International journal of Science Commerce and Humanities
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Photodynamic therapy as a promising treatment of burn wounds after calf dehorning F.P. Sellera1; M.R. Azedo2; L.C.B.A. Silva3; C.H. Seino1; R.G. Gargano1*; C.F. Batista1; A.M.M.P. Della Libera1; F.J. Benesi1; F.C. Pogliani1 1
University of São Paulo, School of Veterinary Medicine and Animal Husbandry, Department of Internal Medicine, São Paulo, SP, 05508-270, Brazil. 2
Metropolitan University of Santos, School of Veterinary Medicine, Santos, SP, 11080-300, Brazil. 3 University of Guarulhos, School of Veterinary Medicine, Guarulhos, SP, 07030-010, Brazil. *Corresponding author. Tel.: +55 11 30911269. E-mail:
[email protected]. Address: School of Veterinary Medicine and Animal Husbandry, Department of Internal Medicine, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP.: 05508-270, São Paulo, SP, Brazil.
Abstract In recent years, photodynamic therapy (PDT) has been highlighted as a treatment of skin infections, since it’s considered one of the main alternatives to the use of antimicrobials and healing drugs. With the aim to evaluate the response and applicability of PDT in burn wound healing, six calves were undergone dehorning by cauterization and treated. Lesions treated with PDT showed less healing time and reduction of symptoms of inflammation. PDT was an effective alternative method in treating burns in calves, providing better response when compared to the previous proposed topic treatment with zinc oxide ointment. Keywords: Calves; Photodynamic therapy; Burn wound treatment; Dehorning; Healing time.
Introduction Dehorning has been a routine procedure in dairy farming with the aim to reduce the risk of injury to handlers and other animals. In addition to the fact of behavioral and physiological researches have already determined that dehorning is a painful experience, regardless of the method used [1,2], the thermal trauma to the skin disrupts the barriers that serve as protection to the invasion of external agents and the large amount of dead tissue provides a nutritional environment favorable for bacterial growth. However, the occlusion of blood supply interrupts the provision of humoral and cellular defense mechanisms, and systemic antibacterial drugs in the burned area, limiting the effectiveness of antibiotics to its topical application. Therefore, the widely most used treatment of burn wounds and, by extension, of dehorning by cauterization, is based on a topical formof zinc oxide [3]. Photodynamic therapy (PDT) was first described back in 1900 by Oscar Raab and his advisor Herman Von Tappeiner. Few years later, in 1903, Niels Finsen won the Physiology and Medicine Nobel Prize with the use of this technique [4]. In the last years, PDT has been one of the most studied therapeutic modalities for the treatment of superficial tumors [5].The effects of PDT result from the activation of a photosensitizer (PS) by a source of light in a specific wavelength and the further generation of a cascade of reactive oxygen species (ROS) [6]. The release of ROS changes fundamental cellular components and produces a cytotoxic effect on the target cells without mutagenic or genotoxic effects, which prevents the development of microbial resistance [7]. Therefore, PDT has also been suggested as an alternative treatment for local infections, since a range of microorganisms are inactivated by this technique [8-13]. Mostly due to the immediate accessibility to the site both by the PS and the light source, superficial infections following burns are particularly treatable through PDT. In addition, the radiated light on the tissue can act in the process of tissue repair [14].Therefore, the objective of this study was to evaluate the use of PDT as an alternative treatment of wounds caused by dehorning by cauterization in calves. Materials and methods All procedures were previously approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of University of Sao Paulo, Brazil. Six male Holstein calves, aged between four and five months, were submitted to the technique of dehorning by cauterization. Concisely, local anesthesia was performed through anesthetic blockade in which the needle was inserted close to the frontal bone, about 2 to 3 cm in front of the base of the horn, and 10 mL of 2% lidocaine hydrochloride was injected for cornual nerve block and for infiltrative circular anesthesia on the base of the horn. After antisepsis with 2% chlorhexidine, bilateral cauterization of germinal buds was made (Fig. 1a). At the end of the procedure, all animals received a single application of PDT in the lesion of the right side, while lesions in the left side were daily treated with topic zinc oxide ointment (20%)(Fig. 1b).
