Extracorporeal perfusion of isolated organs of large animals - Bridging the gap between in vitro and in vivo studies

Since the early 20th century extracorporeal perfusion of large animal organs has been used to investigate a broad variety of research questions, thereby overcoming common draw backs of in vitro studies without suffering from ethical concerns associated with live animal research. The technique is in accordance with the 3R principles and represents an excellent opportunity to investigate in detail the physiology of organs under standardised conditions. It is also suitable for the translation of basic pre-clinical research into a more relevant arena prior to or avoiding altogether live animal research. Furthermore, organ perfusion has also been an important tool in developing methods of organ preservation for transplantation surgery. Yet due to the nature of the experiments only short term observations can be made and while cells are still exposed to their regional secretome, the whole organ itself is isolated from the body and correlations between organ-systems cannot be taken into consideration. This review gives an overview over the history of extracorporeal perfusion of large animal organs and limbs highlighting major achievements in the field and discussing different experimental set-ups. Advantages and limitations of this technique are presented. Prospective future research perspectives, which might include tracking of specific cell types and study of their distinct behaviour towards different stimuli, are given to illustrate the relevance of this method.</p

The effect of exercise on cytokine concentration in equine autologous conditioned serum

Background: Autologous conditioned serum (ACS) is a commonly administered intra-articular treatment for the management of osteoarthritis in athletic horses. Objectives: To investigate the influence of exercise on the concentration of cytokines in a non-commercial method of ACS production. Study design: Non-randomised cross over design. Methods: Whole blood was obtained from eight healthy Standardbred horses immediately prior to, 1 h and 24 h following a single bout of exhaustive exercise. Blood was processed using a non-commercial method of ACS production. Fluorescent microsphere immunoassay (FMIA) analysis was performed to quantify Interleukin 1 receptor antagonist (IL-1Ra), Interleukin-10 (IL-10), Interleukin 1β (IL-1β) and tumour necrosis factor α (TNF-α) concentrations at each time point. Mixed effect repeated measures analysis of variance (ANOVA) was used to compare the pre-exercise and post-exercise cytokine concentrations. Significance was set at P &lt; 0.05. Results: A reduced concentration of IL-1Ra (median 584.4, IQR 81.9–5098 pg/ml, p = 0.004) and an increased concentration of TNF-α (11.92, 9.28–39.75 pg/ml, P =.05) at 1 h post-exercise were observed when compared with baseline values (IL-Ra 7349, 1272–10 760 pg/ml; TNF TNF-α 11.16, 8.36–32.74 pg/ml). No difference in cytokine concentrations of IL-10 or IL-1β were found between any of the time points. Main limitations: The large biological variability and small sample size represents limitations of this study. Conclusions: These results suggest that a single bout of intense exercise can reduce the concentration of the anti-inflammatory cytokine IL-1Ra and increase the concentration of the pro-inflammatory cytokine TNF-α, reducing the 'anti-inflammatory' cytokine composition of ACS. Our findings suggest that collection of blood for ACS production should be performed no sooner than 24 h following a single episode of intense exercise.</p

Comparison of interfragmentary compression across simulated condylar fractures repaired using four techniques

