Salivary pH, calcium, phosphorus and selected enzymes in healthy dogs: A pilot study

Abstract Background Saliva in dogs, as in humans, is a complex fluid secreted by different salivary glands in the oral cavity to protect the oral mucosa and teeth. The use of saliva as a substitute for blood in diagnosing and prognosticating disease in humans is widely accepted. Salivary biochemistry has also been used as a marker for periodontal disease in humans. No studies have as yet investigated the relation between salivary biochemistry and periodontal disease in dogs, however; neither has the salivary composition of healthy dogs with no oral disease been assessed. The purpose of this study was to obtain an overview on pH distribution and a set of salivary biochemical analytes (calcium, phosphorus, lactate dehydrogenase, lysozyme and amylase) commonly related to oral health in humans in a subset population of healthy young dogs with no periodontal disease or previous oral disease. Data were analyzed to gather salivary reference ranges for pH and each parameter and to assess a possible correlation between salivary and serum analytes. Results Twenty-nine adult client-owned dogs were recruited for the study. Lactate dehydrogenase and lysozyme showed higher concentrations in saliva than in serum, whereas amylase showed the contrary. Salivary biochemistry values did not differ between males and females or between non-neutered and neutered individuals. No significant correlations between salivary and serum calcium, phosphorus, lactate dehydrogenase, amylase and lysozyme were identified in this study. Data allowed intervals for the salivary pH and other analytes investigated to be obtained from healthy dogs with healthy oral conditions. Conclusions These preliminary data can contribute to enlarge our understanding of the functional role of saliva and its relation to oral health in dogs

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p < 0.00) and healthy (p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations

Efficacy of conventional versus innovative therapies for treating skin wounds in veterinary medicine

open16siINTRODUCTION: The skin is the largest organ of mammals. The loss of skin integrity may induce important dysfunctions or even death. For superficial wounds, the endogenous healing mechanisms in combination with traditional wound care are sufficient to achieve functional repair. In contrast, in larger wounds, like third and fourth degree burns, chronic wound or deep ulcers it is difficult to obtain the restitutio ad integrum and fibrosis and/or scar tissue develops1,2. The aim of this study was to verify the efficacy of conventional and innovative topic treatments on skin regeneration, induced experimentally in sheep. To achieve this goal different types of investigations (clinical, molecular, histological, immunohistochemical) were performed. METHODS: Six skin lesions (4x4cm) were surgically created on the back of six healthy adult sheep; every single wound was destined, in a randomized way, to one of the following treatments: Acemannan gel, Manuka Honey, hyaluronic acid, Plasma3 (ionized gas), allogeneic mesenchymal stem cells isolated from peripheral blood (PB-MSCs). The sixth wound was the placebo. Biopsies were collected with a surgical punch (0,6x0,6 cm) at time T0, T15 and T40 days. Lesions were clinically evaluated considering the presence and color of wound fluid, the state of hydration, the wound surface/surroundings and other parameters. Histological examinations considered crust formation, re-epithelization and epidermal thickness, dermis edema, extension of granulation tissue, acute and chronic inflammation. Immunohistochemistry for evaluation of inflammation, vascularization and cell proliferation was performed using CD3, CD20, MHCII, von Willebrand factor (vWF) and KI67 antibodies. Furthermore, Real time-PCR investigated genes as V ascular endothelial growth factors (VEGF), Transforming growth factor beta 1(TGFβ1), Vimentin (VIM), Collagen 1α1 (Col1α1) and hair Keratin (hKER). RESULTS: Clinically, the lesions treated with plasma healed more rapidly respect to other treatments and a reduced bacterial load was observed. At T7 wounds treated with stem cells and plasma were less macerated than lesions treated with other therapies. At T15 the wounds treated with hyaluronic acid showed a normal state of hydration while lesions treated with Manuka Honey exhibited a normal hydration from the third week only (Acemannan gel at fourth week). From the second week onwards all wounds did not show presence of fluid and exhibited a dry and clean secondary layer. All lesions, excluded wounds treated with acemannan gel, presented a red (hyaluronic acid and plasma) and dark red (Manuka Honey, PB-MSCs) granulation tissue starting from the first week. Molecular analysis showed a correspondence between clinical and molecular/histologic results. For instance, VEGF mRNA expression confirms angiogenetic events observed at histological level while TGF-β, CD3 and CD20 mRNA/protein expression indicated the presence/absence of inflammation in the used treatments. DISCUSSION & CONCLUSIONS: Innovative therapies led to surprising results regarding regeneration of mammalian skin. Indeed, on the basis of clinical analysis, wounds treated with plasma and MSC healed more rapidly. Further examinations are ongoing in order to elucidate possible mechanisms explaining these differences. REFERENCES: 1S.Y. Broeckx, S. Maes, T. Martinello, et al (2014) Equine epidermis: a source of epithelial-like stem/progenitor cells with in vitro and in vivo regenerative capacities Stem Cells Dev, pp 1134-48. 2J.H. Spaas, C. Gomiero, S.Y. Broeckx, et al (2016) Wound healing markers after autologous and allogeneic epithelial-like stem cell treatment Cytotherapy 2016 (in press). 3E. Martines, M. Zuin, R. Cavazzana, et al. (2009) A novel plasma source for sterilization of living tissues, New J. Phys. 11, 115014.openPatruno, MARCO VINCENZO; Gomiero, Chiara; Martinello, Tiziana; Perazzi, Anna; Gemignani, F; DE BENEDICTIS, GIULIA MARIA; Ferro, Silvia; Zuin, M; Martines, E; Cordaro, Luigi; Brun, Paola; Maccatrozzo, Lisa; Broeckx, Sy; Spaas, Jh; Chiers, K; Iacopetti, IlariaPatruno, MARCO VINCENZO; Gomiero, Chiara; Martinello, Tiziana; Perazzi, Anna; Gemignani, F; DE BENEDICTIS, GIULIA MARIA; Ferro, Silvia; Zuin, M; Martines, E; Cordaro, Luigi; Brun, Paola; Maccatrozzo, Lisa; Broeckx, Sy; Spaas, Jh; Chiers, K; Iacopetti, Ilari

