Nerve Injury Following Regional Nerve Block: A Literature Review of Its Etiologies, Risk Factors, and Prevention.

PURPOSE OF REVIEW: Postoperative nerve injury after nerve block is complex and multifactorial. The mechanisms, etiologies, and risk factors are explored. This review article conducts a literature search and summarizes current evidence and best practices in prevention of nerve injury. RECENT FINDINGS: Emerging technology such as ultrasound, injection pressure monitors, and nerve stimulators for peripheral nerve block have been incorporated into regular practice to reduce the rate of nerve injury. Studies show avoidance of intrafascicular injection, limiting concentrations/volumes of local anesthetic, and appropriate patient selection are the most significant controllable factors in limiting the negative consequences of nerve block. Peripheral nerve injury is an uncommon occurrence after nerve block and is obscured by surgical manipulation, positioning, and underlying neural integrity. Underlying neural integrity is not always evident despite an adequate history and physical exam. Surgical stress, independently of nerve block, may exacerbate these neurologic disease processes and make diagnosing a postoperative nerve injury more challenging. Prevention of nerve injury by surgical teams, care with positioning, and avoidance of intrafascicular injection with nerve block are the most evidence-based practices.

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice.

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the affected cells located in different sites. Intravenous injection is a widely used systemic delivery route in preclinical studies that assess treatments intended for body-wide administration. In adult mice, it involves the intravenous administration of the therapeutic agent into the mouse's lateral tail veins. When mastered, tail-vein injections are safe and fast, and only require simple and commonly available tools. However, tail-vein injections are technically challenging and require extensive training and continuous practice to ensure the accurate delivery of the intended dose. Here we describe a detailed, optimized, lateral tail-vein injection protocol that we have developed based on our experience and on recommendations that had been previously reported by other groups. Other than the mouse restrainers and insulin syringes, this protocol requires only reagents and equipment that are readily available in most labs. We found that following this protocol results in consistently successful intravenous delivery of adeno-associated virus (AAV) into the tail veins of unsedated 7-9 week-old mice. Additionally, we describe the optimized protocols for the histological detection of fluorescent reporter proteins and vector genome per diploid genome (vg/dg) quantification used to assess AAV transduction and biodistribution. The goal of this protocol is to aid experimenters in easily performing tail-vein injections successfully and consistently, which can reduce the practice time needed to master the technique.

A humanized knock-in Col6a1 mouse recapitulates a deep-intronic splice-activating variant.

Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating, COL6A1 variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeutics in vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as "h"), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen a1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as "h+189T"). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen a1(VI) protein. In homozygous Col6a1 h+189T/h+189T mice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients' muscle biopsies. While Col6a1h/h mice do not show any phenotype compared to wildtype animals, Col6a1 h/h+189T and Col6a1 h+189T/h+189T mice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients' biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that the COL6A1 N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce other COL6A1 pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.

The recurrent deep intronic pseudoexon-inducing variant COL6A1 c.930+189C>T results in a consistently severe phenotype of COL6-related dystrophy: Towards clinical trial readiness for splice-modulating therapy.

Collagen VI-related dystrophies (COL6-RDs) manifest with a spectrum of clinical phenotypes, ranging from Ullrich congenital muscular dystrophy (UCMD), presenting with prominent congenital symptoms and characterised by progressive muscle weakness, joint contractures and respiratory insufficiency, to Bethlem muscular dystrophy, with milder symptoms typically recognised later and at times resembling a limb girdle muscular dystrophy, and intermediate phenotypes falling between UCMD and Bethlem muscular dystrophy. Despite clinical and immunohistochemical features highly suggestive of COL6-RD, some patients had remained without an identified causative variant in COL6A1, COL6A2 or COL6A3. With combined muscle RNA-sequencing and whole-genome sequencing we uncovered a recurrent, de novo deep intronic variant in intron 11 of COL6A1 (c.930+189C>T) that leads to a dominantly acting in-frame pseudoexon insertion. We subsequently identified and have characterised an international cohort of forty-four patients with this COL6A1 intron 11 causative variant, one of the most common recurrent causative variants in the collagen VI genes. Patients manifest a consistently severe phenotype characterised by a paucity of early symptoms followed by an accelerated progression to a severe form of UCMD, except for one patient with somatic mosaicism for this COL6A1 intron 11 variant who manifests a milder phenotype consistent with Bethlem muscular dystrophy. Characterisation of this individual provides a robust validation for the development of our pseudoexon skipping therapy. We have previously shown that splice-modulating antisense oligomers applied in vitro effectively decreased the abundance of the mutant pseudoexon-containing COL6A1 transcripts to levels comparable to the in vivo scenario of the somatic mosaicism shown here, indicating that this therapeutic approach carries significant translational promise for ameliorating the severe form of UCMD caused by this common recurrent COL6A1 causative variant to a Bethlem muscular dystrophy phenotype.

Two novel arenaviruses detected in pygmy mice, Ghana.

Two arenaviruses were detected in pygmy mice (Mus spp.) by screening 764 small mammals in Ghana. The Natal multimammate mouse (Mastomys natalensis), the known Lassa virus reservoir, was the dominant indoor rodent species in 4 of 10 sites, and accounted for 27% of all captured rodents. No rodent captured indoors tested positive for an arenavirus.

Exon-Skipping for a Pathogenic COL6A1 Variant in Ullrich Congenital Muscular Dystrophy.

