Dataset Information

Temporally & Spatially-Resolved Microproteomics and MALDI MSI Lipidomic Analysis for Traumatic Brain Injury

ABSTRACT: Using an experimental TBI rat model of mild/moderate Controlled Cortical Impact (CCI) injury, we combined large-scale proteomics identification and relative quantification using Spatially-Resolved Microproteomics with MALDI MS Imaging of Lipids. Spatially by studying different regions in the brain post injury in a coronal view, with main focus on the injury site itself. Temporally by studying the acute and subacute phase post injury, including injured rat brains at 1 day, 3 days, 7 days, and 10 days post injury. Direct on-tissue micro-digestion followed by micoextraction from 1 mm2 surface area within the injured cortical tissue were subjected to LC-MS & MS/MS analysis using HR MS. In addition, several identified potential biomarkers within our study were used to stimulate dorsal root ganglion (DRG), astrocyte, and macrophage cell lines to obtain a better understanding of their role and contribution in the injury.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain, Astrocyte, Macrophage

DISEASE(S): Wounds And Injuries

SUBMITTER: Jusal Quanico

LAB HEAD: Michel Salzet

PROVIDER: PXD011262 | Pride | 2019-06-19

REPOSITORIES: Pride

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Publications

Mapping Spatiotemporal Microproteomics Landscape in Experimental Model of Traumatic Brain Injury Unveils a link to Parkinson's Disease.

Mallah Khalil K Quanico Jusal J Raffo-Romero Antonella A Cardon Tristan T Aboulouard Soulaimane S Devos David D Kobeissy Firas F Zibara Kazem K Salzet Michel M Fournier Isabelle I

Molecular & cellular proteomics : MCP 20190616 8

Traumatic brain injury (TBI) represents a major health concerns with no clinically-approved FDA drug available for therapeutic intervention. Several genomics and neuroproteomics studies have been employed to decipher the underlying pathological mechanisms involved that can serve as potential neurotherapeutic targets and unveil a possible underlying relation of TBI to other secondary neurological disorders. In this work, we present a novel high throughput systems biology approach using a spatiall ...[more]

PMID: 31204315

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Project description:Purpose: Spinal cord injury (SCI) is a devastating neurological disease without effective treatment. To generate a comprehensive view of the mechanisms involved in SCI pathology, we applied RNA-sequencing (RNA-seq) technology to characterize the temporal changes in global gene expression after contusive SCI in mice. Method Part1: A total of 27 female C57BI/6J mice (10-16 weeks of age; 20-25g; The Jackson Laboratory, Bar Harbor, ME) were used with 9 mice in each of following groups: shame control, 2 and 7 days after SCI. The surgical procedure for SCI were described previously [PMID:23289019]. Briefly, after anesthetization with a mixed solution of ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip), mice received a dorsal laminectomy at the 9th thoracic vertebral (T9) level to expose the spinal cord and then a moderate T9 contusive injury using an Infinite Horizons impactor (PrecisionSystems and Instrumentation) at 60 kdyn with the spinestabilized using steel stabilizers inserted under the transverse processes one vertebra above and below the injury [PMID:19196178] . The shame control mice received only a dorsal laminectomy without contusive injury. Afterwards, the wound was sutured in layers, bacitracin ointment(Qualitest Pharmaceuticals,Huntsville, AL) was applied tothe wound area, 0.1mL of a 20 mg/ml stock of gentamicin(ButlerSchein, Dublin, OH) was injected subcutaneously, and the animals recovered on a water-circulating heating pad. Then mice received analgesic agent, buprenorphin(0.05 mg/kg, SQ; Reckitt Benckise, Hull, England)twice a day for two days. Bladders were manually expressed until automatic voiding returned spontaneously, which generally was within 7 days. At 2 or 7 days after SCI, the mice were anesthetized again with ketamine and xylazine and perfused briefly with normal physical saline. The injured spinal cords were then dissected and three 0.5 mm pieces of spinal cord were cut in the injured epicenter. All spinal cords were immediately frozen in liquid nitrogen and processed for RNA isolation. The spinal cords from three mice were combined into one biological replicate for RNA extraction. Three biological replicates were used. Method Part2: RNA-Seq was performed on the polyadenylated fraction of RNA isolated from tissue samples of acute (2D) and subacute phase (7D) and normal tissues (control, denoted as CTR hereafter). Three biological replicates were used for each phase.150-300 ng total RNAs were used for each sequencing library. RNA samples were polyA selected and paired-end sequencing libraries were constructed using TruSeq RNA Sample Prep Kit as described in the TruSeq RNA Sample Preparation V2 Guide (Illumina).The samples were then sequenced using the Illumina HiSeq sequencer. More than 30 million 100bp paired-end reads were generated from each biological replicate. Method Part3: Read mapping and Transcriptome construction were done by using optimized pipeline which integrate Tophat followed by Cufflinks. Result: We sequenced tissue samples from acute and subacute phases (2 days and 7 days after injury) and systematically characterized the transcriptomes with the goal of identifying pathways and genes critical in SCI pathology. The top enriched functional categories include ‘inflammation response’, ‘neurological disease’, ‘cell death and survival’ and ‘nervous system development’. The top enriched pathways include LXR/RXR Activation and Atherosclerosis Signaling etc. Furthermore, we developed a systems-based analysis framework in order to identify key determinants in the global gene networks of the acute and sub-acute phases. Some candidate genes that we identified have been shown to play important roles in SCI, which demonstrates the validity of our approach. There are also many genes whose functions in SCI have not been well studied and can be further investigated by future experiments. We have also incorporated pharmacogenomic information into our analyses. Among the genes identified, the ones with existing drug information can be readily tested in SCI animal models. Conclusion: in this study we have described an example of how global gene profiling can be translated to screening genes of interest and generating new hypotheses. Additionally, the RNA-seq enables splicing isoform identification and the estimation of expression levels, thus providing useful information for increasing the specificity of drug design and reducing potential side effect. In summary, these results provide a valuable reference data resource for a better understanding of the SCI process in the acute and sub-acute phases. mRNA profiles of Acute/subacute phase Spinal Cord Injury sample from mice were generated by RNA-sequencing using Illumina HiSeq.

Dataset Information

Temporally & Spatially-Resolved Microproteomics and MALDI MSI Lipidomic Analysis for Traumatic Brain Injury

Publications

Mapping Spatiotemporal Microproteomics Landscape in Experimental Model of Traumatic Brain Injury Unveils a link to Parkinson's Disease.

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