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PDT was made with the PS methylene blue (aqueous solution, 60 µM). PS was topically applied on the lesions (Fig. 1c). After a pre-irradiation period of five minutes, lesions were irradiated with diode laser (λ = 660 nm, 40mW) with an energy density equal to 180J/cm2 per point (Fig.1e). A total of 5 points were irradiated in each lesion (Fig.1d). Daily antisepsis and examination of all wounds was performed.Macroscopic aspect of lesions, inflammatory symptoms, and healing times were assessed by simple descriptive analysis for a period of 30 days. Results and discussion Routine procedures, such as dehorning by cauterization, cause injuries that are time-consuming and require special care, mainly in the control of secondary infections. Hence, it’s imperative that any form of intervention comes against these adversities. The aim of this experimental model was to demonstrate the effectiveness of therapy by comparing similar lesions in the same animal, discarding individualized responses. To test the post-surgical indication of PDT, each animal had a lesion treated once with PDT and a lesion daily treated with zinc oxide ointment (control). All evaluated animals responded to the two proposed treatments. However, it was possible to see differences in relation to healing time and macroscopic aspect of lesions treated with PDT. As a result, PDT was more efficient and practical, since it was not repeated during the study period. Fifteen days after dehorning, the lesions treated with PDT showed no signs of inflammation and it was possible to see their complete healing. On the other hand, lesions treated daily with ointment had not completely healed and showed signs of local inflammation (Fig. 1f).
Fig. 1.Treatment of lesions after dehorning by cauterization with photodynamic therapy (PDT) or topic ointment.a – postdehorning lesions. b – treatment of lesion in the left side with topic zinc oxide ointment (20%). c-e – treatment of lesion in the right side with PDT. c – topic application of methylene blue (aqueous solution, 60 µM). d – irradiation points (energy density = 180 J/cm2 per point). e – irradiation with diode laser (λ = 660 nm, 40 mW) after a pre-irradiation period of five minutes. f – aspect of lesions 15 days post-dehorning.
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In recent years, studies involving the process of tissue repair with the use of PDT have aroused the interest of many researchers [14], including treatment of wounds with delayed recovery, such as burns [15]. A thermal trauma to the skin produces a series of local changes that will result in the development of pain [1]. Moreover, burns determine great metabolic changes resulting from the combination of stress-induced release of inflammatory mediators and hormonal response. In this type of trauma, the release of cellular and humoral mediators alters capillary permeability and determines metabolic and immunologic changes, leading to electrolyte disturbance, malnutrition and infection. In fact, patients with severe burns have immune suppression and increased susceptibility to infections due to the destruction of skin barriers that serve as protection to the invasion of external agents [16]. Furthermore, denatured proteins present in the burn location provide a nutritional environment favorable to microbial development. Consequently, treatment of animals with burns is based on supportive treatment and use of antimicrobials. According to the extent of the injury, quickly fluid replacement to prevent dehydration and subsequent acute renal failure, a common complication in burned patients, also should be considered. In veterinary medicine, another treatment that is widely used to improve the healing time is a topical form of zinc oxide [3]. On the other hand, regulatory and research agencies have given special attention to the risks to human health represented by direct exposure to antibiotic residues in food of animal origin. Furthermore, since