Percutaneous aspiration-injection-reaspiration (PAIR) for the treatment of abdominal cysts: Initial report in sheep and goat.

Percutaneous aspiration-injection-reaspiration (PAIR), also called sclerotherapy, is a minimally invasive, inexpensive and safe technique for the treatment of abdominal cysts in humans. A study was planned to evaluate the feasibility of this procedure in the management of abdominal cysts in sheep and goat. Adult ewes (n = 5) and one doe (n = 1) found to have abdominal cysts (one cyst/animal) on repeated survey ultrasonography (USG) were included in the study. The animals were restrained in standing position. A hypodermic needle (G-18) securely attached to a 10-mL Dispovan syringe was carefully passed under ultrasound guidance into the abdominal cyst in all these animals. Depending on the size of the cyst, 1.0-5.5 mL fluid was aspirated, and 0.5-2.0 mL of 20% hypertonic saline solution infused. The needle was thereafter kept in situ for 10 min. The maximum possible volume of the cyst content was reaspirated and the needle withdrawn. On day 7, sclerotherapy was repeated in five animals showing no appreciable reduction in cyst size by USG. USG was repeated on days 30 and 90. All the cysts except one responded to PAIR during this period. From this study it can be concluded that sclerotherapy using hypertonic saline (20%) is a minimally invasive, inexpensive, effective and safe interventional ultrasonographic technique for the treatment of abdominal cysts in sheep and goats. However, the procedure needs further evaluation after using different sclerotic agents of varying concentrations and duration of their retention in the cysts in a sufficient number of animals with cysts.

Intra-articular xenogeneic mesenchymal stem cell-based therapy increases CD4+CD25+ cells in synovial fluid.

Osteoarthritis (OA) is a chronic joint disease afflicting a substantial portion of the world's population with no currently available cure. Mesenchymal stem cell (MSC)-based therapies have been observed to have a mild beneficial effect in OA but the mechanism behind their action remains unclear. This study aimed to identify the lymphocytic response to a xenogeneic human umbilical cord-derived MSC-based cell therapy. A unilateral medial meniscal release model was employed in an ovine model of post-traumatic OA, with the contralateral limb employed as the control. A dose of 1.0 × 107 MSCs was administered to a subset of the OA group as well as to a normal sham-operated group. Synovial fluid was aspirated periodically for 13 weeks for flow cytometry analysis. At the termination of the study the stifle joints were collected and analyzed for potential pathologic changes. Cell therapy induced a transient influx of CD4+ leukocytes; there was a similar significant increase in the proportion of CD4+CD25+ and CD4+CD25hi leukocytes in response to cell therapy, the latter being a subset that may be composed of regulatory T cells. There was no significant effect of the cell therapy treatment on the proportion of synovial fluid-derived CD8+ cells or BAQ44A+ B cells. iNOS expression of intimal lining macrophages was evident but reduced in the cell therapy OA group suggesting macrophage phenotype transformation. There were no inflammatory or histological changes that could be attributed to the cell therapy. Cell therapy induced chemotaxis of CD4+ cells to the joint but these cells were not associated with pathological changes, despite their expression of activation markers (CD25+).

Ultra Short Echo Time MRI of Iron-Labelled Mesenchymal Stem Cells in an Ovine Osteochondral Defect Model.

Multipotent Mesenchymal Stem/Stromal Cells (MSCs) are widely used in cellular therapy for joint repair. However, the use of MSC therapies is complicated by a lack of understanding of the behaviour of cells and repair within the joint. Current methods of MSC tracking include labelling the cells with Super Paramagnetic Iron Oxide nanoparticles (SPIOs). However, standard acquisition sequences (T2 and T2*) give poor anatomical definition in the presence of SPIOs. To avoid anatomical compromise in the presence of SPIOs, we have investigated the use of Ultra-short Echo Time (UTE) MRI, using a 3D cones acquisition trajectory. This method was used to track SPIO labelled MSC injected into joints containing osteochondral defects in experimental sheep. This study demonstrates that multiple echo times from UTE with 3 T MRI can provide excellent anatomical detail of osteochondral defects and demonstrate similar features to histology. This work also monitors the location of SPIO-labelled cells for regenerative medicine of the knee with MRI, histology, and Prussian blue staining. With these methods, we show that the SPIOs do not hone to the site of defect but instead aggregate in the location of injection, which suggests that any repair mechanism with this disease model must trigger a secondary process.

