.6 5.9 3.6 4.2 5.7 4.6 1.2 2.2 4 CG 7.4 5.3 7.6 7.4 7.5 5.5 3.4 7.5 5.5 7.7 6 CG 8.3 7.0 7.0 7.0 5.6 4.9 5.8 5.0 5.3 5.1 8 CG 5.8 4.4 5.6 5.1 4.2 4.2 4.2 3.5 3.4 3.5 9 CG 6.6 6.4 7.1 6.5 7.5 6.4 6.4 5.9 6.1 2.8 1 TG 5.1 5.3 5.2 3.5 2.7 2.2 2.4 2.3 1.2 1.4 2 TG 5.5 1.8 1.7 1.4 1.0 0.3 1.7 0.9 0.1 0.1 5 TG 5.8 5.8 6.7 5.9 5.9 5.4 4.9 4.3 4.8 3.9 7 TG 5.4 4.5 4.7 4.4 4.5 4.6 4.1 4.0 4.1 3.9 V= visit, CG=control group (sham laser), TG=treatment group (laser).
76 Table 8. Lesion size (cm2) of acral lick dermatitis for all dogs in control (sham laser) and treatment (laser) groups over 10 visits.
Dog Group V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 3 CG 2.4 2.4 2.0 0.8 0.5 0.6 0.6 0.6 0.6 0.6 4 CG 0.9 1.9 2.2 2.1 1.8 1.8 1.6 1.5 0.9 1.0 6 CG 39.6 43.4 39.9 42.9 41.0 35.4 40.0 39.1 38.9 39.9 8 CG 3.3 4.0 4.2 3.8 2.7 2.5 2.5 2.1 2.1 2.1 9 CG 8.3 10.0 9.8 9.5 9.2 8.4 8.2 8.0 7.4 6.4 1 TG 13.9 12.1 12.8 11.6 11.7 13.8 13.1 11.7 9.5 6.0 2 TG 7.2 6.8 6.4 5.6 4.4 4.7 4.4 3.1 3.7 3.1 5 TG 44.1 43.5 40.5 44.1 31.3 43.1 49.9 50.2 50.4 57.1 7 TG 5.1 5.0 4.7 4.5 4.5 4.1 4.1 3.2 3.1 3.4 V=visit, CG=control group (sham laser), TG=treatment group (laser)
77 Table 9: Composite ulcer size (cm2) of acral lick dermatitis lesions for all dogs in control
(sham laser) and treatment (laser) groups over 10 visits.
Dog Group V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 3 CG 0.4 0.7 0.6 0.4 0.2 0.2 0.1 0.1 0.2 0.1 4 CG 0.2 0.3 0.8 0.7 0.3 0.3 0.3 0.2 0.2 0.2 6 CG 33.9 36.8 31.2 35.0 28.9 28.9 34.3 27.4 33.8 33.7 8 CG 1.4 2.5 1.6 1.4 1.4 1.5 0.9 0.8 0.3 0.8 9 CG 8.1 9.9 9.6 9.3 9.2 8.3 8.0 7.5 7.1 5.8 1 TG 1.3 1.4 1.4 1.7 1.6 1.1 1.1 0.4 0.4 0.5 2 TG 6.1 5.9 5.7 4.8 4.0 4.1 3.8 3.1 3.3 2.7 5 TG 37.8 35.5 38.8 33.9 25.9 31.4 40.2 41.6 41.8 49.0 7 TG 0.9 1.3 1.4 1.3 1.1 1.0 0.7 0.5 0.5 0.5 V=visit, CG=control group (sham laser), TG=treatment group (laser).
78
79
80 Figure 6. Mean Licking Visual Analog Scale for dogs in control (laser sham) and treatment (laser) group.
CG=control group, TG=treatment group. LVAS = licking visual analog scale. The numbers on the horizontal axis represent each visit.
