CRISPR/Cas9 mediated generation of an ovine model for infantile neuronal ceroid lipofuscinosis (CLN1 disease).

The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child's lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.

Genome wide transcriptomic analysis identifies pathways affected by the infusion of Clostridium perfringens culture supernatant in the duodenum of broilers in situ.

Clostridium perfringens type A is the main etiological factor for necrotic enteritis, a multifactorial enteric disease that penalizes performance, health, and welfare of poultry. Lack of knowledge of host responses and disease pathogenesis is slowing down progress on developing therapies for disease control. A combined genomewide and targeted gene approach was used to investigate pathways and biological functions affected by the infusion of C. perfringens culture supernatant in the duodenum of broilers in 2 experiments. An in situ isolated loop of duodenum was prepared in anesthetized broilers of 3 wk of age (Exp. 1) and was infused either with crude C. perfringens culture supernatant (n = 7; treated), positive for necrotic enteritis B-like toxin (NetB) as determined by a cytotoxicity assay, or with a control preparation (n = 6; control). Birds were maintained alive for 1 h and then euthanized for tissue recovery. The use of the Affymetrix chicken genome array on RNA samples from loop tissue showed top biological functions affected by culture supernatant infusion included cell morphology, immune cell trafficking, and cell death; pathways affected included death receptor signaling, inflammatory response, and nuclear factor (NF)-κB signaling. In a second in situ study (Exp. 2), broilers were maintained alive for 4 h to monitor temporal expression patterns of targeted genes. Duodenal tissue was removed at 0.5, 1, 2, and 4 h post-infusion with culture supernatant (n = 9) or a control preparation (n = 5) for histology and gene expression analysis. Genes encoding proinflammatory cytokines, such as interferon γ (IFNγ), cell trafficking, such as neuroblastoma 1 (NBL1) and B cell CLL/Lymphoma 6 (BCL6), and cell death, such as Fas cell surface death receptor (FAS) and GTPase IMAP family member 8 (GIMAP8), were differentially expressed in the duodenum of treated and control broilers (P < 0.05). We have demonstrated that C. perfringens culture supernatant (NetB positive) infusion resulted in histological and gene expression changes consistent with necrotic enteritis in the duodenum of broilers. In the absence of live bacteria, crude culture supernatant resulted in early immunomodulation, inflammation, and cell death in the duodenum. The pathways identified here can be targeted for the development of new drugs, vaccines, and novel therapies for necrotic enteritis in broilers.

Comparison of anaesthesia 'Day 1 skills' expectations between veterinary anaesthetists and general practitioners.

Day One Skills (DOS) were introduced by the Royal College of Veterinary Surgeons (RCVS) in 2006 as a guideline for minimum skills required by a veterinary graduate. However, the RCVS anaesthesia DOS are broad and do not specify differences in skills required for different species. The aims of this study were: (1) to determine which anaesthesia skills were considered essential for day one practice by UK-based veterinary practitioners (GPs) and anaesthetists; and (2) to explore current opinions on veterinary undergraduate anaesthesia training. Questionnaires for veterinary GPs (QGPs) and veterinary anaesthetists (QVAs) were developed which asked general information on expectations of anaesthesia skills as well as specific expectations for the common veterinary species. Fifty-five UK-based members of the Association of Veterinary Anaesthetists responded, with a random sample of veterinary practices stratified by UK county generating 234 responses and a convenience sample targeted at more specialist veterinary specialities in the UK generating 161 responses. There was close overall agreement between the two groups of GPs and anaesthetists on essential anaesthesia DOS. However, expectations varied with species-greatest in cats and dogs, lowest in exotics. Many respondents commented that new veterinary graduates lack practical skills and should not be expected to be omnicompetent across all species. In conclusion, anaesthesia undergraduate training should prioritise essential practical DOS.

Investigation of the relationship between transcranial impedance and intracranial pressure.