IntroductionEquine condylar fractures are commonly repaired using cortex screws applied in lag fashion. Inadequate interfragmentary compression can lead to post-operative complications.MethodsLateral condylar fractures were simulated in 21 cadaver limbs (8 third metatarsals, 13 third metacarpals). In each limb, pressure-sensitive film (Prescale®, Fuji Photo Film Co.) was placed in each osteotomy prior to repair with 4.5 mm diameter cortex screws placed in lag fashion. Screws were placed in linear (L), triangular (T), linear plus a washer (LW) and sequentially tightened triangular configurations (TD1). All screws were tightened to a torque of 4 Nm. Pressure prints obtained were scanned using dedicated software (Fuji FPD-8010E, Fuji Photo Film Co.). A Bayesian Network (BN) model was developed to investigate the impact and interrelationship of each factor on interfragmentary compression. Sixty-three repairs (20*L, 24* T, 11*TD1, and 8*LW) performed on 21 limbs were included in the analysis.ResultsThe BN predicted mean contact area (±s.d.) for pressures within the operating range of the prescale film [≥2.5 Megapascals (MPa) ≤ 10 MPa] by L, T, TD1 and LW repairs were 403mm2 ± (140), 411 mm2 ± (120), 403 mm2 ± (120), and 366mm2 ± (70). The mean contact area (± s.d.) created by L, T, TD1 and LW repairs at pressures &gt;10 MPa were 112 mm2 ± (48), 167 mm2 ± (67), 142 mm2 ± (50), and 100mm2 ± (27). When pressures ≥2.5 MPA to ≤10 MPa were considered, the construct (T or L), washer and screw tightening sequence variables had a very low effect on interfragmentary contact area. At pressures &gt;10 MPa BN sensitivity findings were 16.3, 5.03, and 0.133% for construct, washer and screw tightening sequence. The BN model indicated that triangular repair configuration had a weak influence in the ≥2.5 MPa ≤ 10 MPa range and a moderate influence in the &lt;10 MPa range, on interfragmentary compression. The addition of a washer and the screw tightening sequence had a weak influence on interfragmentary compression at all pressure ranges.DiscussionThe results show that triangular repairs create larger interfragmentary contact areas at greater interfragmentary pressure in simulated condylar fractures, however it is unknown if this results in improved repair stability in the clinical scenario

Ex vivo effect of gold nanoparticles on porcine synovial membrane

Gold nanoparticles (AuNPs) have great potential as carriers for local drug delivery and as a primary therapeutic for treatment of inflammation. Here we report on the AuNP-synovium interaction in an ex vivo model of intra-articular application for treatment of joint inflammation. Sheets of porcine femoropatellar synovium were obtained post mortem and each side of the tissue samples was maintained in a separate fluid environment. Permeability to AuNPs of different sizes (5−52 nm) and biomarker levels of inflammation were determined to characterize the ex vivo particle interaction with the synovium. Lipopolysaccharide or recombinant human interleukin-1β were added to fluid environments to assess the ex vivo effect of pro-inflammatory factors on permeability and biomarker levels. The synovium showed size selective permeability with only 5 nm AuNPs effectively permeating the entire tissues’ width. This process was further governed by particle stability in the fluid environment. AuNPs reduced matrix metalloproteinase and lactate dehydrogenase activity and hyaluronic acid concentrations but had no effect on prostaglandin E(2) levels. Exposure to pro-inflammatory factors did not significantly affect AuNP permeation or biomarker levels in this model. Results with ex vivo tissue modeling of porcine synovium support an anti-inflammatory effect of AuNPs warranting further investigation

Intratumoural tigilanol tiglate in the multicentre treatment of equine sarcoids and cutaneous melanomas

BACKGROUND: Intralesional chemotherapeutic administration represents an important treatment option for equine cutaneous neoplasia. Tigilanol-tiglate (TT), a novel molecule extracted from Fontainea picrosperma, an Australian rainforest plant, is registered for intratumoural treatment of canine MCT, leading to rapid oncosis and tumour slough. Evidence from horses is limited but suggests that efficacy may be similar. OBJECTIVES: To evaluate the response to intratumoural TT treatment in horses with sarcoids (fibroblastic/nodular) and cutaneous melanomas. STUDY DESIGN: Two noncontrolled prospective multicentre clinical trials, one for each of sarcoids and melanomas. METHODS: Cases were enrolled across multiple sites and treated by the same site-specific clinician with intralesional TT (sarcoids: 0.35 mg/cm3; melanomas: 0.2 mg/cm3 of tumour volume - Tvol; max dose 2 mg). Quantitative (Tvol regression) and qualitative outcomes (likely tumour free (LTF) per expert opinion) were recorded, and potential determinants of efficacy were assessed using random effects logistic models. A full clinical response was complete Tvol regression and a LTF treatment site. RESULTS: Forty-one sarcoids and 97 melanomas were enrolled and treated. 73/74% of treated sarcoids/melanomas showed complete Tvol regression. 64/61% (sarcoids/melanomas) showed a full clinical response at medians of 546/247 days post final treatment. For both tumour types, this response was dependent on initial tumour volume (Psarcoids = 0.006; Pmelanomas <0.001). The predicted probability of a full clinical response was 6 times greater for initially small sarcoids (Tvol = 1 cm3) than for the maximum study volume (Tvol = 6 cm3). For melanomas in the perineal region, this was 11 times greater for Tvol ≤0.3 cm3 than for tumours ≥2.0 cm3. For melanomas, tumour location further affected treatment efficacy = 0.005). In total, 5 adverse events were reported. MAIN LIMITATIONS: Lack of treatment control and histologic/biomolecular follow-up data. CONCLUSIONS: The observed therapeutic efficacy of TT supports clinical use as well as early interventions in horses. Successful use necessitates knowledge of the drug's mode of action and management of associated local site responses