Effect of supportive therapy on the pharmacokinetics of intravenous marbofloxacin in endotoxemic sheep.

The purpose of this study was to determine the influences of supportive therapy (ST) on the pharmacokinetics (PK) of marbofloxacin in lipopolysaccharide (LPS)-induced endotoxemic sheep. Furthermore, minimum inhibitory concentration (MIC) of marbofloxacin against Escherichia coli, Mannheimia haemolytica, Pasteurella multocida, Klebsiella pneumoniae, Salmonella spp., and Staphylococcus aureus was determined. The study was performed using a three-period cross PK design following a 15-day washout period. In the first period, marbofloxacin (10 mg/kg) was administered by an intravenous (IV) injection. In the second and third periods, marbofloxacin was co-administered with ST (lactated ringer + 5% dextrose + 0.45% sodium chloride, IV, 20 ml/kg, dexamethasone 0.5 mg/kg, SC) and ST + LPS (E. coli O55:B5, 10 µg/kg), respectively. Plasma marbofloxacin concentration was measured using HPLC-UV. Following IV administration of marbofloxacin alone, the t 1 / 2 λ z , AUC0-∞ , ClT , and Vdss were 2.87 hr, 34.73 hr × µg/ml, 0.29 L hr-1  kg-1 , and 0.87 L/kg, respectively. While no change was found in the MBX + ST group in terms of the PK parameters of marbofloxacin, it was determined that the ClT of marbofloxacin decreased, AUC0-∞ increased, and t 1 / 2 λ z and MRT prolonged in the MBX + ST + LPS group. MIC values of marbofloxacin were 0.031 to >16 µg/ml for E. coli, 0.016 to >16 µg/ml for M. haemolytica, 0.016-1 µg/ml for P. multocida, 0.016-0.25 µg/ml for K. pneumoniae, 0.031-0.063 µg/ml for Salmonella spp., and 0.031-1 µg/ml for S. aureus. The study results show the necessity to make a dose adjustment of marbofloxacin following concomitant administration of ST in endotoxemic sheep. Also, the PK and pharmacodynamic effect of marbofloxacin needs to be determined in naturally infected septicemic sheep following concomitant administration of single and ST.

Influence of Radial Pressure Wave Therapy (RPWT) on collagenase-induced Achilles tendinopathy treated with Platelet Rich Plasma and Autologous Adipose Derived Stem Cells.

Tendinopathy treatment poses a current challenge for sport medicine due to unique physiology and biomechanics of tendons. The goal of this work was to compare the efficacy of the addition of the radial pressure wave therapy (RPWT) treatment to injection of autologous Adipose Derived Stem Cells (ADSCs) or Platelet Rich Plasma (PRP) in the therapeutic procedure for collagenase induced Achilles tendinopathy in sheep. 14 sheep (aged 5 and 6 years, Polish Mountain Sheep breed, weight 60-70 kg) were injected bacterial collagenase type 1A-S (Clostridium histolyticum, C-5894, Sigma Aldrich, Poznan, Poland) bilaterally to Achilles tendons. Subsequently, the animals were injected with PRP (7 sheep) or ADSCs (7 sheep) to previously induced tendinopathy foci. Left limbs of all the animals were additionally treated with RPWT focused above the tendinopathy origins. Treatment progress was controlled by ultrasound scans, and tendon samples were taken on the 126th day of the experiment. Tendon samples taken from the sheep treated with RPWT+ADSCs showed lower cellularity and the highest number of thick collage fibers. Samples taken from the sheep treated with RPWT+PRP showed an elevated rate of neovascularization. Addition of the RPWT to ADSCs injections in the treatment of induced Achilles tendinopathy in sheep resulted in good quality of the tissue regeneration. Dual therapy with RPWT+PRP injection can lead to neovascularization in the tendon tissue.

Systemic inflammatory response to the Radial Pressure Wave Therapy (RPWT) in collagenase-induced Achilles tendinopathy treated with Adipose Derived Stem Cells or Platelet Rich Plasma.

