Protective effects of mesenchymal stem cells-derived extracellular vesicles against ischemia-reperfusion injury of hearts donated after circulatory death: Preliminary study in a pig model.

INTRODUCTION: Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after Circulatory Death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitable more extended period of warm unprotected ischemia between circulatory arrest and graft procurement. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown remarkable protective effects against ischemia-reperfusion injury. Thus, we aimed to enhance grafts preservation from DCD donors, through treatment with MSC-EVs. METHODS: Female pigs were euthanized by barbiturate overdose and after 20 min of a flat EKG, the chest was opened, the heart harvested and subsequently connected to an extracorporeal perfusion machine. MSC-EVs, isolated by ion exchange chromatography, were added to the perfusion solution (1×1011 particles) and the heart was perfused for 2 h. Then, heart tissue biopsies were taken to assess histological changes, mitochondrial morphology, antioxidant enzyme activity and inflammation mediators' expression. Biochemical parameters of myocardial viability were assessed in the perfusate. RESULTS: The treatment with MSC-EVs significantly prevented mitochondria swelling, mitochondrial cristae loss and oxidative stress in cardiac tissue. The protective effect of MSC-EVs was confirmed by the delayed increase of the cardiac-specific enzymes CK and TnC in the perfusate and the reduction of caspase-3+ cells in tissue sections. CONCLUSION: MSC-EVs improve graft quality by preserving the mitochondrial ultrastructure protecting the myocardium against oxidative stress, reducing apoptosis of cardiac cells and preventing the increase of pro-inflammatory cytokines.

Origami-inspired soft fluidic actuation for minimally invasive large-area electrocorticography.

Electrocorticography is an established neural interfacing technique wherein an array of electrodes enables large-area recording from the cortical surface. Electrocorticography is commonly used for seizure mapping however the implantation of large-area electrocorticography arrays is a highly invasive procedure, requiring a craniotomy larger than the implant area to place the device. In this work, flexible thin-film electrode arrays are combined with concepts from soft robotics, to realize a large-area electrocorticography device that can change shape via integrated fluidic actuators. We show that the 32-electrode device can be packaged using origami-inspired folding into a compressed state and implanted through a small burr-hole craniotomy, then expanded on the surface of the brain for large-area cortical coverage. The implantation, expansion, and recording functionality of the device is confirmed in-vitro and in porcine in-vivo models. The integration of shape actuation into neural implants provides a clinically viable pathway to realize large-area neural interfaces via minimally invasive surgical techniques.

Isolation and characterization of mammary epithelial cells derived from Göttingen Minipigs: A comparative study versus hybrid pig cells from the IMI-ConcePTION Project.

The value of pig as "large animal model" is a well-known tool for translational medicine, but it can also be beneficial in studying animal health in a one-health vision. The ConcePTION Project aims to provide new information about the risks associated with medication use during breastfeeding, as this information is not available for most commonly used drugs. In the IMI-Conception context, Göttingen Minipigs have been preferred to hybrid pigs for their genetic stability and microbiological control. For the first time, in the present research, three primary cell cultures of mammary epithelial cells were isolated and characterized from Göttingen Minipigs (mpMECs), including their ability to create the epithelial barrier. In addition, a comparative analysis between Göttingen Minipigs and commercial hybrid pig mammary epithelial cells (pMECs) was conducted. Epithelial markers: CKs, CK18, E-CAD, ZO-1 and OCL, were expressed in both mpMECs and pMECs. RT2 Profiler PCR Array Pig Drug Transporters showed a similar profile in mRNA drug transporters. No difference in energy production under basal metabolic condition was evidenced, while under stressed state, a different metabolic behaviour was shown between mpMECs vs pMECs. TEER measurement and sodium fluorescein transport, indicated that mpMECs were able to create an epithelial barrier, although, this turned out to be less compact than pMECs. By comparing mpMECs with mammary epithelial cells isolated from Hybrid pigs (pMECs), although both cell lines have morphological and phenotypic characteristics that make them both useful in barrier studies, some specific differences exist and must be considered in a translational perspective.

Can environmental nebulization of lavender essential oil (L. angustifolia) improve welfare and modulate nasal microbiota of growing pigs?