Studies on piglets have shown that cranial bioimpedance (Z) measurements correlate well with invasively measured intracranial pressure (ICP). We have tested the feasibility of collecting transcranial impedance from a clinical device for measuring whole-body water content (ImpediMed SFB7). In the clinical study, 50 normal healthy volunteers had transcranial impedance measured using nine different head montages (forehead to mastoid (left/right), temporal to mastoid (left/right), forehead to temporal (left/right), forehead to occipital (left/right) and temporal to temporal). Impedance was measured 20 times over a frequency range per montage and ANOVA used to test for effects of electrode position upon recorded value. For the experimental study, five sedated and ventilated Marino sheep were instrumented for intraventricular ICP and transcranial impedance measurement. Measures of ICP were recorded while ICP was increased from baseline to greater than 50 mmHg in five steps using an intraventricular infusion of mock CSF. There is a significant effect of electrode position and gender upon transcranial impedance (p < 0.001). The temporal-mastoid electrode position had significantly lower impedance values in keeping with its shorter path length. ICP correlated with craniospinal compliance measurements and Impedance vs Freq by ICP step shows a clear ICP dependence (p = 0.007) across the sheep.

The stress response to bilateral femoral fractures: a comparison of primary intramedullary nailing and external fixation.

OBJECTIVES: Concern has recently been expressed with regard to the physiologic effects of primary intramedullary femoral nailing in seriously injured patients. "Damage control orthopaedics" techniques have been proposed, which comprise principally the use of primary external fixation. The aim of this study was to compare the physiologic effects of external femoral fixation with those of intramedullary stabilization over the first 24 hours after femoral fracture using an established large animal (ovine) trauma model. METHODS: Under terminal anesthesia, bilateral high-energy femoral fractures and hypovolemic shock were produced using a pneumatic actuator. Twenty-four sheep were randomized into 4 groups and monitored for 24 hours. Group 1--control, group 2--trauma only, group 3--trauma and external fixation, and group 4--trauma and reamed intramedullary nailing. Outcome measures included the following: pulmonary embolic load (transesophageal echocardiography), metabolic base excess, plasma coagulation markers, and polymorphonuclear cell counts obtained from bronchoalveolar lavage samples. RESULTS: The total embolic load was significantly higher (P < 0.001) in the intramedullary nailing group. All trauma groups had a significant increase (P < 0.05) in prothrombin times with a fall in antithrombin III and fibrinogen levels. However, the type of fracture stabilization used did not significantly affect any of the other outcome measurements. CONCLUSIONS: A higher pulmonary embolic load can be expected during early intramedullary femoral fracture stabilization compared with primary external fixation. However, the degree of stimulation to systemic coagulation and pulmonary inflammation by each type of surgery was comparable.

Temporal relationship between proliferating and apoptotic hormone-producing and endothelial cells in the equine corpus luteum.

The temporal relationship between endothelial cell death, vascular regression and the death of hormone-producing cells in the mare has not been established. To determine the dynamics of cell proliferation and death throughout the luteal phase, corpora lutea were studied at the early, mid- and late luteal phase, and after treatment with cloprostenol in the mid-luteal phase to induce premature luteolysis. Changes in cell proliferation and apoptosis were investigated utilising specific markers (phosphorylated histone-3 and activated caspase-3 respectively). Histone-3 positive cells were most abundant during the early luteal phase, and were mainly present in endothelial cells. Histone-3 activity significantly increased in hormone-producing cells 36 h after cloprostenol treatment. Frequency of activated caspase-3 staining peaked on day 14, and was induced by 36 h after cloprostenol administration in mid-luteal phase. However, cell death occurred simultaneously in the endothelial and hormone-producing cells. These results show that a subset of hormone-producing cells enter the early stages of cell division around luteolysis, while the majority of cells are undergoing cell death. Natural and induced functional and structural luteal regression in the mare can be at least partially attributed to simultaneous apoptosis of endothelial and hormone-producing cells. However, there is no evidence that endothelial cell death is the trigger for naturally occurring luteolysis.