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BACKGROUND: Castration of the stallion is one of the most frequently performed surgical procedures in the horse. Recently barbed suture materials for surgical wound closure were introduced to the market with manufacturers claiming that these sutures enhance speed and security as they eliminate the need to tie knots. Recently, it has been suggested that this type of suture may increase postoperative complications. This study aimed at investigating and comparing a bidirectional absorbable knotless barbed suture (KBS) to a conventional smooth suture (SS) for wound closure of inguinal castrations in the horse. This was done by evaluating short-term and post-discharge complications and by comparing the time spent on suturing the surgical wounds after bilateral inguinal castration, which was performed on 45 horses undergoing castration at The Large Animal Teaching Hospital at University of Copenhagen from September 2017 to May 2019. RESULTS: Short-term complications were few; at 24 h minor swelling occurred in 29 and 33% of horses sutured with KBS and SS respectively and cutaneous dehiscence during recovery occurred in two horses of each group. Post-discharge follow-up revealed that three horses needed veterinary attention for treatment of complications (scrotal swelling (n = 1, KBS); wound exudation (n = 1, SS) and wound dehiscence after return to pasture (n = 1, SS)). Wound closure was achieved 6 min faster with KBS than with SS (P < 0.0001). CONCLUSIONS: Use of the KBS suture did not result in increased occurrence of postoperative complications. Wound closure was faster with KBS than with SS in equine bilateral inguinal castration. Our results show that KBS can safely be used in the horse following bilateral inguinal castrations without adverse effects and with a reduction in suturing time.
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Caballos/cirugía , Orquiectomía/veterinaria , Suturas/veterinaria , Animales , Masculino , Tempo Operativo , Orquiectomía/efectos adversos , Orquiectomía/instrumentación , Complicaciones Posoperatorias/veterinaria , Técnicas de Sutura/veterinariaRESUMEN
BACKGROUND: The re-epithelialization process in equine wound healing is incompletely described. For epithelial cells to migrate during embryogenesis they undergo epithelial-to-mesenchymal transition (EMT); this phenotypic transition occurs during wound healing in humans and rodents, but it has not been investigated in horses. HYPOTHESIS/OBJECTIVES: To investigate keratinocyte differentiation and EMT in equine experimental excisional limb and body wounds healing by second intention. ANIMALS: Six adult research horses. METHODS AND MATERIALS: Immunohistochemical analysis was used to detect expression of the differentiation markers cytokeratin (CK)10, CK14, loricrin and peroxisome proliferator-activated receptor alpha (PPAR-α), and of the EMT markers E-cadherin and N-cadherin in normal limb and body skin, and biopsies from limb and body wounds. RESULTS: Loricrin and CK10 were expressed in normal skin and periwound skin but not in migrating epithelium of body and limb wounds. However, they reappeared at the migrating epithelial tip of body wounds only. CK14 and PPAR-α had uniform distribution throughout the migrating epithelium. N-cadherin was not expressed in normal unwounded skin but was detected in periwound skin adjacent to the wound margin. E-cadherin expression decreased at the wound margin. CONCLUSIONS AND CLINICAL IMPORTANCE: Presence of N-cadherin suggests that cadherin switching occurred during wound healing, this may be an indication that EMT occurs in horses. To the best of the authors' knowledge, this has never been described in horses before and warrants further investigation to assess the clinical implications. The tip of the migrating epithelium in body wounds appeared more differentiated than limb wounds, which could be part of the explanation for the superior healing of body wounds.
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Diferenciación Celular/fisiología , Extremidades/lesiones , Caballos/lesiones , Queratinocitos/fisiología , Cicatrización de Heridas/fisiología , Animales , Biomarcadores/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Transición Epitelial-Mesenquimal , Regulación de la Expresión Génica , Inmunohistoquímica , Queratinas/genética , Queratinas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismoRESUMEN
Objective: Biofilm infections in chronic wounds are common and pose a significant clinical challenge. This challenge was addressed by developing the SoftOx Biofilm Eradicator (SBE) composed of hypochlorous acid (HOCl) and acetic acid with strong broad-spectrum antimicrobial activity. Approach: First-in-human study investigating the safety and tolerability as primary endpoints and wound size effect and antimicrobial efficacy as secondary endpoints of SBE treatment in chronic leg wound patients. The study was divided into two as follows: a randomized, double-blinded, Single Ascending Dose (SAD) phase (n = 16 SBE; n = 4 placebo), where patients were treated with SBE or saline (placebo) only once, followed by an open-label, Multiple Ascending Dose (MAD) phase (n = 8), where patients were treated with SBE once daily or twice daily over five days. Reporting is according to CONSORT guidelines. Results: SBE was safe and well-tolerated in chronic leg wound patients. There were no significant differences in pain during and after treatment with SBE or the placebo. The SBE treatment reduced bioburden in wounds compared to baseline, with 98% and 49% median reduction after SBE or placebo treatment, respectively. A dose-dependent trend in absolute wound size reduction was observed in the MAD groups with a median (min, max) change of -2.99 (-14.25, -1.5) cm2 in the once-daily and -10.48 (-17.95, -0.38) cm2 in the twice-daily group, respectively. Innovation and Conclusion: This study demonstrated the safe use of HOCl-based SBE in chronic leg wounds with promising trends of immediate antimicrobial action and beneficial effect on wound healing.
