The Inflammatory Response after Moderate Contusion Spinal Cord Injury: A Time Study
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. CSF Collection
2.2. Animals
2.3. Induction of Spinal Cord Injury (SCI)
2.4. Basso Mouse Scale (BMS)
2.5. Human SCI Tissue
2.6. Tissue Processing
2.7. Gene Analysis
2.8. In Situ Hybridization for Tnf mRNA
2.9. Protein Purification
2.10. Electrochemiluminescence Analysis
2.11. CSF ELISA
2.12. Immunohistochemistry for TNF
2.13. Immunofluorescence Staining
2.14. Flow Cytometry
2.15. Statistical Analysis
3. Results
3.1. SCI Leads to Significant Changes in Locomotor Function
3.2. SCI Results in Glial Scar Formation
3.3. Tnf mRNA Synthesis Increases in the Acute Phase after SCI
3.4. TNF Is Increased on Glial Cells after SCI
3.5. TNFR1 and TNFR2 Expression Increases in the Lesioned Spinal Cord
3.6. SCI Results in Increased Levels of Inflammatory Cytokines
3.7. SCI Results in Microglial Activation and Immune Cell Infiltration into Spinal Cord
3.8. The Cellular Source of TNF and Its Receptors in Human Traumatic Spinal Cord Injury
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Controls | Sub-Acute | Early Chronic | Late Chronic | |
---|---|---|---|---|
Number of cases | 5 | 12 | 10 | 11 |
Sex, n (%) men | 4 (80) | 11 (92) | 10 (100) | 11 (100) |
Age, years, median (IQR) | 31 (23.0; 44.5) | 30.5 (28.3; 47.0) | 30.0 (26.8; 36.3) | 45.0 (39.0; 54.0) |
Case | Age/Sex | Level of Injury | Cause of Injury | Post-SCI Survival Time |
---|---|---|---|---|
#1 | 80/F | C6–T1 | Fall | 15 h |
#2 | 61/M | C1–2 | Dive accident | 2 weeks |
#3 | 43/M | C7 | Fall | 16 days |
#4 | 33/M | C6–7 | MVA | 3 weeks |
#5 | 65/M | C4 | MVA | 5 weeks |
#6 | 67/M | C5–7 | Fall | 6 weeks |
Gene | Primer Sequences (5′-3′) | Accession No. |
---|---|---|
Tnf | F- AGGCACTCCCCCAAAAGATG | NM_001278601.1 |
R- TCACCCCGAAGTTCAGTAGACAGA | ||
Tnfrsf1a | F- GCCCGAAGTCTACTCCATCATTTG | NM_011609.4 |
R- GGCTGGGGAGGGGGCTGGAGTTAG | ||
Tnfrsf1b | F- GCCCAGCCAAACTCCAAGCATC | NM_011610.3 |
R- TCCTAACATCAGCAGACCCAGTG | ||
Il1b | F- TGCCACCTTTTGACAGTGATG | NM_008361.4 |
R- CAAAGGTTTGGAAGCAGCCC | ||
Il6 | F- AGGATACCACTCCCAACAGA | NM_001314054.1 |
R- ACTCCAGGTAGCTATGGTACTC | ||
Il10 | F- GCCAGGTGAAGACTTTCTTTCAAAC | NM_010548.2 |
R- AGTCCAGCAGACTCAATACACAC | ||
Cxcl1 | F- GCTGGGATTCACCTCAAGAAC | NM_008176.3 |
R- TGTGGCTATGACTTCGGTTTG | ||
Itgam | F- GCCTGTCACACTGAGCAGAA | NM_008401.2 |
R- TGCAACAGAGCAGTTCAGCA | ||
Cx3cr1 | F- TCCCATCTGCTCAGGACCTC | NM_009987.4 |
R- GGCCTCAGCAGAATCGTCAT | ||
Trem2 | F- TGCTGGAGATCTCTGGGTCC | NM_031254.3 |
R- AGGTCTCTTGATTCCTGGAGGT | ||
Arg1 | F- ATGAAGAGCTGGCTGGTGTG | NM_007482.3 |
R- CCAACTGCCAGACTGTGGTC | ||
P2ry12 | F- GCCAGTGTCATTTGCTGTCAC | NM_027571.4 |
R- TAGATGCCACCCCTTGCACT | ||
Hprt1 | F- TCCTCAGACCGCTTTTTGCC | NM_013556.2 |
R- TCATCATCGCTAATCACGACGC |
Antibody | Conjugated | Host (Clone) | Dilution | Source (cat. no.) |
---|---|---|---|---|
Anti-TNF | Unconjugated | Rabbit | 1:200 | Thermo Fischer Scientific (P-350) |
Anti-TNFR1 | Unconjugated | Rabbit (H-271) | 1:50 | Santa Cruz (sc-7895) |
Anti-TNFR2 | Unconjugated | Rabbit | 1:200 | Sigma Aldrich (HPA004796) |
Anti-TNFR2 | Unconjugated | Goat | 1:50 | R&D Systems (AF-426-PB) |
Anti-MAP2 | Unconjugated | Chicken | 1:100 | Abcam (ab5392) |
Anti-CD68 | Unconjugated | Rat | 1:400 | Bio-Rad (MCA1957) |
Anti-CD68 | Unconjugated | Mouse (PG-M1) | 1:100 | Abcam (ab783) |
