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BACKGROUND The cytobrush technique is commonly used to sample the equine ocular surface. Impression cytology (IC) is an innovative noninvasive method, which allows for the collection of superficial layers of ocular epithelium. OBJECTIVES The aims of this study were to compare the cytobrush and IC techniques on healthy equine ocular surfaces, to assess the agreement between observers with different levels of expertise, and to test the preservability of filters over time. METHODS Twenty-four horses were sampled within 10 minutes of slaughter using IC on the left eye and the cytobrush technique on the right eye. May-Grunwald-Giemsa stained specimens were evaluated by two observers with different levels of expertise. Morphologic features were evaluated using a 4-grade system. The IC samples were re-evaluated after 6 months to examine filter preservation. RESULTS In IC samples, corneal and conjunctival cells were clearly separated. Goblet cells were found in five and 17 filters by observer 1 and 2, respectively. Using the cytobrush technique, corneal and conjunctival cells were present but mixed. Goblet cell cellularity, preservation, and enumeration were higher with the IC technique compared with the cytobrush technique (P = 0.013; P = 0.004; P = 0.031, respectively). The inter-observer agreement for the IC technique was moderate to fair. In 7/24 IC samples re-evaluated after 6 months, cellular morphology was impaired, and the overall score was significantly lower. CONCLUSIONS IC is an innovative noninvasive method, which allows for sample collection with higher cellularity and preservation. Moreover, the identification of goblet cells is easier. For these reasons, IC could be interesting and useful as a complementary diagnostic cytologic method in clinical practice.

Background Cornea is a composite tissue exhibiting nonlinear and time-dependent mechanical properties. Corneal ulcers are one of the main pathologies that affect this tissue, disrupting its structural integrity and leading to impaired functions. In this study, uniaxial tensile and stress-relaxation tests are developed to evaluate stress-strain and time-dependent mechanical behaviour of porcine corneas. Results The samples are split in two groups: some corneas are analysed in an unaltered state (healthy samples), while others are injured with alkaline solution to create an experimental ulcer (lesioned samples). Furthermore, within each group, corneas are examined in two conditions: few hours after the enucleation (fresh samples) or after 7 days in a specific culture medium for the tissue (cultured samples). Finally, another condition is added: corneas from all the groups undergo or not a cross-linking treatment. In both stress-strain and stress-relaxation tests, a weakening of the tissue is observed due to the imposed conditions (lesion, culture and treatment), represented by a lower stiffness and increased stress-relaxation. Conclusions Alkali-induced corneal stromal melting determines changes in the mechanical response that can be related to a damage at microstructural level. The results of the present study represent the basis for the investigation of traditional and innovative corneal therapies.

Skin wound healing may sometimes lead to open sores that persist for long periods and expensive hospitalization is needed. Among different kinds of therapeutic innovative approaches, mesenchymal stem cells (MSCs) and low-temperature atmospheric pressure cold plasma (ionized gas) have been recently tested to improve this regenerative process. To optimize wound healing the present study intended to combine, for the first time, these two novel approaches in a large size animal wound healing model with the aim of assessing the putative dual beneficial effects. Based on clinical, histopathological, and molecular results a synergistic action in a second intention healing wound in sheep has been observed. Experimental wounds treated with cold plasma and MSCs showed a slower but more effective healing compared to the single treatment, as observed in previous studies. The combined treatment improved the correct development of skin appendages and structural proteins of the dermis showing the potential of the dual combination as a safe and effective tool for skin regeneration in the veterinary clinical field.

Platelet-rich plasma (PRP) is known to play a crucial role in skin wound healing, in both Human and Veterinary Medicine. Remarkably, until now, no studies have reported PRP treatment in subacute full-thickness skin wounds of the dog. The aim of this study was to evaluate the effects of two consecutive applications of autologous PRP, with the second application after 15 days, in 6 dogs showing large subacute skin wounds. The percentage of contraction, re-epithelialization and healing in all treated patients indicated that no complications or side effects, associated with consecutive PRP treatments, occurred in any patient and all wounds achieved complete closure and re-epithelialization. Our results suggest a positive effect of repeated autologous topical PRP treatments in large cutaneous subacute wounds of different etiology. Therefore, this PRP treatment could represent a simple, cost-effective, and valid alternative to promote healing processes in subacute large wounds cases in dogs.