The use of phytoextracts has been proposed as a method to improve animal welfare, also in pigs, by reducing stress and anxiety and improving performances. Lavandula angustifolia (Miller) essential oil (LaEO) is an interesting calming phytoextract that could be administered by inhalation for prolonged periods of time to help pigs coping with on-farm conditions. The aim of this study was to assess the effects of daily inhalation of vaporized LaEO on pigs' welfare and health indicators, and nasal microbiota, trying to understand whether this phytoextract represents a feasible tool to improve animal welfare under intensive farming conditions. Eighty-four crossbred barrows were randomly divided into 3 experimental groups: control (C); lavender (L): 3 vaporization sessions of 10 min each of a custom made 1% solution of LaEO; sham (S): same vaporization sessions of L group but only using the solution vehicle. Experimental readouts included growth parameters, behavioural traits, tail and skin lesions, hair steroids and nasal microbiota. L group animals did not show altered growth performance and seemed calmer (increased recumbency time), with decreased amount of skin lesions also associated with lower severity class for tail lesions. They also showed decreased CORT/DHEA ratio, potentially suggesting a beneficial effect of LaEO. Inhalation of LaEO significantly affected the nasal pig microbiome by reducing its diversity. Overall, the study suggests how inhalation of Lavender essential oil may be capable of improving welfare in growing pigs, yet it is pivotal to consider the microbial modulatory capabilities of essential oils before exploiting them on larger scale.

The p-ERG spatial acuity in the biomedical pig under physiological conditions.

Pigs are becoming an important pre-clinical animal species for translational ophthalmology, due to similarities with humans in anatomical and physiological patterns. Different models of eye disorders have been proposed, and they are good candidates to assess biocompatibility/functionality of retinal prostheses. Electroretinography is a common tool allowing to gain information on retinal function, with several types of electroretinogram (ERG) been implemented including full field (ff-ERG), multifocal (mf-ERG) and pattern (p-ERG). p-ERG represents a valuable tool to monitor Retinal Ganglion Cells (RGCs) activity and can be used to calculate p-ERG spatial acuity. Unfortunately, scarce methodological data are available regarding recording/interpretation of p-ERG and retinal acuity in biomedical pigs yet enhancing knowledge regarding pig vision physiology will allow for more refined and responsible use of such species. Aim of this study was to record p-ERG in juvenile pigs to functionally assess visual acuity. Six female hybrid pigs underwent two p-ERG recording sessions at 16 and 19 weeks of age. Photopic ff-ERG were also recorded; optical coherence tomography (OCT) and histology were used to confirm retinal integrity. ff-ERG signals were repeatable within/across sessions. All p-ERG traces consistently displayed characterizing peaks, and the progressive decrease of amplitude in response to the increment of spatial frequency revealed the reliability of the method. Mean p-ERG spatial acuities were 5.7 ± 0.14 (16 weeks) and 6.2 ± 0.15 cpd (19 weeks). Overall, the p-ERG recordings described in the present work seem reliable and repeatable, and may represent an important tool when it comes to vision assessment in pigs.

Lithotripsy of Calcified Aortic Valve Leaflets by a Novel Ultrasound Transcatheter-Based Device.

The increasing incidence of calcific aortic valve disease necessitates the elaboration of new strategies to retard the progression of the pathology with an innovative solution. While the increasing diffusion of the transcatheter aortic valve replacements (TAVRs) allows a mini-invasive approach to aortic valve substitution as an alternative to conventional surgical replacement (SAVR) in an always larger patient population, TAVR implantation still has contraindications for young patients. In addition, it is liable to undergo calcification with the consequent necessity of re-intervention with conventional valve surgery or repeated implantation in the so-called TAVR-in-TAVR procedure. Inspired by applications for non-cardiac pathologies or for vascular decalcification before stenting (i.e., coronary lithotripsy), in the present study, we show the feasibility of human valve treatment with a mini-invasive device tailored to deliver shockwaves to the calcific leaflets. We provide evidence of efficient calcium deposit ruptures in human calcified leaflets treated ex vivo and the safety of the treatment in pigs. The use of this device could be helpful to perform shockwaves valvuloplasty as an option to retard TAVR/SAVR, or as a pretreatment to facilitate prosthesis implantation and minimize the occurrence of paravalvular leak.

Biocompatibility of a Conjugated Polymer Retinal Prosthesis in the Domestic Pig.

