Localizing the L5 Vertebra Using Nerve Morphology on MRI: An Accurate and Reliable Technique.

BACKGROUND AND PURPOSE: Multiple methods have been used to determine the lumbar vertebral level on MR imaging, particularly when full spine imaging is unavailable. Because postmortem studies show 95% accuracy of numbering the lumbar vertebral bodies by counting the lumbar nerve roots, attention to lumbar nerve morphology on axial MR imaging can provide numbering clues. We sought to determine whether the L5 vertebra could be accurately localized by using nerve morphology on MR imaging. MATERIALS AND METHODS: One hundred eight cases with full spine MR imaging were numbered from the C2 vertebral body to the sacrum with note of thoracolumbar and lumbosacral transitional states. The origin level of the L5 nerve and iliolumbar ligament were documented in all cases. The reference standard of numbering by full spine imaging was compared with the nerve morphology numbering method. Five blinded raters evaluated all lumbar MRIs with nerve morphology technique twice. Prevalence and bias-adjusted κ were used to measure interrater and intrarater reliability. RESULTS: The L5 nerve arose from the 24th presacral vertebra (L5) in 106/108 cases. The percentage of perfect agreement with the reference standard was 98.1% (95% CI, 93.5%-99.8%), which was preserved in transitional and numeric variation states. The iliolumbar ligament localization method showed 83.3% (95% CI, 74.9%-89.8%) perfect agreement with the reference standard. Inter- and intrarater reliability when using the nerve morphology method was strong. CONCLUSIONS: The exiting L5 nerve can allow accurate localization of the corresponding vertebrae, which is essential for preprocedure planning in cases where full spine imaging is not available. This neuroanatomic method displays higher agreement with the reference standard compared with previously described methods, with strong inter- and intrarater reliability.

Functional Validation of Apoptosis Genes IAP1 and DRONC in Midgut Tissue of the Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) by RNAi.

Culicoides biting midges transmit multiple ruminant viruses, including bluetongue virus and epizootic hemorrhagic disease virus, causing significant economic burden worldwide. To further enhance current control techniques, understanding vector-virus interactions within the midge is critical. We developed previously a double-stranded RNA (dsRNA) delivery method to induce RNA interference (RNAi) for targeted gene knockdown in adult Culicoides sonorensis Wirth & Jones. Here, we confirm the C. sonorensis inhibitor of apoptosis 1 (CsIAP1) as an anti-apoptotic functional ortholog of IAP1 in Drosophila, identify the ortholog of the Drosophila initiator caspase DRONC (CsDRONC), and demonstrate that injection of dsRNA into the hemocoel can be used for targeted knockdown in the midgut in C. sonorensis. We observed CsIAP1 transcript reduction in whole midges, with highest transcript reduction in midgut tissues. IAP1knockdown (kd) resulted in pro-apoptotic caspase activation in midgut tissues. In IAP1kd midges, midgut tissue integrity and size were severely compromised. This phenotype, as well as reduced longevity, was partially reverted by co-RNAi suppression of CsDRONC and CsIAP1. Therefore, RNAi can be directed to the midgut of C. sonorensis, the initial site of virus infection, using dsRNA injection into the hemocoel. In addition, we provide evidence that the core apoptosis pathway is conserved in C. sonorensis and can be experimentally activated in the midgut to reduce longevity in C. sonorensis. This study thus paves the way for future reverse genetic analyses of midgut-virus interactions in C. sonorensis, including the putative antiviral properties of RNAi and apoptosis pathways.

Inducing RNA interference in the arbovirus vector, Culicoides sonorensis.

Biting midges in the genus Culicoides are important vectors of arboviral diseases, including epizootic haemorrhagic disease, bluetongue and most likely Schmallenberg, which cause significant economic burdens worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the difficulty of consistent culturing of certain species and the absence of molecular techniques such as RNA interference (RNAi). Here, we report the establishment of RNAi as a research tool for the adult midge, Culicoides sonorensis. Based on previous research and transcriptome analysis, which revealed putative small interfering RNA pathway member orthologues, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to perform RNA silencing. Injection of control double-stranded RNA targeting green fluorescent protein (dsGFP), into the haemocoel of 2-3-day-old adult female midges resulted in survival curves that support virus transmission. dsRNA injection targeting the newly identified C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) orthologue resulted in a 40% decrease of transcript levels and 73% shorter median survivals as compared with dsGFP-injected controls. These results reveal the conserved function of IAP1. Importantly, they also demonstrate the feasibility of RNAi by dsRNA injection in adult midges, which will greatly facilitate studies of the underlying mechanisms of vector competence in C. sonorensis.