Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and ß4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and ß4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

Ultrasound-guided caudal quadratus lumborum block combined with the greater ischiatic notch plane block as motor-protective analgesia for the pelvic limb in dogs.

OBJECTIVE: To develop an ultrasound-guided caudal quadratus lumborum block (C-QLB) technique in canine cadavers and to compare sensory and motor blockade resulting from the combination of ultrasound-guided greater ischiatic notch (GIN) plane and C-QLB approaches (GIN-CQLB group) versus a lumbosacral plexus (LSP group) approach [combination of lateral pre-iliac (LPI) and parasacral (PS) techniques] in dogs. STUDY DESIGN: Descriptive anatomical study and prospective randomized, blinded, experimental crossover trial. ANIMALS: A total of six canine cadavers and six adult Beagle dogs. METHODS: Phase I: following ultrasound-guided C-QLB injections of 0.3 mL kg-1 of dye, using the interfascial plane located lateral to the quadratus lumborum muscle at the level of the sixth lumbar vertebra (L6) as injection point, the spread of injectate and nerve staining was evaluated using gross anatomical dissection. PHASE II: sensory and motor blockade achieved with the GIN-CQLB or LSP blocks in Beagle dogs were evaluated and compared. The assigned technique was performed with 2% lidocaine: 0.2 mL kg-1 for the GIN and PS approaches and 0.3 mL kg-1 for the C-QLB and LPI approaches. RESULTS: Dissection revealed distribution of dye around the lumbar hypaxial musculature, extending into the paravertebral spaces, with staining of 3 (2-4) [median (interquartile range)] spinal nerves, spanning L3 to L6. The median motor blockade in the GIN-CQLB and LSP groups was 7 (7-8) versus 16 (10-16) (p = 0.026), whereas the median sensory blockade was 5 (4-5) versus 3 (3-3) (p = 0.025), respectively. CONCLUSION AND CLINICAL SIGNIFICANCE: The GIN-CQLB approach desensitized the thigh dermatomes effectively. Compared with the LSP approaches, GIN-CQLB exhibits a motor-protective effect by preserving tonic muscle function.

Ventilator output splitting interface 'ACRA': Description and evaluation in lung simulators and in an experimental ARDS animal model.

The current COVID-19 pandemic has led the world to an unprecedented global shortage of ventilators, and its sharing has been proposed as an alternative to meet the surge. This study outlines the performance of a preformed novel interface called 'ACRA', designed to split ventilator outflow into two breathing systems. The 'ACRA' interface was built using medical use approved components. It consists of four unidirectional valves, two adjustable flow-restrictor valves placed on the inspiratory limbs of each unit, and one adjustable PEEP valve placed on the expiratory limb of the unit that would require a greater PEEP. The interface was interposed between a ventilator and two lung units (phase I), two breathing simulators (phase II) and two live pigs with heterogeneous lung conditions (phase III). The interface and ventilator adjustments tested the ability to regulate individual pressures and the resulting tidal volumes. Data were analyzed using Friedman and Wilcoxon tests test (p < 0.05). Ventilator outflow splitting, independent pressure adjustments and individual tidal volume monitoring were feasible in all phases. In all experimental measurements, dual ventilation allowed for individual and tight adjustments of the pressure, and thus volume delivered to each paired lung unit without affecting the other unit's ventilation-all the modifications performed on the ventilator equally affected both paired lung units. Although only suggested during a dire crisis, this experiment supports dual ventilation as an alternative worth to be considered.

Evaluation of electrical nerve stimulation to confirm sacrococcygeal epidural needle placement in dogs.