Background To optimize wound healing, the use of low-temperature atmospheric pressure plasma (ionized gas) has been proposed as an innovative therapeutic method for treating extensive and chronic wound. The aim of this study was to evaluate the specific effects of an indirect helium plasma treatment in a sheep surgical wound model based on clinical, histopathological and molecular analyses. This study was part of a larger study aimed at assessing five different wound healing improvement methods. Methods Six sheep were used in this study. Six square wounds were performed on the back of each sheep. Five of the lesions were used to analyze the effect of five different treatments, one of which was the plasma treatment. The sixth lesions was used as control. A 2-min plasma treatment was daily performed, using a radiofrequency helium plasma source for indirect treatments, until complete wound healing was obtained. Biopsies were performed at two and six weeks. The plasma effects were tested through clinical evaluation, histopathological and immunohistological evaluation, real time PCR analysis, bacteriological evaluation and intracellular ROS evaluation. Results Wound closure time for the plasma-treated wounds was quicker than that of the control group. Plasma treatment drastically reduced the bacterial load in the wounds. A strong increase in intracellular ROS was observed. After six weeks, the inflammatory process was significantly reduced in the plasma-treated case with respect to the control. The plasma treatment was found to lead to an anticipated induction of blood vessel formation, as detected through mRNA expression of VEGF. The high Ki67-positivity at two weeks indicated a strong stimulation of cell proliferation induced by the plasma treatment. The expression of the hair keratine (hKER) at six weeks indicated that the plasma was promoting hair regrowth. The plasma treatment induced an increased rate of reformation of cutaneous adnexa at six weeks. Conclusions The obtained results suggest that the plasma action, at the used dose, induces an increase in cell proliferation, a reduction of inflammation, a reduction of the bacterial load, a stimulation of blood vessel formation, and an improvement in the formation of cutaneous adnexa with a positive consequence on hair regrowth.

Healing of open wounds is of great medical importance. Wound healing is a complex process that aims to restore the function and structure of damaged tissue. This study was conducted to compare secondary intention healing of wounds treated daily with a topical application of commercially available hyaluronic acid (HA), Manuka honey (MH), Acemannan gel (AG), or a placebo. Bilateral wounds were surgically created on the backs of six sheep. At two and six weeks post-wound creation, biopsies were obtained to perform histological, immunohistochemical, and molecular analyses of the wound site. Daily clinical evaluations were performed and weekly photographs were taken of the wounds. HA treatment promoted a physiological progression of the healing process in all wound healing phases, while stimulating an abundant cutaneous adnexa and promoting rapid healing, representing the most compelling treatment. MH-treated wounds were slightly dry. However, the main effect of MH was to promote cell proliferation and neovascularization, with an overall pro-inflammatory effect. Results suggest that MH treatment enhances the healing process. AG treatment dehydrated the wounds and stimulated late granulation tissue and cell proliferation. Moreover, AG-treated wounds produced a mild late pro-inflammatory and neovascularization effect. Our data indicate that AG treatment can have a positive influence on moist wounds with abundant granulation tissue and exudate.

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.

Research or diagnostic conditions may require the storage of salivary samples for long periods before analysis is processed. The aim of this study was to evaluate the stability of canine salivary α-amylase, lysozyme, lactate dehydrogenase (LDH), and total calcium and phosphorus after storage for 1, 3 and 6 months at -20°C. Available saliva samples were 75 immediately after collection (T0) and 46 at 1 month (T1), 31 at 3 months (T3) and 18 at 6 months (T6) of storage, according to the number of aliquots collected from each saliva sample. Compared to T0, LDH declined by 92.3% after 1 month of storage (P