Utilizzo di concentrato piastrinico (PRP) autologo per il trattamento di lesioni tenodesmiche: esperienze cliniche e sperimentali

In veterinary as well as human medicine the tenodesmic lesions play a great interest because of their high incidence, the difficult wound healing and therefore an incomplete full functional recover with long periods of inactivity. The therapeutical protocols recently used in the treatment of these lesions are not able to reach a real “restitutio ad integrum”. In sport horses, tendon and ligament injuries are a frequent cause of lameness and entail long periods of rest. Often the healing process does not achieve a complete recovery of the functional and morphologic characteristics of these structures, especially if the entity of the lesion is severe. In equine medicine, there is a continuous research of therapies that enhance a correct regeneration of the injured tissue, stimulating the physiological response on a molecular and cellular level, with the aim of improving the quality of the healing response. The ultimate aim of current research is to obtain a total recovery of the animal’s athletic performance, meanwhile reducing the risk of relapses. During recent years great attention has been focused on verifying the possibility of using Platelet Rich Plasma (PRP) as a treatment for musculoskeletal lesions in the horse: platelet rich plasma contains a high concentration of platelets, which, once activated, are able to secrete a great quantity of growth factors. For years it has been proven that growth factors enhance the healing response of tissues. In this thesis it will be described two clinical studies and one experimental research approach carried out in the Department of Clinical Science in collaboration with the Laboratory of Anatomy of the Department of Experimental Veterinary Sciences of the University of Padova, the Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna of Brescia, the Clinica dell’Ippodromo of Merano and veterinary doctor. Aim of the first clinical study has been to evacuate the clinical effectiveness of intralesional ultrasonographic injections of autologous platelet rich plasma for the treatment of tendon and ligament injuries was evaluated in 9 horses. In all the horses treated (100%) no side effects, local or systemic, were recorded following the injection of platelet rich plasma. In all cases, successive ultrasonographic evaluations, performed at regular intervals, confirmed the favourable progression of the tissue’s healing process with a correct re-alignment of the new fibres. In all cases, the formation of scar tissue or of adherences between the damaged tendon or ligament and the adjacent tissues was not reported. At the end of the rehabilitation period (9-12 months) 7 of the 9 horses treated (77.7%) returned to competition, displaying athletic performances that are comparable to the ones reached before the occurrence of injury. The second clinical study had the aim to evaluate if a combined use of platelet rich plasma and autologous mesenchimal stem cells could determine a better effect on tissutal regenerative response. The use of mesenchimal stem cells is very promising in the veterinary field since their potency has been documented in the tissue engineering research. Recently, several studies proposed the use of platelet rich plasma as a matrix for the application of mesenchmal stem cells knowing that platelets might induce multipotent cells thanks to the great amount of growth factors delivered by these cells. Indeed platelet growth factors showed the ability to differentiate stem cells towards the proper fate and to induce angiogenesis. The combination of platelet rich plasma and stem cells should improve the quality of the regenerative response of the tissue. Our study has been conducted on a group of 17 horses affected by tenodesmic lesions. The combined association of platelet rich plasma and mesenchimal stem cells did not cause collateral side effects; the follow up, evidenced by clinical and ecographic observations, showed an amelioration of the subjects injuries. At the end of the reabilitation period 13 of 17 horses returned to the sport activity and no sign of scar or adherences were observed at the level of new formed tissue. The results obtained from these two clinical studies substain the use of platelet rich plasma as a therapeutic useful tool since is preparation is easy and allow a better