Genetic deletion of the glucocorticoid receptor in Cx3cr1+ myeloid cells is neuroprotective and improves motor recovery in female mice after 75 kdyne T9 contusion spinal cord injury

DOI:10.34945/F56887

DATASET CITATION

Madalena K. M., Brennan F. H., Popovich P. G. (2022) Genetic deletion of the glucocorticoid receptor in Cx3cr1+ myeloid cells is neuroprotective and improves motor recovery in female mice after 75 kdyne T9 contusion spinal cord injury. ODC-SCI:734 http://doi.org/10.34945/F56887

ABSTRACT

STUDY PURPOSE: Glucocorticoid receptors (GRs), part of the nuclear receptor superfamily of transcription factors (TFs), are ubiquitously expressed in all cell types and regulate cellular responses to glucocorticoids (e.g., cortisol in humans; corticosterone in rodents). In myeloid cells, glucocorticoids binding to GRs can enhance or repress gene transcription, thereby imparting distinct and context-dependent functions in macrophages at sites of inflammation. In experimental models and in humans, glucocorticoids are widely used as anti-inflammatory treatments to promote recovery of function after SCI. Thus, we predicted that deleting GR in mouse myeloid lineage cells (i.e., microglia and monocyte-derived macrophages) would enhance inflammation at the site of injury and worsen functional recovery after traumatic spinal cord injury (SCI).

DATA COLLECTED: The dataset includes n=29 female mice with genotype of GRf/f (Macrophage GR intact) or Cx3cr1-Cre;GRf/f (Macrophage GR deleted). Mice received a 75 kdyne T9 IH contusion SCI or sham (laminectomy only) surgery. The Basso Mouse Scale (BMS) main score and sub-score were used to assess motor function. At 35 dpi, animals were perfused with 0.1M PBS followed by 4% PFA, and spinal tissues were cryopreserved in OCT at -80oC. Tissues were cut in serial sections on a cryostat, 12 um thick along the coronal axis. Spared myelin (EC), spared axons (NFH), and vessel sparing (CD31) were assessed using immunohistochemical staining. GFAP, Iba1, MBP, CD68, and Oil Red O staining was performed in coronal spinal histological sections at 35 days post-injury (dpi). Injury parameters are included for all injured mice. For validation of GR knockout in myeloid cells, we used additional male and female uninjured Cx3cr1-Cre;TdT mice (n=6) and uninjured TdTomato controls (n=4).

CONCLUSIONS: Contrary to our prediction, the intraspinal macrophage response to a moderate (75 kdyne) spinal contusion SCI was reduced in Cx3cr1-Cre;GRf/f conditional knockout mice (with GR specifically deleted in myeloid cells). This phenotype was associated with improvements in hindlimb motor recovery (increased BMS motor scores and subscores; 57% some-most coordination in macrophage GR deleted vs. 0% in macrophage GR intact, 71% paws parallel in macrophage GR deleted vs. 13% in macrophage GRintact, and 43% severe trunk in macrophage GR deleted vs. 62% in macrophage GR intact). GR depletion in myeloid cells also improved myelin sparing (47% more erichrome cyanine staining in lesion epicenter), axon preservation/regeneration (34% more neurofilament staining in lesion epicenter), and microvascular protection/plasticity (33% more CD31 staining) relative to SCI mice with normal myeloid cell GR expression. Further analysis of phagocytic macrophage morphology and myelin basic protein engulfment using CD68 and MBP staining revealed that macrophage GR deletion impairs lipid and myelin phagocytosis and presence of macrophages with a 'foamy' morphology. Together, these data reveal endogenous GR signaling as a key pathway that normally inhibits mechanisms of macrophage-mediated repair after SCI.

KEYWORDS

Behavior; Spinal Cord Injury; microglia; Macrophage; Astrocyte; Glucocorticoid; Demyelinated; axon regeneration

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DATASET INFO

Contact: Popovich Phillip (phillip.popovich@osumc.edu)

Lab: Phillip Popovich

ODC-SCI Accession:734

Records in Dataset: 39

Fields per Record: 114

Last updated: 2022-05-13

Date published:

Downloads: 18

Files: 2

LICENSE

Creative Commons Attribution License (CC-BY 4.0)

FUNDING AND ACKNOWLEDGEMENTS

The National Institutes of Health grants R01NS099532 (PGP), R01NS083942 (PGP), R35NS111582 (PGP), 5R01NS094566-02 (PGP), P30NS104177 (OSU Core lab facility), Ray W. Poppleton Endowment (PGP), Wings for Life Harnessing microglia to improve neurological recovery from spinal cord injury (FHB)

CONTRIBUTORS

Madalena, Kathryn M. [ORCID:0000-0002-3477-5993]

Neuroscience Graduate Program, The Ohio State University, Columbus, OH, USA; Department of Neuroscience, The Ohio State University Wexner Medical Center, Columbus, OH 43210 USA; Belford Center for Spinal Cord Injury, Center for Brain and Spinal Cord Repair, The Ohio State University Wexner Medical Center, Columbus, OH 43210 USA

Brennan, Faith H. [ORCID:0000-0002-9201-2476]

Department of Neuroscience, The Ohio State University Wexner Medical Center, Columbus, OH 43210 USA; Belford Center for Spinal Cord Injury, Center for Brain and Spinal Cord Repair, The Ohio State University Wexner Medical Center, Columbus, OH 43210 USA

Popovich, Phillip G. [ORCID:0000-0003-1329-7395]

Department of Neuroscience, The Ohio State University Wexner Medical Center, Columbus, OH 43210 USA; Belford Center for Spinal Cord Injury, Center for Brain and Spinal Cord Repair, The Ohio State University Wexner Medical Center, Columbus, OH 43210 USA