Ablation of VEGFA following a lumbar intervertebral disc injury attenuates intradiscal neurovascular features and prevents chronic low back pain symptoms

Authors
Ryan S Potter, Hong J Moon, Sade W Clayton, Remy E Walk, Addison L Liefer, Liufang Jing, Amber N Stratman, Lori A Setton, Munish C Gupta, Simon Y Tang


Lab

Journal
bioRxiv

Abstract
Post co-culture experiments, HMEC-1 and SH-SY5Y cells were lysed (TRIzol reagent, Fisher Scientific), and total RNA was extracted (Direct-zol RNA MicroPrep Kit, Zymo Research). Samples were prepared according to library kit manufacturer’s protocol, indexed, pooled, and sequenced on an Illumina NovaSeq X Plus. Basecalls and demultiplexing were performed with Illumina’s DRAGEN and BCLconvert version 4.2.4 software. RNA-seq reads were then aligned to the Ensembl release 101 primary assembly with STAR version 2.7.9a1. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 2.0.32. Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.5.23. Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 4.04. All gene counts were then imported into the R/Bioconductor package EdgeR5 and TMM normalization size factors were calculated to adjust for samples for differences in library size. Ribosomal genes and genes not expressed in the smallest group size (minus one sample greater than one count-per-million) were excluded from further analysis. The TMM size factors and the matrix of counts were then imported into the R/Bioconductor package Limma6. Weighted likelihoods based on the observed mean-variance relationship of every gene and sample were then calculated for all samples and the count matrix was transformed to moderated log 2 counts-per-million with Limma’s voomWithQualityWeights7. The performance of all genes was assessed with plots of the residual standard deviation of every gene to their average log-count with a robustly fitted trend line of the residuals. Differential expression analysis was then performed to analyze differences between conditions and the results were filtered for only those genes with Benjamini-Hochberg false-discovery rate adjusted p-values less than or equal to 0.05. The raw data files are available on the Gene Expression Omnibus database: GSE294577

Keywords/Topics
VEGFA; intervertebral disc injury; low back pain; neurovascular features; preclinical model

BIOSEB Instruments Used:
Cold Hot Plate Test (BIO-CHP),Electronic Von Frey - Wireless (BIO-EVF-WRS),Electronic Von Frey 5 with embedded camera (BIO-EVF5),Grip strength test (BIO-GS4),Rotarod for rats and mice (BX-ROD),Von Frey Filaments (BIO-VF-M)

Source :

https://pmc.ncbi.nlm.nih.gov/articles/PMC12157658/

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