Novel tendinopathy treatment protocols should be assessed for safety. The goal of this work was to compare differences in selected systemic inflammatory marker concentrations after two treatment protocols for collagenase induced Achilles tendinopathy in sheep. 14 sheep (aged 5 and 6 years, Polish Mountain Sheep breed, weight 60-70kg) were injected with bacterial collagenase type 1A-S (Clostridium histolyticum, C-5894, Sigma Aldrich, Poznan, Poland) bilaterally to Achilles tendons. Subsequently, the animals were injected with Platelet Rich Plasma (7 sheep) or Adipose Derived Stem Cells (7 sheep) to induced tendinopathy foci. Left limbs of all sheep were additionally treated with Radial Pressure Wave Therapy (RPWT) focused above the tendinopathy origins. Treatment progress was controlled by ultrasound scans, and tendon samples were taken on the 126th day of the experiment. Serum Amyloid A (SAA) concentration showed mild elevation before the experiment (2 sheep from group I, 4 sheep from group II) and two days after the intratendinous growth factors injection ( 4 sheep from group I, 3 sheep from group II) combined with RPWT (mean 22,63 mg/L and 53, 6 mg/L respectively). Haptoglobine (Hp) concentration increased from 0 to 0,01 g/L in 2 animals from group I two days after injection. These values declined to 0 during the course of the treatment. Fibrinogen (Fb) concentrations were within reference levels throughout the research, although mild elevation was observed before the treatment course in 6 sheep from group I and 1 sheep from group II. In conclusion, addition of RPWT to growth factors injections in the treatment of yatrogenic Achilles tendinopathy in sheep did not induce systemic inflammatory response.

Development of a CRISPR/Cas9 system against ruminant animal brucellosis.

BACKGROUND: Brucellosis, caused by several Brucella species, such as the bacterium Brucella melitensis, is considered one of the most severe zoonotic diseases worldwide. Not only does it affect ruminant animal populations, leading to a substantial financial burden for stockbreeders, but also poses severe public health issues. For almost four decades in southern Europe and elsewhere, eradication of the disease has been based on ambiguously effective programs, rendering massive sanitation of livestock urgent and indispensable. Gene therapy, which has been proved effective in the clinic, could possibly constitute an alternative option towards a permanent cure for brucellosis, by aiding in the deletion or inactivation of genes associated with the replication of Brucella within the host cells. RESULTS: We infected ovine macrophages with B.melitensis, to simulate the host cell/microorganism interaction in vitro, and transduced the infected cells with CRISPR/Cas9 lentiviral vectors that target Brucella's RNA polymerase subunit A (RpolA) or virulence-associated gene virB10 at a multiplicity of infection of 60. We demonstrate a significant decrease in the bacterial load per cell when infected cells are transduced with the RpolA vector and that the number of internalized brucellae per cell remains unaffected when macrophages are transduced with a conventional lentiviral vector expressing the green fluorescence protein, thus underlining the bactericidal effect of our CRISPR/Cas9 system. CONCLUSIONS: Pending in vivo verification of our findings, overall, these results may prove critical not only for the treatment of human brucellosis, but for other infectious diseases in general.

[Therapeutic options of obstructive urolithiasis in small ruminants]. / Therapeutische Möglichkeiten bei obstruktiver Urolithiasis des kleinen Wiederkäuers.

The diagnosis of obstructive urolithiasis in small ruminants frequently results in a multitude of decisions that have to be made by the consulted practitioner. Factors that influence the decision for therapy (or euthanasia) are the type of the animal's use, economic aspects and specific options of the veterinarian practice as well as emotional aspects depending on the owners of small ruminants kept as companion animals. The present article aims to present the currently available methods of therapy to facilitate a decision by the practicing veterinarian based on the present state of the science. Naturally, the individual method of choice may differ from the scientific point of view depending on the practitioner's evaluation.

Variables of initial examination and clinical management associated with survival in small ruminants with obstructive urolithiasis.