81 Figure 7:
Examples of acral lick dermatitis lesions in dog 4 in the control group (sham laser) at enrollment (a) and study completion (b) and dog 2 in the treatment group (laser) at enrollment (c) and study completion (d). Red lines represent outline of overall lesion, yellow lines represent outline of ulcers.
82 Figure 8: Mean lesion size (cm2) of acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser).
CG=control group, TG=treatment group. The mean lesion size on the vertical axis is measured in cm2. The numbers on the horizontal axis represent each visit.
83 Figure 9: Mean lesion size (cm2) of acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser) group with dog 5 data removed.
CG=control group, TG=treatment group. The mean lesion size on the vertical axis is measured in cm2. The numbers on the horizontal axis represent each visit.
84 Figure 10: Mean composite ulcer size (cm2) of acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser).
CG=control group, TG=treatment group. The mean composite ulcer size on the vertical axis is measured in cm2. The numbers on the horizontal axis represent each visit.
85 Figure 11: Mean composite ulcer size (cm2) of acral lick dermatitis lesions for dogs in control (laser sham) and treatment (laser) group with dog 5 data removed.
CG=control group, TG=treatment group. The mean composite ulcer score on the vertical axis is measured in cm2. The numbers on the horizontal axis represent each visit.
86
Chapter 5
Conclusions and Future Directions
Canine ALD is a frustrating disease that is difficult to treat. There are no recent studies evaluating new treatment options for this disease. LLLT has been used to treat
ALD in dogs, but response has been anecdotal and there are no published prospective clinical trials. In veterinary medicine, LLLT has been evaluated for treatment of pedal pruritus,31 post-operative hemilaminectomy pain,103 wound healing,104 non-inflammatory alopecia,105 and pre-operative for TPLO surgery.106 These studies use different devices, protocol and dosing, making comparison difficult. Ours is the first prospective, double- blinded, sham-controlled study using LLLT in dogs with ALD. The results of our preliminary work indicate that LLLT along with systemic antibiotics and trazodone can be used as an adjunctive treatment for canine ALD. It is important to note that dogs in both control and treatment groups improved over the course of the study, emphasizing the importance of treating the underlying secondary infection appropriately and using anti- anxiety medications. Although the number of dogs was too low to evaluate statistically, the dogs in the treatment group that received the LLLT had a greater percentage of improvement in LVAS, overall lesion size and composite ulcer size compared to the dogs in the control group. A future study with a larger number of dogs is needed to assess if
LLLT significantly improves LVAS, lesion size and ulcer size in dogs with ALD over conventional therapy alone. 87 In all dogs, whether in the treatment or control group, LVAS decreased after visit
1. However, in two dogs (dogs 1 and 2 in the treatment group) there was a transient increase in LVAS after visit 6, which then decreased at visit 8 through to the end of the study, visit 10. This increase in LVAS may have been due to the change in frequency of treatments, which decreased from three times weekly to twice weekly after visit 6. A similar trend was seen in dog 5 in the treatment group, however, both lesion and ulcer size increased after visit 6 and continued to increase until the end of study. In this dog the lesion and ulcer size were larger at the end of the study than at baseline. Since some dogs in the treatment group had a transient increase in LVAS and a continued increase in lesion and ulcer size corresponding to a decrease in frequency of LLLT treatments, future studies should consider continuing three-times weekly treatments for more than 2 weeks before decreasing frequency as well as lengthening the overall duration of the study.
While the setting of the laser used in this study was based on manufacturer recommendation, it is uncertain if choosing a different setting would be more effective.
Setting 1 (50Hz) has anti-inflammatory effects and deep penetration and is used for controlling inflammation, infection, and initiation of healing.147 As ALD lesions are inflamed and usually infected, this setting combined with the anti-inflammatory and antibacterial properties of red and blue LED is likely the appropriate setting for treatment of canine ALD.
This is the first study to report the use of trazodone for treatment of canine ALD.