Background Skin wound healing includes a system of biological processes, collectively restoring the integrity of the skin after injury. Healing by second intention refers to repair of large and deep wounds where the tissue edges cannot be approximated and substantial scarring is often observed. The objective of this study was to evaluate the effects of mesenchymal stem cells (MSCs) in second intention healing using a surgical wound model in sheep. MSCs are known to contribute to the inflammatory, proliferative, and remodeling phases of the skin regeneration process in rodent models, but data are lacking for large animal models. This study used three different approaches (clinical, histopathological, and molecular analysis) to assess the putative action of allogeneic MSCs at 15 and 42 days after lesion creation. Results At 15 days post-lesion, the wounds treated with MSCs showed a higher degree of wound closure, a higher percentage of re-epithelialization, proliferation, neovascularization and increased contraction in comparison to a control group. At 42 days, the wounds treated with MSCs had more mature and denser cutaneous adnexa compared to the control group. The MSCs-treated group showed an absence of inflammation and expression of CD3+ and CD20+. Moreover, the mRNA expression of hair-keratine (hKER) was observed in the MSCs-treated group 15 days after wound creation and had increased significantly by 42 days post-wound creation. Collagen1 gene (Col1α1) expression was also greater in the MSCs-treated group compared to the control group at both days 15 and 42. Conclusion Peripheral blood-derived MSCs may improve the quality of wound healing both for superficial injuries and deep lesions. MSCs did not induce an inflammatory response and accelerated the appearance of granulation tissue, neovascularization, structural proteins, and skin adnexa.

The existence of progenitor cells that can readily differentiate into a specific cell type is a common cellular strategy for physiological tissue growth and repair mechanisms. In the mammalian cornea, many aspects regarding the nature and location of these cells are still unclear. In the human limbus (peripheral area of the cornea) progenitor cells have been found and characterized but in non-human mammals, the picture is not so clear. In this review, we examine current knowledge about the morphology of limbus and the localization of corneal epithelial stem cells in all species studied so far, comparing data with humans. We have also explored different research directions in the veterinary field in order to discuss the: i) currently used protocols and ii) best range of treatments for ocular pathologies in which corneal stem cells are involved.

The aim of this retrospective study was to evaluate the results obtained in 353 dogs (420 eyes) using two different surgical techniques for correction of a prolapsed gland of the third eyelid: the Morgan's pocket technique and a technique combining Morgan's approach with a slightly modified periosteal anchoring technique of Stanley and Kaswan. The pocket technique was used in 234 eyes and the combined technique in 186 eyes. Successful repositioning was obtained in 95% of all cases, with recurrence occurring in 5%. The recurrence rate in large breed dogs such as the English Bulldog and Boxer was lower with the combined technique than with the pocket technique.

The aim of this preliminary study was to investigate the effect of Multiwave Locked System (MLS(®)), a particular model of low-level laser, in the acute phase of collagenase-induced tendon lesions in six adult sheep randomly assigned to two groups.Tendon injuries are common among human athletes and in sport horses, require a long recovery time, and have a high risk of relapse. Many traditional treatments are not able to repair the injured tendon tissue correctly. In recent years, the use of low-level laser therapy (LLLT) produced interesting results in inflammatory modulation in different musculoskeletal disorders.Group 1 received 10 treatments of MLS laser therapy at a fluence of 5 J/cm(2) on the left hindlimb. Group 2 received 10 treatments of MLS laser therapy at a fluence of 2.5 J/cm(2) on the left hindlimb. In every subject in both groups, the right hindlimb was considered as the control leg.Clinical follow-up and ultrasonography examinations were performed during the postoperative period, and histological examinations were performed at day 30 after the first application of laser therapy. In particular, results from histological examinations indicate that both treatments induced a statistically significant cell number decrease, although only in the second group did the values return to normal. Moreover, the MLS laser therapy dose of 2.5 J/cm(2) (group 2) caused a significant decrease of vessel area.In this study, clinical and histological evaluation demonstrated that a therapeutic dose5 J/cm(2) furnished an anti-inflammatory effect, and induced a decrease of fibroblasts and vessel area. Overall, our results suggest that MLS laser therapy was effective in improving collagen fiber organization in the deep digital flexor tendon.

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.