BACKGROUND: Obstructive urolithiasis is a common disease associated with a guarded prognosis in small ruminants. HYPOTHESIS/OBJECTIVE: The results of physical examination, laboratory analyses, and clinical management of male small ruminants presented to 2 referral clinics were investigated to identify variables significantly associated with disease outcome, so as to provide better recommendations to animal owners regarding the management of these patients. ANIMALS: Two-hundred ten small ruminants (130 sheep and 80 goats) with confirmed diagnosis of obstructive urolithiasis. METHODS: Clinical findings (including diagnostic imaging) and laboratory results of the 210 animals were reviewed, and relevant information regarding clinical and laboratory variables recorded upon admission and clinical management was retrieved. The association of the different variables with nonsurvival was investigated by univariable and multivariable logistic regression models. RESULTS: Only 39% of all patients considered for treatment and 52% of those undergoing tube cystostomy survived to be released from the clinic. Nonsurvival was strongly associated with a very poor clinical condition upon presentation, obesity, castration, and evidence of uroperitoneum. Among blood variables, abnormal PCV, severely increased serum creatinine concentrations, and increased activity of the creatine kinase were associated with increased risk of nonsurvival. Presence of signs of colic or macroscopic appearance of urine was not significantly associated with outcome. CONCLUSIONS AND CLINICAL IMPORTANCE: The prognosis of obstructive urolithiasis was guarded with survival rates of 39% (overall) to 52% (after tube cystostomy). Intact young males with normal body condition presented early in the course of disease had the best chances of survival.

Comparative Efficacy of Autologous Stromal Vascular Fraction and Autologous Adipose-Derived Mesenchymal Stem Cells Combined With Hyaluronic Acid for the Treatment of Sheep Osteoarthritis.

The current study explored whether intra-articular (IA) injection of autologous adipose mesenchymal stem cells (ASCs) combined with hyaluronic acid (HA) achieved better therapeutic efficacy than autologous stromal vascular fraction (SVF) combined with HA to prevent osteoarthritis (OA) progression and determined how long autologous ASCs combined with HA must remain in the joint to observe efficacy. OA models were established by performing anterior cruciate ligament transection (ACLT) and medial meniscectomy (MM). Autologous SVF (1×107 mononuclear cells), autologous low-dose ASCs (1×107), and autologous high-dose ASCs (5×107) combined with HA, and HA alone, or saline alone were injected into the OA model animals at 12 and 15 weeks after surgery, respectively. Compared with SVF+HA treatment, low-dose ASC+HA treatment yielded better magnetic resonance imaging (MRI) scores and macroscopic results, while the cartilage thickness of the tibial plateau did not differ between low, high ASC+HA and SVF+HA treatments detected by micro-computed tomography (µCT). Immunohistochemistry revealed that high-dose ASC+HA treatment rescued hypertrophic chondrocytes expressing collagen X in the deep area of articular cartilage. Western blotting analysis indicated the high- and low-dose ASC+HA groups expressed more collagen X than did the SVF+HA group. Enzyme-linked immunosorbent assay showed treatment with both ASC+HA and SVF+HA resulted in differing anti-inflammatory and trophic effects. Moreover, superparamagnetic iron oxide particle (SPIO)-labeled autologous ASC signals were detected by MRI at 2 and 18 weeks post-injection and were found in the lateral meniscus at 2 weeks and in the marrow cavity of the femoral condyle at 18 weeks post-injection. Thus, IA injection of autologous ASC+HA may demonstrate better efficacy than autologous SVF+HA in blocking OA progression and promoting cartilage regeneration, and autologous ASCs (5×107 cells) combined with HA potentially survive for at least 18 weeks after IA injection.

In vitro efficacy of Duddingtonia flagrans against nematodes of sheep based on in vivo calculations / Eficácia in vitro de Duddingtonia flagrans contra nematoides de ovinos com base em cálculo in vivo

Abstract Duddingtonia flagrans has been tested as an alternative parasite control, but data from in vitro experiments based on in vivo calculations describing nematophagous fungi predation in nematodes are restricted. The objective of this work was to determine the efficacy of D. flagrans against sheep nematode larvae in vitro using in vivo calculations. Fecal samples were introduced to fungi in different concentrations: 0.0/control; 0.05; 0.1; 0.2; 0.4; 0.8; 1.6; 3.2; and 6.4 g corresponding, respectively, to 583.000; 1.166.000; 2.332.000; 4.664.000; 9.328.000; 18.656.000; 37.312.000 and 74.624.000 chlamydospores/kg of body weight. The material was incubated for 14 days, before the larvae recovery (Assay 1). Assay 2 was carried out with the doses of 0.00625; 0.0125; and 0.025 g. The results showed a negative correlation between fungal concentrations and larval numbers for both assays. The fungus demonstrated an efficacy above 89% in both assays. Thus, we consider that the data from in vitro studies based on in vivo calculations may optimize the fungi quantities for field experiments.