Quantitative radiographic interpretation and conservative treatment outcome in horses with osteoarthritis of the distal tarsal joints

Various radiographic rating scales (RRSs) for use in horses with distal tarsal joint osteoarthritis (DTJ OA) have been described in the literature but little information is available on their reliability in use. The aim of the first experiment of the study was to develop a RRS based on the consensus of experts in equine diagnostic imaging and orthopaedics, and to test the RRS for reliability. For this purpose 17 experts were invited to participate in an iterative consultation process (Delphi) designed to develop an agreement on the importance of radiographic features, reported to be consistent with DTJ OA. This process was conducted by electronic questionnaire. Rradiographic features for which an agreement was found, were incorporated in the RRS, which used a visual analogue scale. To test the RRS's reliability nine equine surgeons from two academic institutions applied the RRS on two occasions, and a verbal descriptive rating scale, to three sets of tarsal radiographs, each comprising 4 standard radiographic views. Reliability was assessed using Bland-Altman plots and by calculating the 95% agreement limits. ANOVA was used to identify significant interactions between the ratings of different assessors made from different views and on each occasion. Of 17 invited experts nine participated and completed the consultation process. Seven radiographic features were identified and used in the RRS. Rating of DTJ OA was different for the nine equine surgeons (assessors). The most precise assessor's second ratings were between 16 mm higher and 18 mm lower than the 1st. Significant variables were: "joint", "assessor" and "assessment" (univariable ANOVA); and "joint and assessor" and "assessor and assessment" (multivariable ANOVA). Reliability of the verbal descriptive rating scale was higher than for the RRS. The RRS developed for radiographic interpretation of DTJ OA as a result of the Delphi consultation process was less reliable than the use of a verbal descriptive rating scale. The repeatability of the RRS was not affected by the assessors' professional experience. In conclusion the RRS would not be useful clinically. Osteoarthritis of the DTJ, affecting the distal intertarsal (DIT) and tarsometatarsal (TMT) joint, is a common cause of hindlimb lameness in horses. Management options include i.a. treatment of the affected joints but only anecdotal information is available on the outcome. The aim of the second experiment of this study was to document short and long term treatment outcome in horses receiving i.a. methylprednisolone acetate (MPA; Depo-MedroneV) or triamcinolone acetonide (TR; Adcortyl) with or without hyaluronic acid (HA; Hyonate) as treatment of DTJ OA.Cases were selected by searching medical records. Inclusion criteria included ? 50% improvement in lameness following i.a. analgesia of the DIT and/or TMT joint and i.a. treatment with TR (+/- HA) or MPA. Change in lameness grade between examinations was tested using a Wilcoxon signed rank test for each horse, and between horses, grouped according to radiographic severity of DTJ OA and treatment, using a Mann Whitney test. Significance was set at P<0.05. Long term outcome was assessed using an owner telephone questionnaire. A positive treatment outcome was no lameness with the horse able to perform as intended without NSAID administration. Horses treated once with i.a. MPA or TR (+/- HA) showed improvement in hindlimb lameness after a median of 56 days (P<0.000). No difference was found between the use of MPA and TR (P=0.81). In horses treated twice, no further improvement was seen after the first treatment (P=0.141). Lameness in horses with diffuse increased radiopharmaceutical uptake (IRU) of the DTJ identified at scintigraphy tended to improve, in contrast to horses with focal IRU (Pfocal = 0.1; Pdiffuse = 0.032). Radiographic severity of OA did not affect outcome. 13/34 horses (38.2%) had a positive and 21/34 (61.8%) a negative long term outcome. It was concluded that intra-articular corticosteroids can be effective in the management of DTJ OA in horses