Peripheral Nerve Block with Ropivacaine Ameliorates Acute Compartment Syndrome by Promoting Macrophage M2 Polarization via JAK-STAT Signaling.

Background

Acute compartment syndrome (ACS) is a critical orthopedic emergency where increased pressure in a limb compartment leads to muscle ischemia, necrosis, and lasting dysfunction. While fasciotomy is the standard treatment, it often results in complications like infection and nerve damage, underscoring the need for effective non-surgical therapies to manage the inflammation causing tissue damage. Peripheral nerve block (PNB) with ropivacaine is clinically used for analgesia, but whether it can serve as a disease-modifying adjunct intervention for ACS beyond pain control remains unexplored.

Objective

This study examined the potential of PNB utilizing ropivacaine to enhance outcomes in ACS and explored the underlying mechanisms related to macrophage polarization. We aimed to distinguish two unresolved questions: (1) whether ropivacaine-based PNB confers tissue-protective effects in ACS independent of analgesia; (2) what signaling pathway mediates ropivacaine-driven macrophage phenotypic switch under ACS-relevant inflammatory conditions.

Methods

Using rats, an ACS model was established, with the animals being randomly allocated to five different groups: Control, ACS, sciatic PNB, femoral PNB, and combined PNB. The study assessed functional outcomes, histopathological changes, levels of inflammatory markers (interleukin-6 (IL-6), C-reactive protein (CRP), and markers of macrophage polarization (CD86, CD206). In parallel, RAW264.7 macrophages stimulated with LPS and IFN-γ were treated with ropivacaine (1-10 μg/mL), and M1/M2 markers, pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), migratory capacity, and cytotoxicity were assessed. Differentially expressed genes and enriched pathways were identified through RNA sequencing, followed by mechanistic validation of JAK-STAT signaling using Western blotting, immunofluorescence, immunohistochemistry, and rescue experiments with the JAK2 inhibitor AG490.

Results

PNB significantly improved all functional and histological parameters in ACS rats, with combined blockade demonstrating the greatest efficacy. Ropivacaine at 5 μg/mL promoted M2 macrophage polarization, evidenced by upregulated CD206 and arginase-1 alongside downregulated CD86 and iNOS, while reducing pro-inflammatory cytokine production without cytotoxicity. Ropivacaine also suppressed macrophage migration. Among 83 differentially expressed genes, RNA sequencing pinpointed JAK-STAT as the pathway with the highest level of enrichment. Mechanistically, ropivacaine activated JAK2-STAT3, reflected by increased p-JAK2/JAK2 and p-STAT3/STAT3 ratios. AG490 completely reversed ropivacaine-induced JAK-STAT activation, M2 polarization, cytokine suppression, and migratory inhibition in vitro, and similarly abrogated PNB-mediated functional recovery and histological protection in vivo.

Conclusions

In vitro, ropivacaine directly promoted macrophage M2 polarization via JAK-STAT activation. In vivo, ropivacaine-based PNB ameliorates ACS, which may involve both direct immunomodulatory effects of ropivacaine and indirect biological consequences of peripheral neural blockade. This study provides the first pre-clinical proof-of-concept that ropivacaine PNB can act as an adjunctive disease-modifying strategy for ACS (beyond analgesia), and identifies macrophage JAK2-STAT3 as the critical molecular cascade responsible for this immunomodulatory effect. This identifies a readily translatable adjunctive strategy for ACS and establishes JAK-STAT as a potential pharmacological target for compartment syndrome-associated inflammation.