Chronotherapy of Non-Steroidal Anti-Inflammatory Drugs May Enhance Postoperative Recovery

Abstract

Postoperative pain relief is crucial for full recovery. With the ongoing opioid epidemic and the insufficient effect of acetaminophen on severe pain; non-steroidal anti-inflammatory drugs (NSAIDs) are heavily used to alleviate this pain. However, NSAIDs are known to inhibit postoperative healing of connective tissues by inhibiting prostaglandin signaling. Pain intensity, inflammatory mediators associated with wound healing and the pharmacological action of NSAIDs vary throughout the day due to the circadian rhythm regulated by the clock genes. According to this rhythm, most of wound healing mediators and connective tissue formation occurs during the resting phase, while pain, inflammation and tissue resorption occur during the active period of the day. Here we show, in a murine tibia fracture surgical model, that NSAIDs are most effective in managing postoperative pain, healing and recovery when drug administration is limited to the active phase of the circadian rhythm. Limiting NSAID treatment to the active phase of the circadian rhythm resulted in overexpression of circadian clock genes, such as Period 2 (Per2) at the healing callus, and increased serum levels of anti-inflammatory cytokines interleukin-13 (IL-13), interleukin-4 (IL-4) and vascular endothelial growth factor. By contrast, NSAID administration during the resting phase resulted in severe bone healing impairment.

Figure 1

Diagram of the circadian rhythm in immune cells, pro-inflammatory and anti-inflammatory mediators, bone resorption and formation markers.

Figure 2

Study design and objectives.

Figure 3

Effect of bone fracture surgery timing on pain and bone healing. (a,b) Pain assessment, (c–e) μCT analysis and (f–h) mechanical analysis of tibiae fracture during resting time and active time at day 14 after tibia fracture surgery. The data show that both groups (n = 8 per group) had similar pain behaviour (weight bearing (WB), guarding tests (GT)), bone volume fraction (BV/TV), trabecular number (Tb.N), spacing between the trabecula (Tb.Sp), trabecular thickness (Tb.Th), maximum force (MF), stiffness (S) and work to failure (WF). All data are expressed as Mean ± SE values.

Figure 4

Effect of NSAID dose timing on pain behaviour. Effect of NSAID administration on pain behaviour (n = 8 per group) at 0, 7 and 14 days after surgery. (a) Guarding test mesh for the paw of the fractured leg, (b) guarding test, (c) weight bearing test. Scores are expressed as mean ± SE values *P < 0.05.

Figure 5

Effect of NSAID dose timing on bone healing outcomes. (a) μCT images of tibiae retrieved from the NSAID resting time group (left) and NSAID active time group (right) at day 14 after tibia fracture surgery, dotted line represents the average results without NSAID administration from Fig. 3. The group that received NSAID at the active time had (b) a higher bone volume fraction (BV/TV) and (c) trabecular number (Tb.N), (d) lower spacing between the trabecula (Tb.Sp) and higher trabecular thickness (Tb.Th) compared to the group receiving NSAID at resting time (n = 7 per group). (e) The mechanical stress-strain curve of 3-point bending tests on the fractured bones, (f) maximum force (MF), (g) stiffness (Stiff.) and (h) work to failure (WF) of the fractured tibiae of each group (n = 7 per group), dotted line represent the average results without NSAID administration from the surgery time experiment (Fig. 3). (i–n) Histology Von-Kossa-stained sections: (i,j) mineralization in black at fracture sites in mice (i) receiving NSAID during resting time or (j) during active time. (k) Percentage of mineralized tissue within the callus. Tartrate-resistant acid phosphatase (TRAP) stain (l,m) shows the osteoclasts in the fracture site of mice receiving NSAID (l) during resting time and (m) active time (scale bars represents 200 μm (big image, and 10 μm small image)). (n) Number of osteoclasts per unit area of callus (n = 4 per group). All data are expressed as Mean ± SE values. *P < 0.05.

Figure 6

Effect of NSAID dose timing on systemic inflammatory cytokines and gene expression of the healing callus. (a,b) The concentration of serum cytokines during fracture healing: (a) serum concentrations of pro-inflammatory cytokines, and (b) anti-inflammatory cytokines and growth factors. (c) Gene expression heat map for the top 100 genes that showed at least 1.5-fold difference (P < 0.05), upregulated (red) or downregulated (blue), at day three after fracture surgery for the group receiving NSAID at active time compared to those receiving NSAID at resting time. (d) PANTHER pathway ontology analysis showing significantly enriched Panther pathways (p < 0.05) of differently expressed genes between the group that received NSAID at the active time compared to resting time at day 3 after fracture surgery. Concentrations are expressed as mean ± SE values. *P < 0.05.