OBJECTIVES: To evaluate the use of 0.7 mA as a fixed electrical current to indicate epidural needle placement and to confirm that 0.7 mA is greater than the upper limit of the minimal electrical threshold (MET) for sacrococcygeal epidural needle placement in dogs. STUDY DESIGN: Prospective clinical study. ANIMALS: A group of 20 client-owned dogs. METHODS: During general anesthesia and with standard monitoring, the presence of the patellar reflex was confirmed in all dogs. An insulated needle was inserted through the sacrococcygeal intervertebral junction, and absence of tail movement was confirmed when a fixed electrical current of 0.7 mA was applied. Then, the needle was further advanced toward the epidural space until the expected motor response was obtained - the nerve stimulation test (NST). The NST was considered positive when a motor response of the muscles of the tail was elicited but not the perineal muscles, whereas it was considered negative when no movement of the tail was evoked. The electrical current was turned to 0 mA and then increased by 0.01 mA increments until tail movement was evoked; this was recorded as the MET. In the positive NST cases, 0.05 mL cm-1 occipitococcygeal length of 2% lidocaine or 0.25-0.5% bupivacaine was administered. Epidural blockade was confirmed by the loss of patellar reflex. Descriptive statistics were used to present data. RESULTS: Sacrococcygeal epidural needle placement, corroborated by loss of the patellar reflex, was correctly predicted in 89.5% (95% confidence interval, 68.6-97.1%) of the cases. The MET was 0.22 mA (0.11-0.36). CONCLUSIONS AND CLINICAL RELEVANCE: A current of 0.7 mA is approximately twice the upper limit of the MET for epidural placement. Therefore, this study demonstrates, with a success rate of 89.5%, the adequacy of using 0.7 mA as the fixed electrical current to detect sacrococcygeal epidural needle placement in dogs.

Transversus abdominis plane block in cat cadavers: anatomical description and comparison of injectate spread using two- and three-point approaches.

OBJECTIVE: To describe the sonoanatomy of the abdominal wall in live cats and to compare the distribution pattern of two versus three ultrasound-guided transversus abdominis plane (TAP) injections using clinically applicable volumes of lidocaine-dye solution in cat cadavers. STUDY DESIGN: Prospective anatomical study. ANIMALS: A total of eight client-owned healthy cats and eight cat cadavers. METHODS: Ultrasound anatomy of the abdominal wall, landmarks and sites for needle access were determined in live cats. Ultrasound-guided TAP injections were performed in eight thawed cat cadavers. Volumes of 0.25 or 0.16 mL kg-1 per point of a lidocaine-dye solution were injected using either two [subcostal and preiliac (SP)] or three [subcostal, retrocostal and preiliac (SRP)] injection points, respectively. Each cadaver was then dissected to determine the injectate distribution and the number of thoracolumbar nerves stained with each approach. The target nerves were defined as the ventromedial branches of the thoracic nerves 10 (T10), T11, T12, T13 and lumbar nerves 1 (L1) and L2. RESULTS: Sonoanatomy was consistent with anatomy upon dissection and the TAP was identified in all cadavers. A total of 16 subcostal, 16 preiliac and nine retrocostal TAP injections were performed. The overall staining success rate of the target nerves was 66.7% and 92.6% for the SP and SPR approaches, respectively (p = 0.02). The ventromedial branches of T10, T11, T12, T13, L1 and L2 were stained in 57.1%, 100.0%, 85.7%, 28.6%, 42.9% and 85.7%, and in 66.7%, 100.0%, 100.0%, 100.0%, 88.9% and 100.0% of the cases with the SP and SRP approaches, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The SRP approach allowed a broader distribution around the target nerves, whereas a staining gap was observed at T13 and L1 with the SP approach. Further studies are necessary to investigate the analgesic effect of these approaches in a clinical setting.

Analysis of teaching methods in anaesthesia in the undergraduate curriculum of four veterinary universities.