infections caused by several opportunistic pathogens are among the leading causes of morbidity and mortality in humans and animals, the concern about microbial resistance to antibiotics is a subject of global importance [17]. Hence, the use of PDT becomes a therapeutic modality that meets most of the advantages, mainly in relation to undesirable systemic and microbiological effects, which are found in most usual treatments that involve the use of antibiotics. The effect of PDT results from the combination of a PS activated by a source of light in a specific wavelength. Activation of these PS by the light leads these molecules to large chemical instability that will be stabilized by the transfer of energy from the PS to the molecules of the medium. In the presence of oxygen, these reactions can lead to the formation of free radicals, such as peroxide and superoxide ions and hydroxyl radicals, generating a cascade of ROS [6]. Moreover, photonic therapy interacts in three distinct phases of the healing process, providing positive responses in each one of them [18]. In the inflammatory phase, the cell photobiomodulation occurs. As PDT reduces the number of inflammatory cells, it stimulates their functionality to produce growth factors, triggering the second phase, so called proliferative. In this second step, the neoformation of blood vessels occurs by stimulation of endothelial cells, proliferation of fibroblasts and collagen deposition, contributing to the increased formation of granulation tissue and for effective wound contraction. In the remaining phase of the repair process, tissue remodeling occurs, which consists in the reorganization of the blood vessels and collagen fibers. From the beginning of the healing process of wounds, angiogenesis is crucial in removing debris and restoring the supply of oxygen and nutrients to the new tissue, favoring the increase in metabolic rate and consequent cell growth. Beneficial results were obtained with the interaction of red light therapy in the neoformationof blood vessels both in vitro [19] and in vivo [20]. In the present study, the comparative analysis between the treated wounds showed that lesions treated with PDT had less healing time and localpain. This fact confirms the results reported in studies that attest the effectiveness of PDT on events of tissue repair [11]. The methodology adopted in this experiment does not allow the evaluation of all the mechanisms involved in the treatment of burns in calves using PDT. However, results obtained indicate a new non-invasive therapeutic possibility that meets the desired key features on production systems concerned with animal welfare. Also, this work has been a pioneer, since other studies involving the use of PDT in burns of calves have not been found. Diode lasers have wavelength resonant to the absorption band of most PS currently used.They are small, portable and inexpensive, and have great penetration in biological tissues. Such facts may approximate the technique to the system of creation of calves in the near future. Actually, the feasibility of the use of PDT in veterinary medicine has been under investigation, and there is considerable potential for clinical application in different diseases [21,22]. Along with this potential, the development of new PS and types of light with viable costs will certainly become PDT an important tool for veterinarians. Conclusion Results showed that PDT was efficient in the treatment of burns in calves. More research should be carried out to elucidate the mechanisms involved between PDT and burns in calves and its applicability in calf rearing. Conflict of Interest There is no conflict of interest. References 1. Graf, B., Senn, M., 1999. Behavioural and physiological responses of calves to dehorning by heat cauterization with or without local anesthesia. Appl. Anim. Behav. Sci. 62(2), 153-171.