tissutal regeneration without the formation of a scar. Moreover, the combined use of platelet rich plasma and mesenchimal stem cells resulted to be very promising for ameliorate tenodesmic lesions. Overall it has been shown that platelet rich plasma could be very effective both alone or in combination with mesenchimal stem cells at the tissue level although did not shorten the rehabiitation period. In the experimental approach used in our study we observed the effect the use of platelet rich plasma and platelet rich plasma in association with mesenchimal stem cells in sheep. The tendon of sheep was experimentally injured and after the treatment animals were sacrificed in order to evalute also histologically the treated tissues. The positive results of this research allowed to obatin new information about the early regenerative response in lesions treated with platelet rich plasma and platelet rich plasma associated with mesenchimal stem cells. The latter study will shed light on the real efficacy of novel treatments on injured tendons that recently are becoming very popular also in the vetetinary medicine.In medicina umana e veterinaria le patologie teno-desmiche rivestono grande interesse per l’elevata incidenza con cui si verificano, per la difficoltà di ottenere una guarigione completa con un pieno recupero funzionale delle strutture coinvolte e perché comportano lunghi periodi di inattività. I protocolli terapeutici attualmente utilizzati nel trattamento di queste patologie sono lontani dalla potenzialità di una reale “restitutio ad integrum”. Nel cavallo utilizzato per fini sportivi le lesioni dei tendini e dei legamenti rappresentano la principale causa di zoppia e comportano lunghi periodi di riposo; spesso il processo di guarigione non porta ad un completo recupero delle caratteristiche funzionali e morfologiche di queste strutture, soprattutto se l'entità della lesione è grave. Per tale ragione vi è una continua ricerca di terapie in grado di migliorare la rigenerazione corretta del tessuto danneggiato, stimolando la risposta fisiologica a livello molecolare e cellulare con l'obiettivo di ottimizzare la qualità della risposta riparativa. Il fine delle attuali ricerche è quello di ottenere un recupero totale delle prestazioni atletiche degli animali riducendo al minimo il rischio di recidive. Negli ultimi anni grande attenzione è stata posta nel valutare la possibilità di utilizzare il concentrato piastrinico (PRP) come trattamento per le lesioni muscolo-scheletriche del cavallo: il PRP infatti contiene un'alta concentrazione di piastrine, che, nella loro forma attiva, sono in grado di secernere una grande quantità di fattori di crescita. Già da alcuni decenni infatti è stato dimostrato che i fattori di crescita sono in grado di migliorare la risposta di guarigione dei tessuti. In questa tesi verranno illustrati due studi clinici ed uno sperimentale realizzati presso il Dipartimento di Scienze Cliniche dell’Università di Padova in collaborazione con il laboratorio di Anatomia del Dipartimento di Scienze Sperimentali Veterinarie, l’Istituto Zooprofilattico della Lombardia e dell’Emilia Romagna di Brescia, la Clinica dell’Ippodromo di Merano e veterinari liberi professionisti. Scopo del primo studio è stato di valutare l'efficacia clinica dell’iniezione intralesionale ecoguidata di PRP autologo per il trattamento di lesioni tenodesmiche in 9 cavalli. In tutti i cavalli trattati non sono stati registrati effetti collaterali, né locali né sistemici dopo l'iniezione di PRP. Nei diversi soggetti le valutazioni cliniche ed ecografiche eseguite ad intervalli regolari hanno confermato la progressione favorevole del processo di guarigione del tessuto con un corretto ri-allineamento delle fibre neoformate. In nessun caso è stata rilevata la formazione di tessuto cicatriziale o di aderenze tra il tendine danneggiato ed i tessuti adiacenti. Al termine del periodo di riabilitazione (9-12 mesi) 7 dei 9 cavalli trattati (77.7%) sono tornati alle competizioni, con prestazioni atletiche paragonabili a quelle raggiunte prima del verificarsi del danno tissutale. Il secondo studio clinico si è posto invece l’obiettivo di valutare se un utilizzo combinato di PRP e di cellule staminali mesenchimali autologhe potesse determinare un miglior effetto sinergico sul meccanismo dei guarigione tissutale. L’utilizzo delle cellule staminali come strumento per la riparazione tissutale costituisce