The progressive degeneration of retinal photoreceptors is one of the most significant causes of blindness in humans. Conjugated polymers represent an attractive solution to the field of retinal prostheses, and a multi-layer fully organic prosthesis implanted subretinally in dystrophic Royal College of Surgeons (RCS) rats was able to rescue visual functions. As a step toward human translation, we report here the fabrication and in vivo testing of a similar device engineered to adapt to the human-like size of the eye of the domestic pig, an excellent animal paradigm to test therapeutic strategies for photoreceptors degeneration. The active conjugated polymers were layered onto two distinct passive substrates, namely electro-spun silk fibroin (ESF) and polyethylene terephthalate (PET). Naive pigs were implanted subretinally with the active device in one eye, while the contralateral eye was sham implanted with substrate only. Retinal morphology and functionality were assessed before and after surgery by means of in vivo optical coherence tomography and full-field electroretinogram (ff-ERG) analysis. After the sacrifice, the retina morphology and inflammatory markers were analyzed by immunohistochemistry of the excised retinas. Surprisingly, ESF-based prostheses caused a proliferative vitreoretinopathy with disappearance of the ff-ERG b-wave in the implanted eyes. In contrast, PET-based active devices did not evoke significant inflammatory responses. As expected, the subretinal implantation of both PET only and the PET-based prosthesis locally decreased the thickness of the outer nuclear layer due to local photoreceptor loss. However, while the implantation of the PET only substrate decreased the ff-ERG b-wave amplitude with respect to the pre-implant ERG, the eyes implanted with the active device fully preserved the ERG responses, indicating an active compensation of the surgery-induced photoreceptor loss. Our findings highlight the possibility of developing a new generation of conjugated polymer/PET-based prosthetic devices that are highly biocompatible and potentially suitable for subretinal implantation in patients suffering from degenerative blindness.

Enhancing the Therapeutic Potential of Sulfamidase for the Treatment of Mucopolysaccharidosis IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.

In Vitro Effects of Tea Tree Oil (Melaleuca Alternifolia Essential Oil) and its Principal Component Terpinen-4-ol on Swine Spermatozoa.

The growing interest towards essential oils stems from their biological capabilities that include antibacterial and antioxidant effects. Such properties may be extremely useful in the reproductive field; nonetheless essential oils show toxic effects that can lead to cell disruption. The present study aimed to evaluate and compare the effects of tea tree oil (TTO) and its principal component terpinen-4-ol (TER) on the morpho-functional parameters of swine spermatozoa. Experimental samples were prepared by suspending 15 × 107 spermatozoa in 5 mL of medium with different concentrations of the above-mentioned compounds: from 0.2 to 2 mg/mL at an interval of 0.2 for TTO, while TER concentrations were adjusted according to its presence in TTO (41.5%). After 3 h incubation at 16 °C, samples were analyzed for pH, viability, acrosome status, and objective motility. The results highlighted a concentration-dependent effect of TTO with total motility as the most sensitive parameter. TER was better tolerated, and the most sensitive parameters were related to membrane integrity, suggesting a different pattern of interaction. The study confirms the importance of evaluating the effects of natural compounds on spermatozoa before exploiting their beneficial effects. Spermatozoa seem to be good candidates for preliminary toxicological screenings in the light of their peculiar properties.

Non-clinical Models to Determine Drug Passage into Human Breast Milk.

BACKGROUND: Successful practice of clinical perinatal pharmacology requires a thorough understanding of the pronounced physiological changes during lactation and how these changes affect various drug disposition processes. In addition, pharmacokinetic processes unique to lactation have remained understudied. Hence, determination of drug disposition mechanisms in lactating women and their babies remains a domain with important knowledge gaps. Indeed, lack of data regarding infant risk during breastfeeding far too often results in discontinuation of breastfeeding and subsequent loss of all the associated benefits to the breastfed infant. In the absence of age-specific toxicity data, human lactation data alone are considered insufficient to rapidly generate the required evidence regarding risks associated with medication use during lactation. METHODS: Systematic review of literature to summarize state-of-the art non-clinical approaches that have been developed to explore the mechanisms underlying drug milk excretion. RESULTS: Several studies have reported methods to predict (to some extent) milk drug excretion rates based on physicochemical properties of the compounds. In vitro studies with primary mammary epithelial cells appear excellent approaches to determine transepithelial drug transport rates across the mammary epithelium. Several of these in vitro tools have been characterized in terms of transporter expression and activity as compared to the mammary gland tissue. In addition, with the advent of physiology-based pharmacokinetic (PBPK) modelling, these in vitro transport data may prove instrumental in predicting drug milk concentration time profiles prior to the availability of data from clinical lactation studies. In vivo studies in lactating animals have proven their utility in elucidating the mechanisms underlying drug milk excretion. CONCLUSION: By combining various non-clinical tools (physicochemistry-based, in vitro and PBPK, in vivo animal) for drug milk excretion, valuable and unique information regarding drug milk concentrations during lactation can be obtained. The recently approved IMI project ConcePTION will address several of the challenges outlined in this review.