OBJECTIVE: To design a holistic audit tool to assess the effectiveness of anaesthesia teaching strategies, and thereby to study veterinary undergraduate teaching methods in different geographical areas. STUDY DESIGN: Qualitative study using interviews of university staff and students to identify common themes and differences in teaching veterinary anaesthesia. METHODS: An audit was performed using an audit tool in four veterinary universities (École Nationale Vétérinaire d'Alfort, France; Royal Veterinary College, UK; University of Buenos Aires, Argentina; and Alma mater studiorum - Università di Bologna, Italy). First, an open-question interview of anaesthesia head of service (60-90 minutes) identified the pedagogical strategies in order to conceive a subsequent semi-directive interview formulated as a SWOT analysis (Strength/Weaknesses/Opportunity/Threats). Second, the SWOT reflection was conducted by a second staff member and focussed on: 1) general organization; 2) topics for pre-rotation teaching; 3) teaching methods for clinical rotation; and 4) assessment methods. Qualitative analysis of the interview responses was performed with semi-structured interviews. Finally, the students evaluated their teaching through a students' questionnaire generated from the output of both interviews. RESULTS: A group of nine lecturers and 106 students participated in the study at four different sites. Preclinical teaching ranged from 13 to 24 hours (median 15 hours). Clinical teaching ranged from 4 to 80 hours (median 60 hours). Overall, all faculties perceived time as a limitation and attempted to design strategies to achieve the curriculum expectations and optimize teaching using more time-efficient exercises. Large animal anaesthesia teaching was found to be a common area of weakness. Internal feedback was delivered to each university, whereas generalized results were shared globally. CONCLUSIONS: This preliminary study proved the generalizability of the protocol used. Recruiting a larger pool of universities would help to identify and promote efficient teaching strategies and innovations for training competent new graduates in an ever-expanding curriculum.

Sonographic evaluation of epidural and intrathecal injections in cats.

OBJECTIVES: To describe the ultrasonographic anatomy of the caudal lumbar spine in cats and to detect ultrasound (US) signs associated with epidural or intrathecal injection. STUDY DESIGN: Prospective, clinical study. ANIMALS: Twenty-six client-owned cats. METHODS: Transverse (position 1) and parasagittal (position 2) two-dimensional US scanning was performed over the caudal lumbar spine in all cats. Midline distances between the identified structures were measured. Cats assigned to epidural injection (group E, n = 16) were administered a bupivacaine-morphine combination confirmed by electrical stimulation. Cats assigned to intrathecal injection (group I, n = 10) were administered a morphine-iohexol combination injected at the lumbosacral level and confirmed by lateral radiography. The total volume injected (0.3 mL kg-1 ) was divided into two equal aliquots that were injected without needle repositioning, with the US probe in positions 1 and 2, respectively. The presence or absence of a burst of color [color flow Doppler test (CFDT)], dural sac collapse and epidural space enlargement were registered during and after both injections. RESULTS: US scanning allowed measurement of the distances between the highly visible structures inside the spinal canal. CFDT was positive for all animals in group E. In group I, intrathecal injection was confirmed in only two animals, for which the CFDT was negative; seven cats inadvertently and simultaneously were administered an epidural injection and showed a positive CFDT during the second aliquot injection, and the remaining animal was administered epidural anesthesia and was excluded from the CFDT data analysis. Dural sac collapse and epidural space enlargement were present in all animals in which an epidural injection was confirmed. CONCLUSIONS AND CLINICAL RELEVANCE: US examination allowed an anatomical description of the caudal lumbar spine and real-time confirmation of epidural injection by observation of a positive CFDT, dural sac collapse and epidural space enlargement.

The use of a nerve stimulation test to confirm sacrococcygeal epidural needle placement in cats.