Anti-CD11b | Unconjugated | Rat | 1:500 | Bio-Rad (MCA711) |
Anti-Iba1 | Unconjugated | Rabbit | 1:500 | Wako (019–19741) |
Anti-IBA1 | Unconjugated | Mouse (GT10312) | 1:1000 | Sigma Aldrich (SAB2702364) |
Anti-Galectin-3 | Unconjugated | Rat (M38) | 1:300 | Hakon Leffler’s Lab [38] |
Anti-NF-L | Alexa Fluor-488 | Mouse (N52) | 1:50 | Sigma Aldrich (MAB5266) |
Anti-IL-1β | Unconjugated | Mouse (2E8) | 1:50 | Bio-Rad (MCA5542Z) |
Anti-GFAP | Cy3 | Mouse (G-A-5) | 1:500 | Sigma Aldrich (C9205) |
Anti-GFAP | Alexa Fluor-488 | Mouse (131–17719) | 1:400 | Invitrogen (A21294) |
Anti-Rabbit | Alexa Fluor-594 | Donkey | 1:200 | Invitrogen (A21207) |
Anti-Rabbit | Alexa Fluor-488 | Chicken | 1:200 | Invitrogen (A21441) |
Anti-Rat | Alexa Fluor-594 | Goat | 1:200 | Invitrogen (A11007) |
Anti-rabbit | Alexa Fluor-594 | Goat | 1:200 | Invitrogen (A21207) |
Anti-rabbit | Alexa Fluor-488 | Goat | 1:200 | Invitrogen (A11008) |
Anti-rabbit | Alexa Fluor-568 | Goat | 1:200 | Invitrogen (A11011) |
Anti-Rat | Alexa Fluor-488 | Goat | 1:200 | Invitrogen (A11006) |
Anti-Chicken | Alexa Fluor-488 | Goat | 1:200 | Invitrogen (A11039) |
Anti-mouse | Alexa Fluor-488 | Goat | 1:200 | Invitrogen (A11001) |
Anti-mouse | Alexa Fluor-568 | Goat | 1:200 | Invitrogen (A11004) |
Anti-mouse | Alexa Fluor-555 | Goat | 1:200 | Invitrogen (A21422) |
Anti-goat | Alexa Fluor-594 | Donkey | 1:200 | Invitrogen (A11058) |
Antibody | Conjugated | Host (Clone) | Dilution | Source (cat. no.) |
---|---|---|---|---|
Anti-CD45 | PerCP-Cy5.5 | Rat (30-F11) | 1:100 | BD Biosciences (561869) |
Anti-CD11b | BB515 | Rat (M1/70) | 1:200 | BD Biosciences (564454) |
Anti-Ly-6C | PE-Cy7 | Rat (AL-21) | 1:200 | BD Biosciences (560593) |
Anti-Ly-6G | BV421 | Rat (1A8) | 1:200 | BD Biosciences (562737) |
IgG2b, κ | PerCP-Cy5.5 | Rat (A95–1) | 1:100 | BD Biosciences (550764) |
IgG2b, κ | BB515 | Rat (A95–1) | 1:200 | BD biosciences (564421) |
IgM, κ | PE-Cy7 | Rat (clone R4–22) | 1:200 | BD biosciences (560572) |
IgG2a, κ | BV421 | Rat (clone R35–95) | 1:200 | BD Biosciences (562602) |
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Lund, M.C.; Ellman, D.G.; Nissen, M.; Nielsen, P.S.; Nielsen, P.V.; Jørgensen, C.; Andersen, D.C.; Gao, H.; Brambilla, R.; Degn, M.; et al. The Inflammatory Response after Moderate Contusion Spinal Cord Injury: A Time Study. Biology 2022, 11, 939. https://doi.org/10.3390/biology11060939
Lund MC, Ellman DG, Nissen M, Nielsen PS, Nielsen PV, Jørgensen C, Andersen DC, Gao H, Brambilla R, Degn M, et al. The Inflammatory Response after Moderate Contusion Spinal Cord Injury: A Time Study. Biology. 2022; 11(6):939. https://doi.org/10.3390/biology11060939
Chicago/Turabian StyleLund, Minna Christiansen, Ditte Gry Ellman, Maiken Nissen, Pernille Sveistrup Nielsen, Pernille Vinther Nielsen, Carina Jørgensen, Ditte Caroline Andersen, Han Gao, Roberta Brambilla, Matilda Degn, and et al. 2022. "The Inflammatory Response after Moderate Contusion Spinal Cord Injury: A Time Study" Biology 11, no. 6: 939. https://doi.org/10.3390/biology11060939
APA StyleLund, M. C., Ellman, D. G., Nissen, M., Nielsen, P. S., Nielsen, P. V., Jørgensen, C., Andersen, D. C., Gao, H., Brambilla, R., Degn, M., Clausen, B. H., & Lambertsen, K. L. (2022). The Inflammatory Response after Moderate Contusion Spinal Cord Injury: A Time Study. Biology, 11(6), 939. https://doi.org/10.3390/biology11060939