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Objective: To determine the synovial fluid (SF) concentrations of lidocaine and mepivacaine after intra-articular injection with clinically relevant doses to the distal interphalangeal (DIP), metacarpophalangeal (MCP), middle carpal (MC), and tarsocrural (TC) joint at two different time points after injection in order to be able to compare concentrations with previously established concentrations associated with cytotoxicity and antimicrobial activity. Procedures: In the first of two experiments, 20 joints (5 MC, 5 MCP, 10 DIP joints) of five horses under general anesthesia were injected with clinically referenced doses of 2% lidocaine. Simultaneously, the horses had 19 joints (5 MC, 5 MCP, 9 DIP joints) injected with clinically referenced doses of 2% mepivacaine. Synovial fluid samples were collected ~7 min after injection. In experiment 2, 23 joints of seven horses under standing sedation were injected with clinically referenced doses of 2% lidocaine. Similarly, the horses had 21 joints injected with 2% mepivacaine. Synovial fluid samples were collected ~23 min after injection. The concentration of mepivacaine and lidocaine in the obtained SF samples was assessed using high-performance-liquid-chromatography with mass spectrometry detection (HPLC MS). Results: Synovial fluid was obtained 6.8 ± 1.5 (experiment 1) and 23 ± 4.3 (experiment 2) min following intra-articular injection of mepivacaine and lidocaine. Synovial fluid concentrations of experiment 1 for lidocaine and mepivaciane were 6.46-19.62 mg/mL (mean 11.96 ± SD 3.89 mg/mL) and 5.01-13.38 mg/mL (mean 8.18 ± SD 1.76 mg/mL), respectively. In experiment 2, concentrations were 2.94-10.40 mg/mL (mean 6.31± SD 2.23 mg/mL) for lidocaine and 2.10-8.70 mg/mL (mean 4.97 ± SD 1.77 mg/mL) for mepivacaine. Conclusions and clinical relevance: Intra-articular LA injections in horses resulted in SF concentrations above those previously associated with cytotoxic effects in vitro but also above those associated with beneficial antimicrobial activities. Local anesthetic concentration was 33-60% lower after 23 min (experiment 2) than after 7 min (experiment 1).
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In chronic wounds in humans, biofilm formation and wound chronicity are linked, as biofilms contribute to chronic inflammation and delayed healing. Biofilms are aggregates of bacteria, and living as biofilms is the default mode of bacterial life; within these aggregates, the bacteria are protected from both antimicrobial substances and the immune response of the host. In horses, delayed healing is more commonly seen in limb wounds than body wounds. Chronic inflammation and hypoxia are the main characteristics of delayed wound healing in equine limbs, and biofilms might also contribute to this healing pattern in horses. However, biofilm formation in equine wounds has been studied to a very limited degree. Biofilms have been detected in equine traumatic wounds, and recent experimental models have shown that biofilms protract the healing of equine limb wounds. Detection of biofilms within wounds necessitates advanced techniques that are not available in routine diagnostic yet. However, infections with biofilm should be suspected in equine limb wounds not healing as expected, as they are in human wounds. Treatment should be based on repeated debridement and application of topical antimicrobial therapy.
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Bovine mastitis is one of the most important diseases in the dairy industry and has detrimental impact on the economy and welfare of the animals. Further, treatment failure results in increased antibiotic use in the dairy industry, as some of these mastitis cases for unknown reasons are not resolved despite standard antibiotic treatment. Chronic biofilm infections are notoriously known to be difficult to eradicate with antibiotics and biofilm formation could be a possible explanation for mastitis cases that are not resolved by standard treatment. This paper reviews the current literature on biofilm in bovine mastitis research to evaluate the status and methods used in the literature. Focus of the current research has been on isolates from milk samples and investigation of their biofilm forming properties in vitro. However, in vitro observations of biofilm formation are not easily comparable with the in vivo situation inside the udder. Only two papers investigate the location and distribution of bacterial biofilms inside udders of dairy cows with mastitis. Based on the current knowledge, the role of biofilm in bovine mastitis is still unclear and more in vivo investigations are needed to uncover the actual role of biofilm formation in the pathogenesis of bovine mastitis.