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2. Grondahl-Nielsen, C., Simonsen, H.B., Lund, J.D., Hesselholt, M., 1999. Behavioural, endocrine and cardiac responses in young calves undergoing dehorning without and with the use of sedation and analgesia. Vet. J. 158(1), 14-20. 3. Cangul, I.T., Gul, N.Y., Topal, A., Yilmaz, R., 2006. Evaluation of the effects of topical tripeptide-copper complex and zinc oxide on open wound healing in rabbits. Vet. Dermatol. 17(6), 417-423. 4. Ackroyd, R., Kelty, C., Brown, N., Reed, M., 2001. The history of photodetection and photodynamic therapy.Photochem. Photobiol. 74(5), 656-669. 5. Marmur, E.S., Schmults, C.D., Goldberg, D.J., 2004. A review of laser and photodynamic therapy for the treatment of nonmelanoma skin cancer. Dermatol. Surg. 30(2), 264-271. 6. Maisch, T., 2007. Anti-microbial photodynamic therapy: useful in the future? Lasers Med. Sci. 22(2), 83-91. 7. Konopka, K., Goslinski, T., 2007. Photodynamic therapy in dentistry. J. Dent. Res. 86(8), 694-707. 8. Chabrier-Roselló, Y., Giesselman, B.R., de Jesus-Andino, F.J., Foster, T.H., Mitra, S., Haidaris, C.G., 2010. Inhibition of electron transport chain assembly and function promotes photodynamic killing of Candida. J. Photochem. Photobiol. B. 99(3), 117-125. 9. Lee, R.G., Vecchiotti, M.A., Heaphy, J., Panneerselvam, A., Schluchter, M.D., Oleinick, N.L., Lavertu, P., Alagramam, K.N., Arnold, J.E., Sprecher, R.C., 2010. Photodynamic therapy of cottontail rabbit papillomavirus-induced papillomas in a severe combined immunodeficient mouse xenograft system. Laryngoscope. 120(3), 618-624. 10. Perlin, M., Mao, J.C., Otis, E.R., Shipkowitz, N.L., Duff, R.G., 1987. Photodynamic inactivation of influenza and herpes viruses by hematoporphyrin. Antiviral Res. 7(1), 43-51. 11. Prates, R.A., Yamada, A.M.Jr, Suzuki, L.C., Eiko Hashimoto, M.C., Cai, S., Gouw-Soares, S., Gomes, L., Ribeiro, M.S., 2007. Bactericidal effect of malachite green and red laser on Actinobacillusactinomycetemcomitans. J. Photochem. Photobiol. B. 86(1), 70-76. 12. Zeina, B., Greenman, J., Purcell, W.M., Das, B., 2001. Killing of cutaneous microbial species by photodynamic therapy. Br. J. Dermatol. 144(2), 274-278. 13. Zolfaghari, P.S., Packer, S., Singer, M., Nair, S.P., Bennett, J., Street, C., Wilson, M., 2009. In vivo killing of Staphylococcus aureus using a light-activated antimicrobial agent. BMC Microbiol. 9, 27. 14. Bruscino, N., Lotti, T., Rossi, R., 2011. Photodynamic therapy for a hypertrophic scarring: a promising choice. Photodermatol. Photoimmunol. Photomed. 27(6), 334-335. 15. Garcia, V.G., de Lima, M.A., Okamoto, T., Milanezi, L.A., Junior, E.C., Fernandes, L.A., de Almeida, J.M., Theodoro, L.H., 2010. Effect of photodynamic therapy on the healing of cutaneous third-degree-burn: histological study in rats. Lasers Med. Sci. 25(2), 221-228. 16. Church, D., Elsayed, S., Reid, O., Winston, B., Lindsay, R., 2006. BurnWoundInfections. Clin. Microbiol. Rev. 19(2), 403434. 17. Mateu, E., Martin, M., 2001. Why is anti-microbial resistance a veterinary problem as well? J. Vet. Med. B Infect. Dis. Vet. Public Health. 48(8), 569-581. 18. Yu, W., Naim, J.O., Lanzafame, R.J., 1997. Effects of photostimulation on wound healing in diabetic mice. Lasers Surg. Med. 20(1), 56-63. 19. Kipshidze, N., Nikolaychik, V., Keelan, M.H., Shankar, L.R., Khanna, A., Kornowski, R., Leon, M., Moses, J., 2001. Lowpower helium:neon laser irradiation enhances production of vascular endothelial growth factor and promotes growth of endothelial cells in vitro. Lasers Surg. Med. 28(4), 355-364. 20. Amir, A., Solomon, A.S., Giler, S., Cordoba, M., Hauben, D.J., 2000. The influence of helium-neon irradiation on the viability of skin flaps in the rat. Br. J. Plast. Surg. 53(1), 58-62.
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21. Lucroy, M.D., 2002. Photodynamic therapy for companion animals with cancer. Vet. Clin. North Am. Small Anim. Pract.32(3), 693-702. 22. Lucroy, M.D., Edwards, B.F., Madewell, B.R., 2000. Veterinary photodynamic therapy. J. Am. Vet. Med. Assoc. 216(11), 1745-1751.
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