Neuroprotection and bromodomain and extra-terminal domain (BET) protein inhibition after T8 spinal cord contusion in female rats and mice

DOI:10.34945/F5ZP4P

DATASET CITATION

Cerqueira S. R., Benavides S., Lee H. E., Ayad N. G., Lee J. K. (2022) Neuroprotection and bromodomain and extra-terminal domain (BET) protein inhibition after T8 spinal cord contusion in female rats and mice. ODC-SCI:696 http://doi.org/10.34945/F5ZP4P

ABSTRACT

STUDY PURPOSE: In this study, we tested the therapeutic potential of inhibiting bromodomain and extraterminal domain (BET) proteins after SCI, and investigated the role of the BET protein BRD4 in macrophages during progression of SCI pathology.

DATA COLLECTED: We investigated the anti-inflammatory and neuroprotective effects of BET inhibition in a rat moderate T8 contusion model (IH impactor; 200 kDyn). First, we performed a dose response study (7.5; 25; 75 mg/kg) of two compounds (IBET-151; IBET-762) administered intraperitoneally (IP) at 3 hours post-SCI. At 6 hours post-SCI, 4-mm long samples of spinal cord centered at the injury epicenter were collected to measure expression of proinflammatory cytokines using qPCR (n=6/group). We then performed IP administration of vehicle, IBET-151 or IBET-762 (50mg/kg) at 3 and 18 hours after SCI and verified transcription levels of proinflammatory cytokines at 24 hours post-SCI using RT-qPCR (n=6/group). Based on these data, we performed a long-term study using the same experimental SCI model, and treated animals with vehicle or IBET-762 (3h; 18h; 1, 2 and 3 days after SCI) (n=11-12/group). Rats were scored using the Basso, Beattie and Breshnahan (BBB) scale at day 1, and weekly thereafter. Eight weeks after SCI, animals were perfused, spinal cord injury sites removed and sectioned sagittally. Unbiased stereological quantifications of lesion size (GFAP negative area), fibrotic scar formation (PDGFRb positive area), and immune cell infiltration (CD11b positive area) in the lesion site were done in 3-5 sections per animal. Next, we investigated the role of BRD4 in macrophages after SCI using LysM-Cre-TDT BRD4 KO mice. Female mice (8-10 weeks old) were subjected to a T8 contusion (IH impactor; 65 kDyn) (WT controls n=8; Brd4 KO n=8). Locomotor recovery was assessed at day 1 after SCI and weekly thereafter using the Basso Mouse Scale open field test for mice. Four weeks after SCI, mice were perfused and spinal cord injury sites collected and sectioned sagittally for histological evaluation. Unbiased stereological quantifications of lesion size (GFAP negative area), and macrophage infiltration (TDT positive area) in the lesion site were done in 3-5 sections per animal. Finally, we evaluated the effect of macrophage-targeted BET inhibition using compound delivery via liposomes. Female mice (8-10 weeks old) were subjected to a T8 contusion (IH impactor; 65 kDyn) and liposomes were administered IP at days 1, 3, 6, 11, and 18 (vehicle liposomes n=5; IBET-762 liposomes n=5). Locomotor recovery was assessed at day 1 after SCI and weekly thereafter using the Basso Mouse Scale open field test for mice. Four weeks after SCI, mice were perfused and spinal cord injury sites collected and sectioned sagittally for histological evaluation. Unbiased stereological quantifications of lesion size (GFAP negative area), fibrotic scar (PDGFRb positive area), and macrophage infiltration (CD11b positive area) in the lesion site were done in 3-5 sections per animal.

CONCLUSIONS: Taken together, our data indicate that systemic I-BET762 treatment is neuroprotective, and the histopathological improvement observed is likely to be a result of effects on non-macrophage targets.

KEYWORDS

Spinal Cord Injury; Inflammation; Epigenetic inhibition; BET proteins; BRD4

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DATASET INFO

Contact: Cerqueira Susana (scerqueira@miami.edu)

Lab: Jae Lee Lab

ODC-SCI Accession:696

Records in Dataset: 109

Fields per Record: 46

Last updated: 2022-04-08

Date published: 2022-04-08

Downloads: 15

Files: 2

LICENSE

Creative Commons Attribution License (CC-BY 4.0)

FUNDING AND ACKNOWLEDGEMENTS

National Institutes of Health grants NINDS R01NS081040 JKL and 10OD023579, Department of Defense SCIRP SC160139 JKL; NGA

CONTRIBUTORS

Cerqueira, Susana R. [ORCID: 0000-0002-9412-237X]

The Miami Project to Cure Paralysis, Miller School of Medicine at the University of Miami

Benavides, Sofia

The Miami Project to Cure Paralysis, Miller School of Medicine at the University of Miami

Lee, Ha E.

The Miami Project to Cure Paralysis, Miller School of Medicine at the University of Miami

Ayad, Nagi G. [ORCID: 0000-0002-8249-3392]

The Miami Project to Cure Paralysis, Miller School of Medicine at the University of Miami

Lee, Jae K. [ORCID: 0000-0001-7413-9783]

The Miami Project to Cure Paralysis, Miller School of Medicine at the University of Miami