Resumo Duddingtonia flagrans tem sido testado como uma alternativa no controle de parasitos, entretanto, trabalhos in vitro da predação de nematoides por fungos nematófagos correlacionados com cálculos baseados para testes in vivo são restritos. O objetivo deste trabalho foi determinar a eficácia in vitro de D. flagrans contra larvas de nematoides de ovinos tendo como base cálculos in vivo. Amostras fecais receberam a adição do fungo em diferentes concentrações: 0.0/controle; 0,05; 0,1; 0,2; 0,4; 0,8; 1,6; 3,2 e 6,4 gramas correspondendo, respectivamente, às seguintes dosagens: 583.000; 1.166.000; 2.332.000; 4.664.000; 9.328.000; 18.656.000; 37.312.000 e 74.624.000 clamidósporos/Kg de peso vivo animal. O material foi incubado por 14 dias, para recuperação das larvas (Ensaio 1). O Ensaio 2 foi realizado com concentrações de 0,00625; 0,0125 e 0,025 g. Foi observada correlação negativa entre a concentração fúngica e o número de larvas, nos dois ensaios. O fungo demonstrou eficácia acima de 89% em ambos os ensaios. A partir destes dados, acreditamos que ensaios in vitro baseados em cálculos in vivo podem aprimorar as dosagens para a realização de experimentos a campo.

Control of sheep gastrointestinal nematodes using the combination of Duddingtonia flagrans and Levamisole Hydrochloride 5% / Controle de nematódeos gastrintestinais de ovinos utilizando a combinação de Duddingtonia flagrans e Cloridrato de Levamisole 5%

Abstract The objective was to evaluate the action of D. flagrans pellets in association with Levamisole Hydrochloride 5% for controlling sheep gastrointestinal nematodes in the northeastern Brazil. Three groups of six sheep each were formed: group 1 received 3 g of the pellets (0.6 g of D. flagrans mycelium) for each 10 kg b.w., twice a week for six months, and deworming with Levamisole Hydrochloride 5% when EPG ≥ 1500; group 2 received a dosage of Levamisole Hydrochloride 5% when EPG ≥ 1500; and group 3 received 3 g of pellets without fungi for each 10 kg b.w., twice a week for six months. EPG counts, larval cultures, packed cell volume (PCV) and weighing were performed every 15 days; monthly, samples of grass from each paddock were collected. The mean EPG of the groups began to statistically differ from day 30 (p < 0.05). Group 1 required less deworming with Levamisole Hydrochloride 5% and showed superiority of PCV values ​​throughout the experiment (p < 0.05). There was a significant reduction (p < 0.05) in L3 recovery in the group 1 paddock from day 30 onwards. The use of D. flagrans pellets in association with Levamisole Hydrochloride 5% was effective for controlling gastrointestinal nematodes.

Resumo O objetivo foi avaliar a ação de péletes de Duddingtonia flagrans em associação ao Cloridrato de Levamisole 5% no controle de nematódeos gastrintestinais de ovinos no Nordeste do Brasil. Foram formados três grupos de seis animais cada: grupo 1 recebeu 3 g de péletes (0,6 g de micélio de D. flagrans) para cada 10 kg p.v., duaz vezes por semana durante seis meses, e vermifugações com Cloridrato de Levamisole 5% quando OPG > 1500; grupo 2 recebeu uma dosagem de Cloridrato de Levamisole 5% quando OPG ≥ 1500; e grupo 3 recebeu 3 g de péletes sem fungos para cada 10 kg de p.v., duas vezes por semana durante seis meses. Contagens de OPG, coproculturas, de volumes globulares (VG) e pesagens foram realizadas a cada 15 dias. Mensalmente, amostras de pasto de cada piquete eram coletadasa. A média de OPG dos grupos começou a diferir estatisticamente a partir do dia 30 (p < 0,05). O grupo 1 necessitou de menos vermifugações com Cloridrato de Levamisole 5% e demonstrou superioridade nos valores de VG durante todo o experimento (p < 0,05). Houve redução significativa (p < 0,05) nas L3 recuperadas no piquete do grupo 1 a partir do dia 30. Em conclusão, a utilização de péletes de D. flagrans em associação ao Cloridrato de Levamisole 5% foi eficaz no controle de nematódeos gastrintestinais de ovinos.