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In the present study, we investigated lipid membrane interactions of silica nanoparticles as carriers for the antimicrobial peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES). In doing so, smooth mesoporous nanoparticles were compared to virus-like mesoporous nanoparticles, characterized by a "spiky" external surface, as well as to nonporous silica nanoparticles. For this, we employed a combination of neutron reflectometry, ellipsometry, dynamic light scattering, and ζ-potential measurements for studies of bacteria-mimicking bilayers formed by palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol. The results show that nanoparticle topography strongly influences membrane binding and destabilization. We found that virus-like particles are able to destabilize such lipid membranes, whereas the corresponding smooth silica nanoparticles are not. This effect of particle spikes becomes further accentuated after loading of such particles with LL-37. Thus, peptide-loaded virus-like nanoparticles displayed more pronounced membrane disruption than either peptide-loaded smooth nanoparticles or free LL-37. The structural basis of this was clarified by neutron reflectometry, demonstrating that the virus-like nanoparticles induce trans-membrane defects and promote incorporation of LL-37 throughout both bilayer leaflets. The relevance of such effects of particle spikes for bacterial membrane rupture was further demonstrated by confocal microscopy and live/dead assays on Escherichia coli bacteria. Taken together, these findings demonstrate that topography influences the interaction of nanoparticles with bacteria-mimicking lipid bilayers, both in the absence and presence of antimicrobial peptides, as well as with bacteria. The results also identify virus-like mesoporous nanoparticles as being of interest in the design of nanoparticles as delivery systems for antimicrobial peptides.
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Proteínas de Escherichia coli , Nanopartículas , Proteínas de la Membrana Bacteriana Externa , Escherichia coli , Membrana Dobles de Lípidos , Péptidos , Dióxido de SilicioRESUMEN
OBJECTIVE: To compare the extent of inflammation and catabolic collagen response in the middle carpal joints (MCJs) of healthy horses following intra-articular injection of 2% lidocaine, 2% mepivacaine, lactated Ringer solution (LRS), or 0.1% methyl parahydroxybenzoate. ANIMALS: 17 adult horses. PROCEDURES: In the first of 2 experiments, the left middle carpal joint (MCJ) of each of 12 horses was injected with 10 mL of 2% lidocaine (n = 3), 2% mepivacaine (3), or LRS (control; 6). After a 4-week washout period, the right MCJ of the horses that received lidocaine or mepivacaine was injected with 10 mL of LRS, and the right MCJ of horses that received LRS was injected with 10 mL of 2% lidocaine (n = 3) or 2% mepivacaine (3). In experiment 2, the left MCJ of each of 5 horses was injected with 10 mL of 0.1% methyl parahydroxybenzoate. After a 48-hour washout period, the right MCJ of each horse was injected with 10 mL of LRS. Synovial fluid (SF) samples were aseptically collected before and at predetermined times after each injection. Synovial fluid WBC count, neutrophil percentage, and total protein, neutrophil myeloperoxidase, neutrophil elastase, and Coll2-1 concentrations were compared among treatments. RESULTS: Both lidocaine and mepivacaine induced SF changes indicative of inflammation and a catabolic collagen response, but the magnitude of those changes was more pronounced for lidocaine. Methyl parahydroxybenzoate did not cause any SF changes indicative of inflammation. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that mepivacaine was safer than lidocaine for intra-articular injection in horses.
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Enfermedades de los Caballos/tratamiento farmacológico , Mepivacaína/uso terapéutico , Animales , Biomarcadores , Caballos , Inyecciones Intraarticulares/veterinaria , Lidocaína/uso terapéutico , Líquido SinovialRESUMEN
Objective: Relevant animal models to study effects of bacterial aggregates on wound healing are lacking. We aimed at establishing an equine wound model with bacterial aggregates to investigate the impact of bacterial inoculation on normal (thorax) and impaired (limb) wound healing. Approach: Wounds were created on three limbs and both thorax sides of six horses. Twelve out of 20 wounds per horse were inoculated with 104 Staphylococcus aureus and 105 Pseudomonas aeruginosa on day 4. Healing was monitored until day 27 by clinical assessment, including wound scoring, surface pH measurements, and digital photography for area determination. Biopsies were used for bacterial culture and for peptide nucleic acid fluorescence in situ hybridization to detect bacterial aggregates. Results: Inoculated limb wounds healed slower than noninoculated limb wounds from day 10 onward (p < 0.0001). Inoculated and noninoculated thorax wounds healed equally well and faster than limb wounds. The odds ratio of detecting bacterial aggregates in inoculated limb wounds was 7.1 (2.4-21.0, p = 0.0086) compared with noninoculated limb wounds and 36.2 (3.8-348, p = 0.0018) compared with thorax wounds. Innovation: This equine wound model with bacterial aggregates might be superior to other animal wound models, as both normal and impaired healing can be studied simultaneously. In this model, many aspects of wound healing, including novel treatments, may be studied. Conclusions: The impaired healing observed in inoculated limb wounds may be related to the persistent bacterial aggregates. Both in capability of clearing inoculated bacteria from the wounds and in healing pattern, thorax wounds were superior to limb wounds.