OBJECTIVE: To determine if a nerve stimulation test (NST) could act as a monitoring technique to confirm sacrococcygeal epidural needle placement in cats. STUDY DESIGN: Prospective experimental trial in a clinical setting. ANIMALS: Twenty-four adult cats, scheduled for a therapeutic procedure where epidural anesthesia was indicated. METHODS: Under general anesthesia, an insulated needle was inserted through the S3 -Cd1 intervertebral space guided by the application of a fixed electrical current (0.7 mA) until a motor response was obtained. The NST was considered positive when the epidural nerve stimulation produced a motor response of the muscles of the tail, whereas it was considered negative when no motor response was evoked. In the NST positive cases, 0.3 mL kg(-1) of 0.5% bupivacaine was administrated before needle withdrawal. Ten minutes after injection, epidural blockade was confirmed by the loss of perineal (anal), and pelvic limbs reflexes (patellar and withdrawal). RESULTS: The use of a fixed electrical stimulation current of 0.7 mA resulted in correct prediction of sacrococcygeal epidural injection, corroborated by post bupivacaine loss of perineal and pelvic limb reflexes, in 95.8% of the cases. CONCLUSION AND CLINICAL RELEVANCE: This study demonstrates the feasibility of using, in a clinical setting, an electrical stimulation test as an objective and in real-time method to confirm sacrococcygeal epidural needle placement in cats.

Use of electrical nerve stimulation to monitor lumbosacral epidural needle placement in cats.

OBJECTIVE: To determine the minimal electrical threshold (MET) necessary to elicit muscle contraction of the pelvic limb or tail when an insulated needle is positioned outside (METout) and inside (METin) the lumbosacral epidural space in cats. STUDY DESIGN: Prospective, blinded study. ANIMALS: Twelve mixed-breed healthy adult cats, scheduled for a therapeutic procedure where lumbosacral epidural administration was indicated. METHODS: Under general anesthesia, an insulated needle was advanced through tissues of the lumbosacral interspace until its tip was thought to be just dorsal to the interarcuate ligament. An increasing electrical current (0.1 ms, 2 Hz) was applied through the stimulating needle in order to determine the MET necessary to obtain a muscle contraction of the pelvic limb or tail (METout), and then 0.05 mL kg−1 of iohexol was injected. The needle was further advanced until its tip was thought to be in the epidural space. The MET was determined again (METin) and 0.2 mL kg−1 of iohexol was injected. The cats were maintained in sternal position. Contrast medium spread was determined through lateral radiographic projections. RESULTS: The radiographic study confirmed the correct needle placement dorsal to the interarcuate ligament in all cats. When the needle was placed ventrally to the interarcuate ligament, iohexol was injected epidurally in ten and intrathecally in two cats. The METout and METin was 1.76 ± 0.34 mA and 0.34 ± 0.07 mA, respectively (p < 0.0001). CONCLUSION AND CLINICAL RELEVANCE: Nerve stimulation can be employed as a tool to determine penetration of the interarcuate ligament but not the piercing of the dura mater at the lumbosacral space in cats.

Use of electrical stimulation to monitor lumbosacral epidural and intrathecal needle placement in rabbits.

OBJECTIVE: To determine the minimal electric threshold of neurostimulation dorsally and ventrally to the interarcuate ligament in the lumbosacral area necessary to cause muscle contraction of the hind limb or tail and determine whether a continuous electrical stimulation applied to an insulated needle during lumbosacral epidural needle placement could be used to distinguish the epidural from the intrathecal space in rabbits. ANIMALS: 24 New Zealand white rabbits. PROCEDURES: Rabbits received iohexol (0.2 mL/kg) either dorsally (group 1) or ventrally to the interarcuate ligament in the lumbosacral area (groups 2 and 3). Correct placement of the needle was determined by use of the loss of resistance to injection technique (group 2) or a continuous electrical stimulation (group 3) and confirmed by examination of the iohexol distribution pattern on radiographs. RESULTS: In all rabbits of group 1, iohexol was injected in the lumbosacral area, outside the epidural space. In groups 2 and 3, iohexol was injected intrathecally. No pure iohexol epidural migration of iohexol was observed. Mean ± SD minimal electric threshold to elicit a motor response was 1.2 ± 0.3 mA, 0.3 ± 0.1 mA, and 0.3 ± 0.1 mA in groups 1, 2, and 3, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Neurostimulation was a useful technique to determine correct intrathecal needle placement in rabbits but failed to detect the lumbosacral epidural space when the common technique, used in dogs and cats for the lumbosacral epidural approach, was used.