Tracheal suctioning improves gas exchange but not hemodynamics in asphyxiated lambs with meconium aspiration.

BACKGROUND: Current neonatal resuscitation guidelines recommend tracheal suctioning of nonvigorous neonates born through meconium-stained amniotic fluid. METHODS: We evaluated the effect of tracheal suctioning at birth in 29 lambs with asphyxia induced by cord occlusion and meconium aspiration during gasping. RESULTS: Tracheal suctioning at birth (n = 15) decreased amount of meconium in distal airways (53 ± 29 particles/mm(2) lung area) compared to no suction (499 ± 109 particles/mm(2); n = 14; P < 0.001). Three lambs in the suction group had cardiac arrest during suctioning, requiring chest compressions and epinephrine. Onset of ventilation was delayed in the suction group (146 ± 11 vs. 47 ± 3 s in no-suction group; P = 0.005). There was no difference in pulmonary blood flow, carotid blood flow, and pulmonary or systemic blood pressure between the two groups. Left atrial pressure was significantly higher in the suction group. Tracheal suctioning resulted in higher Pao2/FiO2 levels (122 ± 21 vs. 78 ± 10 mm Hg) and ventilator efficiency index (0.3 ± 0.05 vs.0.16 ± 0.03). Two lambs in the no-suction group required inhaled nitric oxide. Lung 3-nitrotyrosine levels were higher in the suction group (0.65 ± 0.03 ng/µg protein) compared with the no-suction group (0.47 ± 0.06). CONCLUSION: Tracheal suctioning improves oxygenation and ventilation. Suctioning does not improve pulmonary/systemic hemodynamics or oxidative stress in an ovine model of acute meconium aspiration with asphyxia.

Treatment with embryonic stem-like cells into osteochondral defects in sheep femoral condyles.

BACKGROUND: Articular cartilage has poor intrinsic capacity for regeneration because of its avascularity and very slow cellular turnover. Defects deriving from trauma or joint disease tend to be repaired with fibrocartilage rather than hyaline cartilage. Consequent degenerative processes are related to the width and depth of the defect. Since mesenchymal stem cells (MSCs) deriving from patients affected by osteoarthritis have a lower proliferative and chondrogenic activity, the systemic or local delivery of heterologous cells may enhance regeneration or inhibit the progressive loss of joint tissue. Embryonic stem cells (ESCs) are very promising, since they can self-renew for prolonged periods without differentiation and can differentiate into tissues from all the 3 germ layers. To date only a few experiments have used ESCs for the study of the cartilage regeneration in animal models and most of them used laboratory animals. Sheep, due to their anatomical, physiological and immunological similarity to humans, represent a valid model for translational studies. This experiment aimed to evaluate if the local delivery of male sheep embryonic stem-like (ES-like) cells into osteochondral defects in the femoral condyles of adult sheep can enhance the regeneration of articular cartilage. Twenty-two ewes were divided into 5 groups (1, 2, 6, 12 and 24 months after surgery). Newly formed tissue was evaluated by macroscopic, histological, immunohistochemical (collagen type II) and fluorescent in situ hybridization (FISH) assays. RESULTS: Regenerated tissue was ultimately evaluated on 17 sheep. Samples engrafted with ES-like cells had significantly better histologic evidence of regeneration with respect to empty defects, used as controls, at all time periods. CONCLUSIONS: Histological assessments demonstrated that the local delivery of ES-like cells into osteochondral defects in sheep femoral condyles enhances the regeneration of the articular hyaline cartilage, without signs of immune rejection or teratoma for 24 months after engraftment.

Survival of bone marrow mesenchymal stem cells labelled with red fluorescent protein in an ovine model of collagenase-induced tendinitis.

