The effects of anaesthesia on cell death in a porcine model of neonatal hypoxic-ischaemic brain injury

Background: Hypothermia is neuroprotective after neonatal hypoxic-ischaemic brain injury. However, systemic cooling to hypothermic temperatures is a stressor and may reduce neuroprotection in awake pigs. We compared two experiments of global hypoxic-ischaemic injury in newborn pigs, in which one group received propofol–remifentanil and the other remained awake during post-insult hypothermia treatment. Methods: In both studies, newborn pigs were anaesthetised using halothane during a 45-min global hypoxic-ischaemic insult induced by reducing Fio2 and graded hypotension until a low-voltage <7 μV electroencephalogram was achieved. On reoxygenation, the pigs were randomly allocated to receive 24 h of normothermia or hypothermia. In the first study (n=18) anaesthesia was discontinued and the pigs' tracheas were extubated. In the second study (n=14) anaesthesia was continued using propofol and remifentanil. Brain injury was assessed after 72 h by classical global histopathology, Purkinje cell count, and apoptotic cell counts in the hippocampus and cerebellum. Results: Global injury was nearly 10-fold greater in the awake group compared with the anaesthetised group (P=0.021). Hypothermia was neuroprotective in the anaesthetised pigs but not the awake pigs. In the hippocampus, the density of cleaved caspase-3-positive cells was increased in awake compared with anaesthetised pigs in normothermia. In the cerebellum, Purkinje cell density was reduced in the awake pigs irrespective of treatment, and the number of cleaved caspase-3-positive Purkinje cells was greatly increased in hypothermic awake pigs. We detected no difference in cleaved caspase-3 in the granular cell layer or microglial reactivity across the groups. Conclusions: Our study provides novel insights into the significance of anaesthesia/sedation during hypothermia for achieving optimal neuroprotection

Plant production of a virus-like particle-based vaccine candidate against porcine reproductive and respiratory syndrome

Porcine reproductive and respiratory syndrome (PRRS) is a disease leading to spontaneous abortions and stillbirths in sows and lowered life quality and expectancy in growing pigs. PRRS is prevalent worldwide and has significant economic impacts to swine industries around the globe. Co-expression of the two most abundant proteins in the viral envelope, the matrix protein (M) and glycosylated protein 5 (GP5), can produce a neutralizing immune response for the virus providing a potentially effective subunit vaccine against the disease, but these proteins are difficult to express. The goal of this research was to display antigenic portions of the M and GP5 proteins on the surface of tobacco mosaic virus-like particles. A modified tobacco mosaic virus coat protein (TMVc) was transiently expressed in Nicotiana benthamiana leaves and targeted to three subcellular compartments along the secretory pathway to introduce glycosylation patterns important for M-GP5 epitope immunogenicity. We found that accumulation levels in the apoplast were similar to the ER and the vacuole. Because glycans present on plant apoplastic proteins are closest to those present on PRRSV proteins, a TMVcM-GP5 fusion construct was targeted to the apoplast and accumulated at over 0.5 mg/g of plant fresh weight. TMVc virus-like particles self-assembled in plant cells and surface-displayed the M-GP5 epitope, as visualized by transmission electron microscopy and immunogold localization. These promising findings lay the foundation for immunogenicity and protective-immunity studies in animals to examine the efficacy of this vaccine candidate as a measure to control PRRS

Persistent microglial activation and synaptic loss with behavioral abnormalities in mouse offspring exposed to CASPR2-antibodies in utero

Gestational transfer of maternal antibodies against fetal neuronal proteins may be relevant to some neurodevelopmental disorders, but until recently there were no proteins identified. We recently reported a fivefold increase in CASPR2-antibodies in mid-gestation sera from mothers of children with intellectual and motor disabilities. Here, we exposed mice in utero to purified IgG from patients with CASPR2-antibodies (CASPR2-IgGs) or from healthy controls (HC-IgGs). CASPR2-IgG but not HC-IgG bound to fetal brain parenchyma, from which CASPR2-antibodies could be eluted. CASPR2-IgG exposed neonates achieved milestones similarly to HC-IgG exposed controls but, when adult, the CASPR2-IgG exposed progeny showed marked social interaction deficits, abnormally located glutamatergic neurons in layers V-VI of the somatosensory cortex, a 16% increase in activated microglia, and a 15-52% decrease in glutamatergic synapses in layers of the prefrontal and somatosensory cortices. Thus, in utero exposure to CASPR2-antibodies led to permanent behavioral, cellular, and synaptic abnormalities. These findings support a pathogenic role for maternal antibodies in human neurodevelopmental conditions, and CASPR2 as a potential target