In Vivo Effects of Einkorn Wheat (Triticum monococcum) Bread on the Intestinal Microbiota, Metabolome, and on the Glycemic and Insulinemic Response in the Pig Model.

Einkorn wheat (Triticum monococcum) is characterized by high content of proteins, bioactive compounds, such as polyunsaturated fatty acids, fructans, tocols, carotenoids, alkylresorcinols, and phytosterols, and lower α-, ß-amylase and lipoxygenase activities compared to polyploid wheat. These features make einkorn flour a good candidate to provide healthier foods. In the present study, we investigated the effects of einkorn bread (EB) on the intestinal physiology and metabolism of the pig model by characterizing the glycemic and insulinemic response, and the microbiota and metabolome profiles. Sixteen commercial hybrid pigs were enrolled in the study; four pigs were used to characterize postprandial glycemic and insulinemic responses and twelve pigs underwent a 30-day dietary intervention to assess microbiota and metabolome changes after EB or standard wheat bread (WB) consumption. The postprandial insulin rise after an EB meal was characterized by a lower absolute level, and, as also observed for glucose, by a biphasic shape in contrast to that in response to a WB meal. The consumption of EB led to enrichment in short-chain fatty acid producers (e.g., Blautia, Faecalibacterium, and Oscillospira) in the gut microbiota and to higher metabolic diversity with lower content of succinate, probably related to improved absorption and therefore promoting intestinal gluconeogenesis. The observed changes, at both a compositional and metabolic scale, strongly suggest that EB consumption may support a health-promoting configuration of the intestinal ecosystem.

Targeting and silencing of rhodopsin by ectopic expression of the transcription factor KLF15.

The genome-wide activity of transcription factors (TFs) on multiple regulatory elements precludes their use as gene-specific regulators. Here we show that ectopic expression of a TF in a cell-specific context can be used to silence the expression of a specific gene as a therapeutic approach to regulate gene expression in human disease. We selected the TF Krüppel-like factor 15 (KLF15) based on its putative ability to recognize a specific DNA sequence motif present in the rhodopsin (RHO) promoter and its lack of expression in terminally differentiated rod photoreceptors (the RHO-expressing cells). Adeno-associated virus (AAV) vector-mediated ectopic expression of KLF15 in rod photoreceptors of pigs enables Rho silencing with limited genome-wide transcriptional perturbations. Suppression of a RHO mutant allele by KLF15 corrects the phenotype of a mouse model of retinitis pigmentosa with no observed toxicity. Cell-specific-context conditioning of TF activity may prove a novel mode for somatic gene-targeted manipulation.

Bioavailability of Microencapsulated Iron from Fortified Bread Assessed Using Piglet Model.

The aim of this study was to investigate the influence of oral iron supplementation, in the form of fortified breads, on the growth performance, health, iron status parameters, and fecal metabolome of anemic piglets. A study was conducted on 24 hybrid (Large White × Landrace × Duroc) piglets. From day 44, the post-natal 12 piglets were supplemented with 100 g of one of two experimental breads, each fortified with 21 mg of ferrous sulphate, either encapsulated or not. After one week of oral supplementation, hematological parameters (hematocrit value, hemoglobin, and red blood cells) showed statistically significant differences (p ≤ 0.05). Piglets fed with the fortified breads had higher iron concentrations in the heart, liver, and intestinal mucosa compared to anemic piglets fed with control bread. Gene expression of hepcidin, iron exporter ferroportin (IREG1), and divalent metal transporter 1 (DMT1), together with concentrations of plasma ferritin, showed no significant statistical differences between groups. Both fortified breads could be used as sources of bioavailable iron. The seven-day intervention trial showed microencapsulation to have only a mild effect on the effectiveness of iron supplementation in the form of fortified bread.

The biomedical piglet: establishing reference intervals for haematology and clinical chemistry parameters of two age groups with and without iron supplementation.