OBJECTIVE: The aim of this study was to track the survival and efficacy of allogeneic bone marrow mesenchymal stem cells (BM-MSC) marked with red fluorescent protein (BM-MSCRFP) in an ovine model of collagenase-induced tendinopathy. METHODS: Bone marrow was harvested from one donor sheep and BM-MSC were isolated, cultivated and transfected with red fluorescent protein (BM-MSCRFP). Collagenase was injected into both Achilles tendons in the remaining nine sheep. After two weeks the left tendon was injected with a solution of 6 x 106 BM-MSCRFP and fibrin glue, while only fibrin glue was administered to the contra-lateral tendon in each sheep. After three, four and six weeks the tendons were harvested and evaluated for morphology, collagen I deposition, presence of CD34+ cells, and fluorescent labelled BM-MSC. RESULTS: We demonstrated that delivery of BM-MSC into tendon lesions had positive effects on the injured tendons. The BM-MSCRFP survived at three, four and six weeks after treatment, leading to better quality healing of tendons as compared to the controls, where no labelled cells were detected. Interestingly, we demonstrated high expression of CD34+ cells in tendons that had been treated with BM-MSCRFP. CLINICAL RELEVANCE: Mesenchymal stem cell allografts have a positive effect on tendon healing and local injection of BM-MSC directly into the tendon allows the homing of BM-MSC for good efficiency of engraftment.

Histology and immunohistochemistry study of ovine tendon grafted with cBMSCs and BMMNCs after collagenase-induced tendinitis.

OBJECTIVES: The aim of this study was to compare the regeneration abilities of cultured bone marrow mesenchymal cells (cBMSC) and bone marrow mononuclear cells (BMMNC) with fibrin glue, saline solution and sham control in collagenase-induced tendinitis of the Achilles tendon in sheep. METHODS: Six sheep were recruited randomly to each group: cBMSC, BMMNC, fibrin, saline and sham control. Each group received the relative treatment two weeks after inducing lesions (T(0)). After eight weeks (T(8)) of treatment, the tendons were harvested and evaluated for histomorphology, Collagen type I, III, Cartilage Oligomeric Matrix Protein (COMP) and CD34 positive cells expression. RESULTS: Histology and immunohistochemistry showed similar capabilities of cBMSC and BMMNC to restore the architecture of fibres and Extra Cellular Matrix (ECM), with a high expression of collagen type I and COMP and a very low expression of collagen type III in treated tendons. The complete architectural disruption of fibres, dramatic reduction of collagen Type I and COMP expression and increase collagen type III expression were commonly observed in tendons treated with fibrin or saline only. The presence of CD34 positive cells was appreciable in the BMMNC group while few cBMSC showed this cluster of differentiation, not expressed in tendons treated with fibrin or saline. CLINICAL SIGNIFICANCE: The data in this study show the efficacy of cBMSC and BMMNC in regenerating tendon tissue after collagenase-induced tendinitis.

Cell therapy for tendinitis, experimental and clinical report.

To compare cultured bone marrow mesenchymal cells (cBMSC), bone marrow mononucleated cells (BMMNCs), and placebo to repair collagenase-induced tissue damage in an equine model of experimental tendonitis, 6 Standardbred horses with no signs of previous SDF tendon injury have been recruited. Three weeks after collagenase treatment an average of either 5.5 x 10(6) cBMSCs or 122.3 x 10(6) BMMNCs, saline solution (placebo) or fibrin glue were injected intralesionally in random order. Horses were stall rested for 21 weeks, and tendon ultrasound scans performed before and during this period. Horses were euthanized and tendons harvested for histology and immunohistochemistry. Data observed in this study showed effectiveness of cBMSC and BMMNC in regenerating tendon tissue after collagenase -induced tendonitis. Both cBMSC and BMMNC transplantation resulted in qualitatively similar regeneration of tendon extracellular matrix in terms of type I/III collagen ratio, fiber orientation, and COMP expression. After this favourable results, 20 horses were recruited referred for spontaneous lesions of the flexor tendons or the suspensory ligament. Horses were treated with autologous graft of BMMNCs.After treatment the. the exercise program allowed was 8 weeks stall rest, 4 weeks hand walking, 4 weeks trotting, 4 weeks of gradually raising of exercise level then horses were gone back to race. US characteristics of lesions started to improve at T3. CSA-l, FPS and TLS were better in all patients, with an appreciable filling of lesions indicated by a decreasing of CSA-l and increasing of TLS. When horses started the exercise program T8 tendon architecture improved, demonstrated by their longitudinal alignment and length. At T6, and persistently in later follow-up, no lameness was evident by clinical examination. At time of writing 12 patients (60%) were go back to races, while other 8 (40%) are under controlled exercise program. Re-injury rate was assessed at 25%. All the owners judged good to excellent the outcome in term of athletic success.