un’importante prospettiva di ricerca in clinica veterinaria. Sempre con maggior interesse ci si avvicina alle prospettive terapeutiche basate sulla capacità rigenerativa e riparativa di organi e tessuti che le cellule staminali offrono, come la capacità di auto-replicazione e di proliferazione illimitata. La capacità differenziativa di tali cellule ha reso soprattutto nell’ultimo decennio le MSCs una componente potenzialmente promettente nel campo dell’ingegneria tissutale. Recentemente diversi studi hanno proposto l’utilizzo di concentrato piastrinico, sia nella sua forma liquida che in quella gelatinosa, come matrice per l’applicazione di cellule staminali, con l’obiettivo che le piastrine debitamente attivate potessero liberare i diversi fattori di crescita necessari per la differenziazione delle cellule multipotenti. I fattori di crescita liberati dalle piastrine infatti hanno dimostrato di possedere la capacità di favorire la differenziazione delle cellule staminali verso il destino cellulare e di promuovere l’angiogenesi. L’associazione delle cellule mesenchimali con i fattori di crescita piastrinici permette di accelerare l’impianto e la crescita in vivo delle cellule impiantate. Il nostro studio è stato effettuato in un gruppo di 17 cavalli purosangue inglesi affetti da lesioni teno-desmiche. Anche in questo secondo gruppo di cavalli, il protocollo terapeutico scelto non ha evidenziato effetti collaterali locali né sistemici. Il follow up rilevato, in termini di valutazione clinica ed ecografica, ha evidenziato un soddisfacente miglioramento dei soggetti. Non sono state osservati fenomeni cicatriziali né aderenze a livello del tessuto neoformato ed al termine della fase di riabilitazione 13 dei 17 cavalli trattati (76.5%) sono tornati all’attività sportiva con buoni risultati e senza manifestare recidive. I risultati ottenuti durante questi due studi clinici vanno a sostenere l’utilizzo a scopo terapeutico del PRP nel trattamento di lesioni tenodesmiche: infatti PRP non ha causato effetti collaterali locali o sistemici o la comparsa di segni di rigetto, inoltre può essere ottenuto in breve tempo e quindi utilizzato sul paziente in anestesia locale. Inoltre, il PRP ha dimostrato di essere una terapia promettente, in quanto migliora la rigenerazione dei tessuti, favorendo la formazione di tessuto sano e funzionale, invece di tessuto cicatriziale. L’utilizzo combinato di PRP e MSC, sulla base delle valutazioni cliniche, ecografiche e dei risultati ottenuti a breve e a lungo termine risulta essere molto promettente nel trattamento delle lesioni tenodesmiche del purosangue inglese. Comparando i risultati ottenuti nei due studi clinici, possiamo osservare un effettivo apporto benefico del PRP sia da solo che in associazione alle MSCs sia in termini di miglioramento clinico dei soggetti sia in termini di riduzione dei casi di recidiva rispetto ai trattamenti convenzionali. Rimane di fondamentale importanza il protocollo riabilitativo dei cavalli, che deve tener conto dei lunghi tempi di recupero associati alla guarigione del tessuto: i trattamenti studiati nel nostro lavoro, sembrano apportare effettivamente un’ottimale stimolo per la guarigione tissutale per quanto riguarda le caratteristiche del tessuto neoformato ma non consente di accorciare i tempi di recupero dei soggetti trattati. Un importante aspetto di questo progetto è stato infine quello di verificare, in vivo ed in animali sperimentali, l’efficacia da un punto di vista clinico, ecografico ed istologico, dell’applicazione di PRP da solo o in associazione a MSCs autologhe. Questo protocollo è stato utilizzato nel trattamento di lesioni tendinee sperimentalmente indotte nella pecora: uno degli obiettivi che ci siamo posti è di cercare di comprendere maggiormente la reale efficacia di questi trattamenti attraverso approfondimenti istologici ed immunoistochimici dei tessuti trattati. L'esito positivo di tale ricerca, confermato dai rilievi clinici, ecografici ed istologici ha permesso di ottenere maggiori informazioni sull’andamento del processo di guarigione di lesioni trattate con PRP e con PRP e MSCs nel breve periodo. Tale studio potrà quindi gettare le basi per aumentare la casistica e la numerosità sperimentale nell'applicazione di nuove terapie, volte a migliorare sia il tipo che le caratteristiche della guarigione, che sembrano riscuotere, secondo quando riportato in letteratura, risultati sempre più promettenti

