Paolo Franci

Decellularized Allogeneic Heart Valves Demonstrate Self-Regeneration Potential after a Long-Term Preclinical Evaluation

Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (n = 11) with 6 (n = 5) and 15 (n = 6) follow-up months. Repositioned native valves (n = 2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.

Physiological Performance of a Detergent Decellularized Heart Valve Implanted for 15 Months in Vietnamese Pigs: Surgical Procedure, Follow‐up, and Explant Inspection

This study features the longest experimental follow-up for decellularized heart valves implanted in an animal model. Porcine aortic heart valves were decellularized according to a disclosed standardized method in which TRITON X-100 and sodium cholate (TRICOL) are used in succession, followed by a further treatment with the endonuclease Benzonase to completely remove the nucleic acid remnants. Experimental animals (n = 17), represented by Vietnamese pigs (VPs), received a decellularized aortic allograft as a substitute for the replacement of their right ventricular outflow tract. The surgical implantation of the TRICOL-treated aortic valve conduit was successful in 11 VPs, while perioperative or postoperative complications occurred in the remaining six animals. In the sham-operated group (n = 4), the native pulmonary root was excised and immediately reimplanted orthotopically in the same animal. Echocardiography demonstrated a satisfactory hemodynamic performance of the TRICOL-treated valves during follow-up as well as the absence of relevant leaflet alterations concerning thickness and motility or valve insufficiency. At explantation, macroscopic inspection of tissue-engineered heart valve conduits did not evidence calcifications and showed a decreased wall thickness, comparable to that of the reimplanted native pulmonary roots. Noteworthy, extended functional performance, recovery of DNA content, and active extracellular matrix precursor incorporation are apparently compatible with the properties of a living self-supporting substitute.

A retrospective study of efficacy and side effects of intrathecal administration of hyperbaric bupivacaine and morphine solution in 39 dogs undergoing hind limb orthopaedic surgery

OBJECTIVE To evaluate the intraoperative efficacy of intrathecal anaesthesia with hyperbaric bupivacaine 0.5% and morphine 1% solution (HIA) in dogs undergoing hind limb orthopaedic surgery, using the cardiovascular response to surgical stimulation and to report the perioperative side effects. STUDY DESIGN Retrospective clinical study. ANIMALS Forty-three dogs that underwent general anaesthesia for hind limb orthopaedic surgery between 2010 and 2011. METHODS The anaesthesia records of dogs that received HIA were reviewed. The bupivacaine and morphine doses were calculated based on body mass (BM) and spinal cord length (SCL). Cardiovascular response (CR) to surgical stimulation, the incidence of hypotension, bradycardia, urinary retention, pruritus and offset of motor block were all reported. The intraoperative time-to-event probability of CR was analyzed using Kaplan-Meier survival analysis. RESULTS The median (range) bupivacaine dose related to BM was 0.57 (0.40-0.78) mg kg(-1) , while that related to SCL was 0.13 (0.08-0.19) mg cm(-1) . A CR was observed in 3/39 (8%) dogs within the first hour after intrathecal injection (Ii) and in 9/39 (23%) dogs over the entire duration of surgery. At 70 minutes from Ii the event-free probability of CR fell below 80%. Hypotension was observed in 12/39 (31%), bradycardia in 6/39 (15%), pruritus in 3/39 (8%), and urinary retention in 3/39 (8%) dogs respectively. Five hours after Ii, 35/39 (89%) dogs were able to walk with only residual ataxia. CONCLUSIONS AND CLINICAL RELEVANCE Intrathecal anaesthesia with hyperbaric bupivacaine 0.5% and morphine 1% solution provided effective intraoperative antinociception up to 70 minutes in dogs undergoing hind limb surgery. The technique of HIA can provide effective analgesia during short hind limb surgeries in dogs.

Correlation of the ratio of caudal vena cava diameter and aorta diameter with systolic pressure variation in anesthetized dogs

OBJECTIVE To evaluate the correlation coefficient of the ratio between diameter of the caudal vena cava (CVC) and diameter of the aorta (Ao) in dogs as determined ultrasonographically with systolic pressure variation (SPV). ANIMALS 14 client-owned dogs (9 females and 5 males; mean ± SD age, 73 ± 40 months; mean body weight, 22 ± 7 kg) that underwent anesthesia for repair of skin wounds. PROCEDURES Anesthesia was induced. Controlled mechanical ventilation with a peak inspiratory pressure of 8 cm H2O was immediately started, and SPV was measured. During a brief period of suspension of ventilation, CVC-to-Ao ratio was measured on a transverse right-lateral intercostal ultrasonographic image obtained at the level of the porta hepatis. When the SPV was ≥ 4 mm Hg, at least 1 bolus (3 to 4 mL/kg) of Hartmann solution was administered IV during a 1-minute period. Bolus administration was stopped and the CVC-to-Ao ratio measured when SPV was < 4 mm Hg. Correlation coefficient analysis was performed. RESULTS 28 measurements were obtained. The correlation coefficient was 0.86 (95% confidence interval, 0.72 to 0.93). Mean ± SD SPV and CVC-to-Ao ratio before bolus administration were 7 ± 2 mm Hg and 0.52 ± 0.16, respectively. Mean ± SD SPV and CVC-to-Ao ratio after bolus administration were 2 ± 0.6 mm Hg and 0.91 ± 0.13, respectively. CONCLUSIONS AND CLINICAL RELEVANCE In this study, the CVC-to-Ao ratio was a feasible, noninvasive ultrasonographically determined value that correlated well with SPV.