Since trazodone has a quick onset of action with changes in behavior noted within 31-45 minutes,23 it has advantages over other behavioral medications such as fluoxetine and clomipramine, which can take four to six weeks for full efficacy.152 Trazodone also had 88 minimal side effects, with only 2/9 dogs experiencing sedation that resolved with a decrease in dose. These properties and lack of detrimental side effects makes trazodone an attractive option for adjunctive treatment of canine ALD. Based on the results of our study, trazodone, together with systemic antibiotics, is beneficial as adjunctive therapy for treatment of canine ALD.
An alternate avenue of research would be to evaluate the efficacy of newer anti- pruritic medications, such as oclacitinib and lokivetmab for treatment of ALD. As these medications are involved with the itch-nerve pathway,55,63 they may be effective in reducing licking in dogs with ALD. To date, there has only been one published abstract in which the administration of oclacitinib for treatment of ALD resulted in complete remission of the lesion in a single dog after 4 months of treatment.62 Prospective, randomized, double-blinded, placebo-controlled studies should be designed for evaluation of oclacitinib and lokivetmab in decreasing licking behaviors in dogs with
ALD.
Our in vitro study using blue LED confirmed the efficacy of blue LED against
MRSA.6-9,78,119,121 Percent reduction of MRSA in our study reached 100% at 112.5 and
225 J/cm2, and was 93.3% at the lowest dose of 56.25 J/cm2. Unfortunately, these results were not replicated when blue LED was used with MSSP and MRSP. There was minimal reduction of these organisms with blue LED with the highest reduction of only 21.2% with MRSP at the highest dose of 225 J/cm2. Lower doses produced no higher than 10% reduction in colony counts for MSSP and MRSP. There was also no difference in percentage kill of MSSP compared to MRSP. While MRSA infections in veterinary medicine are not common, blue LED may be effective for use in these infections 89 clinically. If used clinically, treatment time needs to be considered as 100% CC reduction was achieved at the 112.5 J/cm2 dose which equates to a 30-minute exposure time. This would be an extended amount of time to hold the device on an area in an unsedated animal. This therapy would be unreasonable for clinical use if multiple areas are affected on an animal or if the infections are generalized, but may be useful for treatment of localized infections. As 93% of the MRSA colonies were killed at the lowest dose or 15- minute exposure time, blue LED may be useful as adjunctive therapy for treatment of these infections.
Intracellular porphyrins are a key factor in the absorption of blue light.122 It is likely that differences in amounts of endogenous porphyrins between MSSP, MRSP and
MRSA are the reason for differences in percentage kill among these bacterial isolates.
Currently there are no published studies measuring porphyrin in S. pseudintermedius. To determine porphyrin concentration, the optical density would need to be determined from nutrient broth of S. pseudintermedius. The porphyrins would then be extracted and fluorescence measured using a spectrometer, as described in previous studies of S. aureus.124 Future studies using this methodology could be performed to measure and then compare concentrations of porphyrins in S. aureus to S. pseudintermedius. It is hypothesized that S. pseudintermedius would have lower concentrations of porphyrins compared to S. aureus based on our previous in vitro results using blue LED.
Future research to evaluate the efficacy of blue LED in killing MSSP or MRSP in vitro would be the use of photodynamic therapy to increase production of endogenous porphyrins. An in vitro study using a photosensitizer such as 5-ALA may increase the porphyrin concentration of MSSP and MRSP, thus increasing the amount of kill using 90 blue LED. 5-ALA in a topical formulation would be added to S. pseudintermedius bacterial isolate suspensions and plated on 35-mm plates. Treatment with blue LED would be at similar doses to our previous in vitro study (56.25, 112.5, 225 J/cm2) to evaluate if the addition of 5-ALA produced a significant percent reduction in colony counts for MSSP and MRSP. If treatment was successful using 5-ALA, lower doses with lower treatment times could also be evaluated so as to be useful clinically. 5-ALA may also have applications when used in vivo. As a topical product, 5-ALA could be applied directly to infected areas prior to treatment with blue LED.
91
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