The loss of skin integrity may induce important dysfunctions and, in large wounds, it is difficult to obtain the restitutioadintegrum. The aim of this study was to verify the efficacy of conventional and innovative topic treatments on skin regeneration. To achieve this goal different types of investigations were performed. Six lesions were surgically created on the back of six healthy adult sheep; every single wound was destined to different conventional (acemannan gel, manuka honey, hyaluronic acid) or innovative treatments such as allogeneic mesenchymal stem cells and plasma gas. The sixth wound was the placebo.Biopsies were collected at time T0, T15 and T40 days. Lesions were clinically evaluated. Histological examinations considered re-epithelization and epidermal thickness. Immunohistochemistry for the evaluation of inflammation, vascularization, and cell proliferation was performed using CD3, CD20, MHCII, von Willebrand factor and KI67 antibodies. Real time-PCR investigated transcripts such as VEGF, TGF-beta 1, Vimentin, Collagen 1α1 and hair Keratin.Clinically, the lesions treated with MSCs healed more rapidly with respect to the placebo. From the second week onwards, all wounds did not show presence of fluid and exhibited a dry and clean secondary layer. MSCs treatment reached the optimal healing since the new tissue presented a mature organization and skin annexes. To sum up, MSCs isolated from peripheral blood can be used in vivo to regenerate wound healing suggesting their involvement not only to improve the healing of primary intention but also for the stimulation of secondary intention.

A 1-year-old sexually intact male Korat cat was referred for ophthalmological consultation due to anisocoria. Mydriasis with external ophthalmoplegia and absence of pupillary light responses in the right eye and nasofacial hypalgesia were seen. Cavernous sinus syndrome (CSS) was suspected. Bilateral deformities of the jaw and phalangeal bones, severe spinal pain and abnormal conformation of the lumbar spine were also present. Radiographic examination revealed several mineralised masses in the appendicular and axial skeleton, indicative of multiple cartilaginous exostoses. For further investigation of the CSS-related neurological deficits, the cat underwent computed tomography (CT) examination of the skull. CT images revealed a non-vascularised, calcified, amorphous mass originating from the right lateral skull base and superimposing on the sella turcica. Based on the severity of diffuse lesions and owing to the clinical signs of extreme pain, the cat was euthanased. A diffuse skeletal and intracranial osteochondromatosis was diagnosed histologically.

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.

Tendon injuries, degenerative tendinopathies, and overuse tendinitis are common in races horses. Novel therapies aim to restore tendon functionality by means of cell-based therapy, growth factor delivery, and tissue engineering approaches. This study examined the use of autologous mesenchymal stromal cells derived from peripheral blood (PB-MSCs), platelet-rich plasma (PRP) and a combination of both for ameliorating experimental lesions on deep digital flexor tendons (DDFT) of Bergamasca sheep. In particular, testing the combination of blood-derived MSCs and PRP in an experimental animal model represents one of the few studies exploring a putative synergistic action of these treatments. Effectiveness of treatments was evaluated at 30 and 120 days comparing clinical, ultrasonographic, and histological features together with immunohistochemical expression of collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP). Significant differences were found between treated groups and their corresponding controls (placebo) regarding tendon morphology and extracellular matrix (ECM) composition. However, our results indicate that the combined use of PRP and MSCs did not produce an additive or synergistic regenerative response and highlighted the predominant effect of MSCs on tendon healing, enhanced tissue remodeling and improved structural organization.

Tendon injuries, degenerative tendinopathies, and overuse tendinitis are common in races horses. Novel therapies aim to restore tendon functionality by means of cell-based therapy, growth factor delivery, and tissue engineering approaches. This study examined the use of autologous mesenchymal stromal cells derived from peripheral blood (PB-MSCs), platelet-rich plasma (PRP) and a combination of both for ameliorating experimental lesions on deep digital flexor tendons (DDFT) of Bergamasca sheep. In particular, testing the combination of blood-derived MSCs and PRP in an experimental animal model represents one of the few studies exploring a putative synergistic action of these treatments. Effectiveness of treatments was evaluated at 30 and 120 days comparing clinical, ultrasonographic, and histological features together with immunohistochemical expression of collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP). Significant differences were found between treated groups and their corresponding controls (placebo) regarding tendon morphology and extracellular matrix (ECM) composition. However, our results indicate that the combined use of PRP and MSCs did not produce an additive or synergistic regenerative response and highlighted the predominant effect of MSCs on tendon healing, enhanced tissue remodeling and improved structural organization.