Use of systolic pressure variation to predict the cardiovascular response to mini-fluid challenge in anaesthetised dogs

Systolic pressure variation (SPV), the maximum variation in systolic pressure values following a single positive pressure breath delivered by controlled mechanical ventilation (CMV), is highly correlated with volaemia in dogs. The aim of this study was to determine an SPV value that would indicate when fluid administration would be beneficial in clinical practice. Twenty-six client-owned dogs were anaesthetised, following which CMV with a peak inspiratory pressure (PIP) of 8 cmH2O was applied. After SPV measurement and recording of heart rate (HR) and blood pressure (BP), 3 mL/kg fluid were administered, then HR and BP were recorded again. Dogs exhibiting a 10% decrease in HR and/or an increase in BP were defined as responders, and their SPV pre-bolus was analysed retrospectively. SPV values > 4 mmHg or >4.5% predicted haemodynamic improvement in dogs with normal cardiovascular function, with a sensitivity of 90% and a specificity of 87%. The area under the curve receiver operating characteristic value for SPV was 0.931 mmHg (95% confidence interval, CI, 0.76-0.99 mmHg) and 0.944% (95% CI 0.78-0.99%). It is proposed that SPV values > 4.5% in dogs with a normal cardiovascular function, anaesthetised with isoflurane in oxygen and air, and on CMV (PIP 8 cmH2O), can be used to predict a cardiovascular response (>10% increase in mean arterial BP and/or >10% decrease in heart rate).

Thoracic Epidural Catheter Placement Using a Paramedian Approach with Cephalad Angulation in Three Dogs

Objective To describe a technique for insertion of a thoracic epidural catheter. Study Design Clinical report. Animals Dogs (n = 3) undergoing thoracic wall resection and thoracotomy. Methods A paramedian approach with cephalic angulation was used to place a 24-g epidural catheter in 3 dogs. Dogs 1 and 2 had left caudal thoracic wall resection and dog 3 had left thoracotomy. In dog 1, the epidural catheter was inserted at L2–L3 intervertebral space and the tip of the catheter advanced to the level of T13 vertebral body. In dog 2, the epidural catheter was inserted at T12–T13 intervertebral space and the tip of the catheter was advanced to the level of T8 vertebral body. In dog 3, the epidural catheter was inserted at T13–L1 intervertebral space and its tip advanced until reaching the vertebral body of T10. All dogs were administered a combination of bupivacaine and morphine through the epidural catheter to provide intra- and postoperative analgesia. Results The peridural space was identified and the tip of the catheter was positioned where intended in all dogs. Dog 1 developed transient Horners syndrome and dog 3 required intraoperative fentanyl during the first part of the procedure. Conclusion Paramedian approach with cephalad angulation is a suitable technique to place thoracic epidural catheters in dogs.OBJECTIVE To describe a technique for insertion of a thoracic epidural catheter. STUDY DESIGN Clinical report. ANIMALS Dogs (n = 3) undergoing thoracic wall resection and thoracotomy. METHODS A paramedian approach with cephalic angulation was used to place a 24-g epidural catheter in 3 dogs. Dogs 1 and 2 had left caudal thoracic wall resection and dog 3 had left thoracotomy. In dog 1, the epidural catheter was inserted at L2-L3 intervertebral space and the tip of the catheter advanced to the level of T13 vertebral body. In dog 2, the epidural catheter was inserted at T12-T13 intervertebral space and the tip of the catheter was advanced to the level of T8 vertebral body. In dog 3, the epidural catheter was inserted at T13-L1 intervertebral space and its tip advanced until reaching the vertebral body of T10. All dogs were administered a combination of bupivacaine and morphine through the epidural catheter to provide intra- and postoperative analgesia. RESULTS The peridural space was identified and the tip of the catheter was positioned where intended in all dogs. Dog 1 developed transient Horners syndrome and dog 3 required intraoperative fentanyl during the first part of the procedure. CONCLUSION Paramedian approach with cephalad angulation is a suitable technique to place thoracic epidural catheters in dogs.