The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis

TAR DNA binding protein of 43 kDa (TDP-43)-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS.Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their fifth to seventh decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies.The G376V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue.The identification of individuals with TDP-43-related myopathy, but not ALS, implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype. Zibold et al. identify a new TDP-43 missense variant (G376V) in two French families affected by late-onset distal myopathy but not ALS. The findings support a primary role for TDP-43 in skeletal muscle pathophysiology and suggest that TARDBP screening should be included in the genetic work-up of patients with distal myopathy

Immune or Genetic-Mediated Disruption of CASPR2 Causes Pain Hypersensitivity Due to Enhanced Primary Afferent Excitability

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/-mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability

Immune or genetic-mediated disruption of CASPR2 causes pain hypersensitivity due to enhanced primary afferent excitability

Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2(-/-)) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2(-/-) mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability

Treating the placenta to prevent adverse effects of gestational hypoxia on fetal brain development.

Some neuropsychiatric disease, including schizophrenia, may originate during prenatal development, following periods of gestational hypoxia and placental oxidative stress. Here we investigated if gestational hypoxia promotes damaging secretions from the placenta that affect fetal development and whether a mitochondria-targeted antioxidant MitoQ might prevent this. Gestational hypoxia caused low birth-weight and changes in young adult offspring brain, mimicking those in human neuropsychiatric disease. Exposure of cultured neurons to fetal plasma or to secretions from the placenta or from model trophoblast barriers that had been exposed to altered oxygenation caused similar morphological changes. The secretions and plasma contained altered microRNAs whose targets were linked with changes in gene expression in the fetal brain and with human schizophrenia loci. Molecular and morphological changes in vivo and in vitro were prevented by a single dose of MitoQ bound to nanoparticles, which were shown to localise and prevent oxidative stress in the placenta but not in the fetus. We suggest the possibility of developing preventative treatments that target the placenta and not the fetus to reduce risk of psychiatric disease in later life

An Interspecific Nicotiana Hybrid as a Useful and Cost-Effective Platform for Production of Animal Vaccines

The use of transgenic plants to produce novel products has great biotechnological potential as the relatively inexpensive inputs of light, water, and nutrients are utilised in return for potentially valuable bioactive metabolites, diagnostic proteins and vaccines. Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines of importance for both animals and humans. Vaccine purification is generally regarded as being integral to the preparation of safe and effective vaccines for use in humans. However, the use of crude plant extracts for animal immunisation may enable plant-made vaccines to become a cost-effective and efficacious approach to safely immunise large numbers of farm animals against diseases such as avian influenza. Since the technology associated with genetic transformation and large-scale propagation is very well established in Nicotiana, the genus has attributes well-suited for the production of plant-made vaccines. However the presence of potentially toxic alkaloids in Nicotiana extracts impedes their use as crude vaccine preparations. In the current study we describe a Nicotiana tabacum and N. glauca hybrid that expresses the HA glycoprotein of influenza A in its leaves but does not synthesize alkaloids. We demonstrate that injection with crude leaf extracts from these interspecific hybrid plants is a safe and effective approach for immunising mice. Moreover, this antigen-producing alkaloid-free, transgenic interspecific hybrid is vigorous, with a high capacity for vegetative shoot regeneration after harvesting. These plants are easily propagated by vegetative cuttings and have the added benefit of not producing viable pollen, thus reducing potential problems associated with bio-containment. Hence, these Nicotiana hybrids provide an advantageous production platform for partially purified, plant-made vaccines which may be particularly well suited for use in veterinary immunization programs