The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury.
Journal of visualized experiments : JoVE
The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.
View details for DOI 10.3791/63590
View details for PubMedID 35969043
Temporal contribution of myeloid-lineage TLR4 to the transition to chronic pain: A focus on sex differences.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs. central), timing (prevention vs. treatment), and sex (male vs. female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-CreERT2-eYFP;TLR4fl/fl (TLR4 cKO) to specifically knock-out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.Significance statementThe contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs. central), timing (prevention vs. treatment), and sex (male vs. female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Altogether we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.
View details for DOI 10.1523/JNEUROSCI.1940-20.2021
View details for PubMedID 33846230
Augmented beta2-adrenergic signaling dampens the neuroinflammatory response following ischemic stroke and increases stroke size.
Journal of neuroinflammation
2019; 16 (1): 112
BACKGROUND: Ischemic stroke provokes a neuroinflammatory response and simultaneously promotes release of epinephrine and norepinephrine by the sympathetic nervous system. This increased sympathetic outflow can act on beta2-adrenergic receptors expressed by immune cells such as brain-resident microglia and monocyte-derived macrophages (MDMs), but the effect on post-stroke neuroinflammation is unknown. Thus, we investigated how changes in beta2-adrenergic signaling after stroke onset influence the microglia/MDM stroke response, and the specific importance of microglia/MDM beta2-adrenergic receptors to post-stroke neuroinflammation.METHODS: To investigate the effects of beta2-adrenergic receptor manipulation on post-stroke neuroinflammation, we administered the beta2-adrenergic receptor agonist clenbuterol to mice 3h after the onset of photothrombotic stroke. We immunostained to quantify microglia/MDM numbers and proliferation and to assess morphology and activation 3days later. We assessed stroke outcomes by measuring infarct volume and functional motor recovery and analyzed gene expression levels of neuroinflammatory molecules. Finally, we evaluated changes in cytokine expression and microglia/MDM response in brains of mice with selective knockout of the beta2-adrenergic receptor from microglia and monocyte-lineage cells.RESULTS: We report that clenbuterol treatment after stroke onset causes enlarged microglia/MDMs and impairs their proliferation, resulting in reduced numbers of these cells in the peri-infarct cortex by 1.7-fold at 3days after stroke. These changes in microglia/MDMs were associated with increased infarct volume in clenbuterol-treated animals. In mice that had the beta2-adrenergic receptor specifically knocked out of microglia/MDMs, there was no change in morphology or numbers of these cells after stroke. However, knockdown of beta2-adrenergic receptors in microglia and MDMs resulted in increased expression of TNFalpha and IL-10 in peri-infarct tissue, while stimulation of beta2-adrenergic receptors with clenbuterol had the opposite effect, suppressing TNFalpha and IL-10 expression.CONCLUSIONS: We identified beta2-adrenergic receptor signaling as an important regulator of the neuroimmune response after ischemic stroke. Increased beta2-adrenergic signaling after stroke onset generally suppressed the microglia/MDM response, reducing upregulation of both pro- and anti-inflammatory cytokines, and increasing stroke size. In contrast, diminished beta2-adrenergic signaling in microglia/MDMs augmented both pro- and anti-inflammatory cytokine expression after stroke. The beta2-adrenergic receptor may therefore present a therapeutic target for improving the post-stroke neuroinflammatory and repair process.
View details for DOI 10.1186/s12974-019-1506-4
View details for PubMedID 31138227