BACKGROUND: The similarities between swine and humans in physiological and genomic patterns, and the great correlation in size and anatomy, make pigs extremely useful in preclinical studies. New-born piglets can represent a model for congenital and genetic diseases in new-born children. It is known that piglets may have significant differences in clinicopathological results compared to adult pigs. Therefore, adult laboratory reference intervals cannot be applied to piglets. The aim of this study was to compare haematological and chemical variables in piglets of two ages and determinate age-related reference intervals for commercial hybrid young pigs. Blood samples were collected under general anaesthesia from 130 animals divided into five- (P5) and 30- (P30) day-old piglets. Only P30 animals were treated with parenteral iron after birth. Samples were analysed using automated haematology (ADVIA 2120) and chemistry analysers, and age-related reference intervals were calculated. RESULTS: Significant higher values of RBC, Hb and HCT were observed in P30 animals when compared to P5, with an opposite trend for MCV. These results were associated with a reduction of the RBC regeneration process and the thrombopoietic response. The TSAT and TIBC were significantly higher in P30 compared to P5; however, piglets remained iron deficient compared to adult reference intervals reported previously. CONCLUSIONS: In conclusion, this paper emphasises the high variability occurring in clinicopathological variables between new-born and 30-day-old pigs, and between piglets and adult pigs. This study provides valuable reference data for piglets at precise ages and could be used in the future as historical control improving the Reduction in animal experiments, as suggested by the 3Rs principle.

Constant-Rate Infusion of Dexmedetomidine to Manage Thiopental Anesthesia during Intracranial Surgery in Cynomolgus Macaques (Macaca fascicularis).

Macaques (Macaca spp.) are often used as animal models in biomedical research involving a neurosurgical approach. The development of new anesthetic techniques is pivotal for these studies. Studies in human anesthesia for intracranial surgery have shown that dexmedetomidine infusion reduces the incidence of cardiocirculatory complications in the perioperative period, reduces the need for supplemental analgesia, and provides an analgesic effect analogous to that of remifentanil. Data regarding the anesthetic effects of dexmedetomidine infusion in NHP including Macaca spp. are currently unavailable. The study population comprised 5 healthy cynomolgus macaques (Macaca fascicularis) that underwent intracranial surgery. On the day of surgery, the subjects were sedated with intramuscular ketamine (8 mg/kg) and dexmedetomidine (0.02 mg/kg). Anesthesia was induced with thiopental (3 mg/kg IV) and maintained by using constant-rate infusion of thiopental (3 mg/ kg/h); analgesia was provided by constant-rate infusion of dexmedetomidine (0.012 mg/kg/h). Atipamezole (0.1 mg/kg IM) was administered at the end of the surgical procedure. The median heart rate increased after sedation, reaching its highest level at 60 min (91.0 6.9 bpm); the highest systolic blood pressure (119.6 10.5 mm Hg) occurred at 75 min. No animal experienced respiratory arrest, and all recovered within 6 min after atipamezole administration. In cynomolgus macaques, dexmedetomidine constant-rate infusion provided adequate analgesia and stable hemodynamic control. Using dexmedetomidine as an adjunct to thiopental-maintained anesthesia may be advantageous in healthy NHP undergoing intracranial surgery.

Non-invasive evaluation of the haemodynamic effects of high-dose medetomidine in healthy cats for semen collection.

OBJECTIVES: This study aimed to assess non-invasively the cardiovascular effects of high-dose medetomidine on healthy male cats undergoing semen collection. METHODS: Haemodynamic evaluations were assessed on the basis of clinical examination, systolic arterial pressure (SAP) and transthoracic echocardiographic examination. Eight client owned, male domestic shorthair cats were sedated with a bolus of medetomidine intramuscularly (IM; 0.13 mg/kg), and semen collection was performed. A second transthoracic echocardiographic examination and SAP measurement were carried out 15 mins after sedation. At the end of the examination, the patients received a bolus of atipamezole (0.3 mg/kg) IM. RESULTS: The cats were deeply sedated, relaxed and laterally recumbent during the entire procedure. No rhythm abnormalities were observed during the examinations and no significant increase in SAP was recorded. Heart rate dropped from 200 ± 33 to 92 ± 13.1 beats per min after sedation. There was a significant increase in left ventricular dimensions and the left atrial area. The parameters of left ventricular systolic function were reduced, as were systemic and pulmonary cardiac outputs. Peak diastolic wave velocities were significantly reduced, while isovolumic contraction and relaxation time of the left ventricle were prolonged. Aortic valve insufficiency was recorded for all cats, while mitral valve insufficiency was noted in five cats. None of the subjects developed systolic anterior motion of the mitral valve. CONCLUSIONS AND RELEVANCE: The protocol allowed us to collect good semen samples in healthy cats. However, high-dose medetomidine induces significant haemodynamic effects on the feline heart, mainly due to a reduced heart rate, an increased cardiac preload and impaired systolic function. The animals recovered from the anaesthesia, after antagonist administration, without showing any clinically relevant consequences.

A Comprehensive Map of CNS Transduction by Eight Recombinant Adeno-associated Virus Serotypes Upon Cerebrospinal Fluid Administration in Pigs.

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.