Tissue-Engineered Heart Valves: Intra-operative Protocol

Tissue engineering of heart valves investigates the possibility to create a fully compatible and biomimetic graft able to provide host cell repopulation like the native living valve. Decellularized aortic and pulmonary valves and synthetic polymers have been used to promote the creation of a native-like scaffold suitable to be colonized by cells either in vitro, in dynamic bioreactors or in vivo using different animal models. The herein presented research provides the intra-operative protocol and details of surgical technique. Porcine aortic valve conduits were decellularized and implanted in the right ventricular outflow tract of Vietnamese pigs.

Lethal septic shock after dental scaling in a healthy dog due to Ochrobactrum anthropi-contaminated propofol

A 10-year-old, 6-kg male Yorkshire terrier dog was scheduled for routine dental cleaning. No significant problem was observed either during anaesthesia, which was induced with propofol, or during recovery. However, 2 hours after discharge, the dogs owner returned to the clinic, complaining that the animal was lethargic and had had bloody diarrhoea. On physical examination the dog was depressed, dyspnoeic, tachycardic and hypoglycaemic. Despite supportive treatment, the dog deteriorated and died within a few hours.A presumed diagnosis of sepsis was confirmed by laboratory testing. Bacteriological and molecular examinations of both premortem blood samples and the anaesthetic, highlighted the presence of Ochrobactrum anthropi, an opportunistic pathogen usually associated with immunocompromised hosts with indwelling medical devices. To the authors’ knowledge, this is the first case of sepsis in a healthy dog due to contamination of an anaesthetic solution by O. anthropi, suggesting a potential role of this microorganism as an emerging pathogen.

Clinical evaluation of an end-tidal target-controlled infusion closed-loop system for isoflurane administration in horses undergoing surgical procedures

A new volatile anaesthetic agent delivery system was tested in 15 horses undergoing scheduled surgical procedures. The delivery system consisted of a laptop computer (with dedicated software), a computer-controlled syringe driver (loaded with liquid isoflurane) connected to the inspiratory arm of a large-animal circle breathing system and a respiratory gas monitor, providing isoflurane end-tidal concentrations (ET(measured)) every 20 s to the computer. Following induction and connection to the breathing system, mechanical ventilation was started. The bodyweight (BW), fresh gas flow, breathing system and ventilator volume, and end-tidal isoflurane target (ET(target)) were entered into the computer. Using Lowes equation, the software calculated the prime dose to be delivered by the syringe driver over 2 min. After this, the system delivered each minute the amount of isoflurane as determined by the following equation: Isoflurane (mL) = {2 × λ(B/G) × (200 × BW(0.75)) × (ET(target) - ET(measured)) + (fresh gas flow - (BW(0.75) × 0.07)) × (ET(measured))}/206. A fresh gas flow of 4 L oxygen min(-1) was administered until the inspired fraction of oxygen reached 0.7, and was then decreased. A target of 1.5% end-tidal isoflurane was initially used and subsequently adjusted to the clinical requirements. The system performance was evaluated using the median prediction error (MDPE) and the median absolute performance error (MDAPE), which were -3.6% and 5.29%, respectively. It was concluded that this system was useful to achieve end-tidal target-controlled infusion of isoflurane during equine anaesthesia.

Respiratory variation in aortic blood peak velocity and caudal vena cava diameter can predict fluid responsiveness in anaesthetised and mechanically ventilated dogs

BACKGROUND AND M&MS Dynamic preload indices, such as systolic pressure variation (SPV), aortic flow peak velocity variation (ΔVpeak) and distensibility index of the caudal vena cava (CVCDI), are reliable indices for predicting fluid responsiveness in humans. This study aimed to investigate the ability of these indices to predict fluid response in 24 healthy dogs undergoing general anaesthesia and mechanical ventilation. Aortic flow peak velocity variation (∆Vpeak), CVCDI, and SPV were calculated before volume expansion (5mL/kg bolus of lactated Ringers solution). The aortic velocity time integral (VTI) was measured before and after volume expansion as a surrogate of stroke volume. Dogs were considered responders (n=9) when the VTI increase was ≥15% and non-responders (n=15) when the increase was <15%. RESULTS AND CONCLUSIONS Before volume expansion, ΔVpeak, CVCDI and SPV were higher in responders than in non-responders (P=0.0009, P=0.0003, and P=0.0271, respectively). Receiver operating characteristic (ROC) curves were plotted for the three indices. The areas under the ROC curves for SPV, ΔVpeak, and CVCDI were 0.91 (CI 0.73-0.99; P=0.0001), 0.95 (CI 0.77-1; P=0.0001), and 0.78 (CI 0.56-0.92; P=0.015), respectively. The best cut-offs were 6.7% for SPV (sensitivity, 77.78%; specificity, 93.33%), 9.4% for ΔVpeak (sensitivity, 88.89%; specificity, 100%), and 24% for CVCDI (sensitivity, 77.78%; specificity, 73.33). In conclusion, ΔVpeak, CVCDI, and SPV are reliable predictors of fluid responsiveness in healthy dogs undergoing general anaesthesia and mechanical ventilation.