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a &ldquo;multi-aspect approach.&rdquo; Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p &lt; 0.00) and healthy (p &lt; 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations

Use of canine sourced platelet-rich plasma in a feline contaminated cutaneous wound

A 4-year-old neutered female domestic shorthaired cat was treated with canine sourced platelet-rich plasma at the Veterinary Hospital at University of Padua for a large skin defect on the left lateral neck region. The wound healed completely within 20 days and no adverse reaction was observed during the healing process

Maxillomandibulocardiac reflex in a dog

Abstract Background The trigeminocardiac reflex (TCR) is a brainstem reflex that may be observed in anaesthesia during surgical procedures stimulating the intracranial or peripheral portion of the trigeminal nerve. The peripheral TCR is divided into the oculocardiac reflex and the maxillomandibulocardiac reflex based on the affected sensory branches of the trigeminal nerve. In veterinary medicine the oculocardiac reflex has been described, however the maxillomandibulocardiac reflex has never been reported. Case presentation A 5-year-old male Epagneul Breton was presented for surgical management of an upper lip mass. During surgery, a sudden severe bradycardia and a decrease in systemic arterial blood pressure developed. The occurrence of a maxillomandibulocardiac reflex was suspected on the basis of the temporary link between surgical stimulation and haemodynamic changes. Three doses of atropine were given before starting a dopamine infusion due to lack of response. The dopamine infusion normalized heart rhythm and blood pressure. The dog recovered uneventfully and he was discharged 24 h later with a sinus rhythm and no sign of recurrence of arrhythmias. Conclusion The TCR is a rare but potentially life-threatening complication of procedures involving the sensory areas innervated by the three branches of the trigeminal nerve and it may cause bradycardia with hypotension. The use of a β1-adrenergic receptor agonist such as dopamine may be indicated in cases of a refractory response to the conventional treatment with atropine