Effects of sainfoin (Onobrychis viciifolia) extract and monomers of condensed tannins on the association of abomasal nematode larvae with fundic explants.

Tannin-rich forages offer an alternative to anthelmintic chemicals to control gastrointestinal nematodes. However, the mode of action of such bioactive plants still needs to be assessed. Previous studies have shown that extracts of tannin-rich plants interfere with the first phase of host invasion, i.e., the exsheathment of infective larvae (L3s). In the current study, we examined the hypothesis that exposure to tannins could also affect the second phase of larval establishment, i.e., the tissue association/penetration of the exsheathed L3s into the digestive mucosae. An in vitro direct challenge technique using fundic explants was applied in this study. The main parasite model was Haemonchus contortus. The objectives were to verify: (i) whether a modification of the association/penetration of L3s with the mucosae occurred after contact with sainfoin extract; (ii) whether this is a dose-dependent phenomenon; (iii) whether tannins were responsible for these effects; (iv) whether these effects were dependent on the parasite species; and (v) how the biochemical structure of tannins might influence these effects. Following 3h contact with sainfoin extract at 1,200 microg/ml, the penetration of exsheathed L3s of H. contortus and Teladorsagia circumcincta into fundic explants was significantly reduced. Moreover, a dose-response relationship was found for H. contortus. For both nematodes, the changes were totally alleviated after addition of polyvinyl polypyrrolidone, an inhibitor of tannins, to the sainfoin extract, suggesting that tannins play a major role in the observed effects. Comparison of results obtained with different monomers of condensed tannins confirms a relationship between structure and activity, the prodelphinidin monomers and galloyl-derivatives being more effective than the procyanidin monomers. Combined with the delay or the inhibition of larval exsheathment previously shown, these effects could explain how tanniniferous plants reduce the establishment of infective larvae in small ruminants.

[Pathological change in hydatid cysts of Echinococcus granulosus treated with high intensity focused ultrasound].

OBJECTIVE: To investigate the pathological change in Echinococcus granulosus hydatid cysts treated with high intensity focus ultrasound (HIFU). METHODS: Thirty cysts with thinner wall and proper elasticity, taken from livers of infected sheep, were randomly divided into three groups. By cyclical multilayer radiation around the cyst wall, two experiment groups were treated with HIFU under 150 W and 250 W sound power respectively. The control group was treated by ordinary ultrasound for 2 min. RESULTS: The inner cyst wall of hydatid treated with HIFU became curved, thicker, stiffer, white and less transparent. The germinal layer was detached mostly from the laminated layers of hydatid in the experiment groups. Images from the transmission electron microscopy showed that in the experiment groups fabric texture of hydatid changed significantly and germinal cells were broken. CONCLUSION: HIFU in a model of cyclical multilayer radiation causes pathological damage of the E. granulosus hydatid.

Cystic hydatic disease in sheep: treatment with percutaneous aspiration and injection with dipeptide methyl ester.

An in vitro and in vivo study was conducted to show the effect of dipeptide methyl ester on the protoscolices of Echinococcus granulosus and in naturally infected sheep. Easily punctured cysts were located by ultrasonography. A PAIR and PAI method were performed by the injection of dipeptide methyl ester into these cysts at a final concentration of 110 mmol/L. Follow-up was conducted monthly by ultrasonography. After injection of the compound, the sheep were sacrificed at different times from 6 to 17 weeks. The size and the morphological aspect of treated cysts were noted. Samples were collected for histology and electron microscopy. In conclusion, these studies revealed significant and rapid detachment of the membrane of the treated cyst and alteration of the inner membrane in less than 5 min after injection of the drug, confirming the effect of the compound on the laminated layer of the parasite.