Single nucleotide variants that alter splice sites or splicing regulatory elements can lead to the skipping of exons, retention of introns, or insertion of pseudo-exons (PE) into the mature mRNA transcripts. When translated, these changes can disrupt the function of the synthesized protein. Splice-switching antisense oligonucleotides (ASOs) are synthetic, modified nucleic acids that can correct these aberrant splicing events. They are currently in active clinical development for a number of conditions and have been approved by regulatory agencies for the treatment of neuromuscular disorders such as Duchenne muscular dystrophy and spinal muscular atrophy. We have previously reported that splice-switching ASOs effectively skip a pathogenic PE that causes Ullrich congenital muscular dystrophy (UCMD). This erroneous PE insertion is caused by a deep-intronic variant located within intron 11 of COL6A1 (c.930+189 C>T). Here, we describe the detailed protocols and workflow that our labs have used to assess the efficacy of ASOs to skip this PE in vitro. The protocols include designing ASOs; isolating, culturing, and transfecting fibroblasts; extracting RNA and protein; and validating splicing correction at the mRNA and protein levels using quantitative reverse transcription PCR (qRT-PCR) and western blot assays, respectively.

Nationwide tracheostomy among neonatal admissions - A cross-sectional analysis.

OBJECTIVE: To describe characteristics and outcomes of infants admitted as neonates requiring tracheostomy placement. METHODS: A cross-sectional analysis of the Kids' Inpatient Database (KID) between 2003 and 2016 included all children admitted within the first 28 days of life that had a tracheostomy placed prior to discharge. Patient characteristics and surgical outcomes were compared between term (≥37 weeks gestation) and preterm (<37 weeks gestation) infants. A subset analysis for Black or African American neonates was performed given disproportional preterm births. RESULTS: An estimated 4268 (95% CI: 4123-4414) tracheostomies were performed in infants admitted as a neonate with preterm infants accounting for 47% (1998/4268). Among preterm children, 20% were Black or African American compared to 12% in the term group (P < .001). More preterm infants had bronchopulmonary dysplasia (46% vs. 14%, P < .001), cardiac defects (66% vs. 58%, P < .001) and developed pneumonia, newborn sepsis, or sepsis during admissions (P < .001). Laryngotracheal anomalies (25% vs. 18%, P < .001) and vocal cord paralysis (11% vs. 4.9%, P < .001) were more common in term infants. Median length of stay (LOS) (154 vs. 100 days, P < .001) and total charges ($1,395,106 vs. $917,478, P < .001) were greater among preterm infants. Mortality was no different between groups (13% vs. 15%, P = .07). Characteristics strongly associated with preterm status were newborn sepsis (OR: 2.31, 95% CI: 1.97-2.72, P < .001), bronchopulmonary dysplasia (OR: 2.17, 95% CI: 1.77-2.65, P < .001) and Black or African American race (OR: 1.78, 95% CI: 1.46-2.17, P < .001). The following factors increased among all neonates between the baseline year 2003 to the final study year 2016: complications of care (OR: 1.9, 95% CI: 1.5-2.5, P < .001); sepsis (OR: 4.1, 95% CI: 3.0-5.5, P < .001); congenital cardiac anomalies (OR: 5.8, 95% CI: 4.5-7.4, P < .001); and respiratory failure (OR: 1.9, 95% CI: 1.5-2.4, P < .001). Compared to other races, median LOS and total charges were greater among Black or African American infants. CONCLUSION: Tracheostomies among preterm infants admitted as neonates reflect a growing and complex group with increased costs and hospitalization lengths. Black or African American children are disproportionately born preterm with higher costs and LOS compared to other racial cohorts. Future work will be necessary to design quality-improvement initiatives to improve outcomes for this vulnerable population.

Combined Environment Simulator for Low-Dose-Rate Radiation and Partial Gravity of Moon and Mars.

Deep space exploration by humans has become more realistic, with planned returns to the Moon, travel to Mars, and beyond. Space radiation with a low dose rate would be a constant risk for space travelers. The combined effects of space radiation and partial gravity such as on the Moon and Mars are unknown. The difficulty for such research is that there are no good simulating systems on the ground to investigate these combined effects. To address this knowledge gap, we developed the Simulator of the environments on the Moon and Mars with Neutron irradiation and Gravity change (SwiNG) for in vitro experiments using disposable closed cell culture chambers. The device simulates partial gravity using a centrifuge in a three-dimensional clinostat. Six samples are exposed at once to neutrons at a low dose rate (1 mGy/day) using Californium-252 in the center of the centrifuge. The system is compact including two SwiNG devices in the incubator, one with and one without radiation source, with a cooling function. This simulator is highly convenient for ground-based biological experiments because of limited access to spaceflight experiments. SwiNG can contribute significantly to research on the combined effects of space radiation and partial gravity.

An effective venipuncture technique and normal serum biochemistry parameters of the captive fat-tailed jird (Pachyuromys duprasi).

Thirty-nine captively reared fat-tailed jirds (Pachyuromys duprasi) were enrolled in a minimally invasive study to determine an effective venipuncture technique and establish normal serum biochemistry parameters. A jugular venipuncture technique using chemical restraint (ketamine, 30 mg/kg; xylazine, 6 mg/kg; acepromazine, 1 mg/kg) administered intraperitoneally was safe and consistently yielded at least 0.3 mL of blood. Of the biochemical indicators measured (glucose, total protein, albumin, globulin, alkaline phosphatase, alanine transferase, total bilirubin, amylase, BUN, creatinine, calcium, phosphorous, sodium and potassium), amylase and glucose levels differed significantly between male and female fat-tailed jirds.