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INTRODUCTION: Information on microscopic anatomy of equine skin is sparse. In horses, limb wounds often become chronic and/or non-healing whereas body wounds heal normally. These dissimilarities in healing patterns might be a product of different phenotypic characteristics of body and limb skin. The objective of this study was to investigate microscopic anatomy, epidermal thickness, keratinocyte proliferation and differentiation as well as the presence of mast cells in normal equine skin of body and limb. MATERIALS AND METHODS: The study involved body and limb skin biopsies from six horses. Histological characteristics of the epidermis were assessed and epithelial thickness measured. Immunohistochemistry was performed to investigate epidermal differentiation patterns of cytokeratin (CK) 10, CK14, CK16, loricrin, and peroxisome proliferator-activated receptor alpha (PPAR-α), epidermal proliferation (Ki-67 immunostaining), and mast cells distribution in the skin. RESULTS: The epidermis was significantly thicker in the limb skin compared to body skin (p<0.01). Epidermal proliferation and CK distribution did not show differences in the two anatomical areas. Loricrin presence was focally found in the spinous layer in four out of six limb skin samples but not in body skin samples. Tryptase positive mast cells were detected in the dermis and their density (cell/mm2) was not different between body and limb. DISCUSSION AND CONCLUSION: Here we report for the first time about the normal distribution of CK10, CK14, CK16, PPAR-α, and loricrin in equine limb and body skin as well as about epidermal proliferation rate and mast cell count. It will be relevant to investigate the distribution of the investigated epithelial differentiation markers and the role of mast cells during equine wound healing and/or other skin diseases.
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Caballos/anatomía & histología , Piel/anatomía & histología , Animales , Diferenciación Celular , Proliferación Celular , Dermis/citología , Células Epidérmicas , Epidermis/anatomía & histología , Epidermis/ultraestructura , Extremidades/anatomía & histología , Inmunohistoquímica , Queratinocitos/ultraestructura , Queratinas/metabolismo , Masculino , Mastocitos/ultraestructura , Proteínas de la Membrana/metabolismo , Piel/citología , Piel/ultraestructuraRESUMEN
To obtain robust data on the toxicity of LAS, tests with the collembolan Folsomia candida L., the oligochaetes Aporrectodea caliginosa Savigny (earthworm) and Enchytraeus crypticus Westheide and Graefe (enchytraeid) were performed in a sandy loam soil. Additionally limited tests with LAS spiked to sewage sludge, and subsequently mixed into soil, were performed. For the endpoint of interest, reproduction in soil, we found an EC10 of 205 mg LAS kg(-1) soil [8.6-401] [95% confidence limits] for F. candida and an EC10 of 46 mg LAS kg(-1) soil [13-80] for A. caliginosa after 28 days. E. crypticus was not affected by concentrations up to 120 mg LAS kg(-1) soil. When adding (low contaminated) non-spiked sludge to soil, high stimulation of reproduction was observed for E. crypticus and A. caliginosa but not for F. candida. We argue that this difference in stimulative response between the tested species is related to the difference in feeding behaviour. Sludge spiked with LAS did not significantly affect the reproduction of F. candida (fertility: number of juvenile offspring) and A. caliginosa (fecundity: number of cocoons) (dose equivalent to 181 g and 91 g LAS kg(-1) sludge, respectively). Significantly reduced reproduction was observed for E. crypticus (at 120 mg LAS kg(-1) soil+sludge corresponding to 72 g LAS kg(-1) sludge) compared to non-spiked sludge. The reproduction by E. crypticus was, however, comparable to the reproduction observed in the control soil without sludge. Compared to LAS directly spiked to soil, the reproductive output of organisms exposed to spiked sludge was either not significantly different (F. candida, E. crypticus) or significantly improved (A. caliginosa). More studies are needed in order to make firm conclusions on the potential effect of artificially contaminated sludge in soil systems.