Dermoid sinus, a congenital malformation of neural tube development, has been reported in humans and several animal species including dogs. It is typically found in the dorsal midline and commonly occurs in the Rhodesian Ridgeback breed. A case of multiple dermoid sinuses in the fronto-occipital region is described. An 11-month-old, intact female Saint Bernard dog was presented with a 2 day history of discharge from a large irregular subcutaneous mass in the fronto-occipital region. The dog was otherwise healthy. The dog had two circular skin lesions (approximately 4 × 4 and 4 × 2 cm diameter) surrounded by multiple irregular elevated masses. The masses had multiple small openings on the skin surface with tufts of hair protruding from the apertures. The masses were surgically removed, and the diagnosis of multiple dermoid sinuses was confirmed by histological examination. Histopathological examination showed multiple, variably sized, spherical to tubular cysts expanding the dermis and subcutis. Cysts were filled with hair shafts and lamellar keratin and were lined by a stratified squamous epithelium. Sebaceous and apocrine gland adnexal structures were also observed. To the best of our knowledge, this is the first reported case of multiple dermoid sinuses of two different types in the head of a Saint Bernard dog.

The “restitutio ad integrum” pursue in the treatment of tenodesmic lesions might represents a tangible target thanks to the increased number of novel cellular-based therapies. In this work, we evaluated the efficacy of the application of platelet-rich plasma (PRP), mesenchymal stem cells (MSCs), and PRP + MSCs to experimentally injured sheep deep digital flexor tendon (DM n° 97/2010-B). Our results indicate that the in vivo integration of injected MSCs was successful as verified by the presence of green fluorescent protein GFP-positive cells. Tissue architecture and the tendon linear fiber pattern were significantly improved on histologic sections, especially after the use of MSCs, and the clinic evaluation was also satisfactory after the use of PRP.

Background To evaluate the efficiency of platelet-rich plasma preparations by means of a double centrifugation tube method to obtain platelet-rich canine plasma at a concentration at least 4 times higher than the baseline value and a concentration of white blood cells not exceeding twice the reference range. A complete blood count was carried out for each sample and each concentrate. Whole blood samples were collected from 12 clinically healthy dogs (consenting blood donors). Blood was processed by a double centrifugation tube method to obtain platelet concentrates, which were then analyzed by a flow cytometry haematology system for haemogram. Platelet concentration and white blood cell count were determined in all samples. Results Platelet concentration at least 4 times higher than the baseline value and a white blood cell count not exceeding twice the reference range were obtained respectively in 10 cases out of 12 (83.3%) and 11 cases out of 12 (91.6%). Conclusions This double centrifugation tube method is a relatively simple and inexpensive method for obtaining platelet-rich canine plasma, potentially available for therapeutic use to improve the healing process.

A large torn wound of the dorsal elbow region was observed in a 17-year-old Arabian mare. Surgical reconstruction was performed with the horse in standing sedation, but suture dehiscence occurred 2 days later. Autologous platelet-rich gel (PRG) was then applied to the wound every 3 weeks for a total of 3 administrations to accelerate good-quality healing. The wound had healed rapidly and completely within 5 months of the first PRG treatment, without chronic effects or formation of exuberant tissue granulation and with minimum scarring. This case report suggests that topical treatment with autologous PRG, as additional therapy, might be considered beneficial in the management of large-wound healing in horses, and it can be regarded as safe and inexpensive treatment that can be used in field.

Summary A ventrally displaced incisive bone fracture was diagnosed in a 3-year-old Andalusian stallion. Symptoms included swelling of the lips, dysphagia and ptyalism. External manipulation revealed pain and crepitus in the gingival region of the rostral maxilla. An intraoral examination revealed upper gingival haematomas, misalignment and malocclusion of the incisors (prognathism). A radiograph of the rostral maxilla confirmed ventrally displaced bilateral fractures of the incisive portion of the maxilla rostral to the canine teeth (Triadan 104/204). The fracture was reduced under general anaesthesia. A methylmethacrylate intraoral splint was used to stabilise the fracture. Post operative radiographs confirmed the fracture reduction. Post operative clinical control confirmed the correct position of the splint. No complications were encountered in the post operative period and good stabilisation was obtained. The splint was removed 60 days post operatively. The use of an acrylic intraoral splint successfully stabilised a bilateral, ventrally displaced incisive bone fracture. Normal occlusion was obtained. The use of an acrylic splint may represent a relatively simple, inexpensive and noninvasive technique for the repair of incisive bone fractures rostral to canine teeth in horses.