Chronic morphine administration delays wound healing by inhibiting immune cell recruitment to the wound site

Abstract

Patients prescribed morphine for the management of chronic pain, and chronic heroin abusers, often present with complications such as increased susceptibility to opportunistic infections and inadequate healing of wounds. We investigated the effect of morphine on wound-healing events in the presence of an infection in an in vivo murine model that mimics the clinical manifestations seen in opioid user and abuser populations. We show for the first time that in the presence of an inflammatory inducer, lipopolysaccharide, chronic morphine treatment results in a marked decrease in wound closure, compromised wound integrity, and increased bacterial sepsis. Morphine treatment resulted in a significant delay and reduction in both neutrophil and macrophage recruitment to the wound site. The delay and reduction in neutrophil reduction was attributed to altered early expression of keratinocyte derived cytokine and was independent of macrophage inflammatory protein 2 expression, whereas suppression of macrophage infiltration was attributed to suppressed levels of the potent macrophage chemoattractant monocyte chemotactic protein-1. When the effects of chronic morphine on later wound healing events were investigated, a significant suppression in angiogenesis and myofibroblast recruitment were observed in animals that received chronic morphine administration. Taken together, our findings indicate that morphine treatment results in a delay in the recruitment of cellular events following wounding, resulting in a lack of bacterial clearance and delayed wound closure.

Figure 1

A: Gross morphology of cutaneous wounds in hind limbs of mice. Cutaneous wounds of mice treated with placebo, morphine, or morphine plus naltrexone in the presence or absence of LPS were photographed on days 1 and 7. Ratios of quantified pixel areas of the wounds were used in determining the rate of wound closure. A marked decrease in wound healing integrity was seen in the morphine-treated groups. Integrity is based on the quality of healing. Wounds of morphine-treated animals presented with increased necrotic tissue, pus, and marked edema/swelling. Co-administration of naltrexone reversed the effects seen in the morphine-treated groups. Following chronic morphine administration, in the presence and absence of LPS, wounds showed a significant decrease in closure rates compared with control groups. B: Quantified analysis of pixel area wound closure events. Percentages of wound areas were quantified to measure closure rates between treatment groups. Areas of wound beds from mice treated with morphine showed significant delay in closure when compared with their respective control groups. Co-administration of naltrexone restored the morphine-mediated delay in wound closure to rates seen in the placebo treated group. Significance of P values are *P ≤ 0.001, **P ≤ 0.01 (n = 4). C: Temporal decrease in wound closure in morphine-treated animals. Wounds were photographed on days 0, 1, 3, 5, 7, and 10 following wounding. Quantified pixel areas of the wound were calculated as percentage of wound area at day 0. Wound closure measured at each time points were significantly lower in the morphine-treated wild-type (WT) mice when compared with all other treatment groups. *P < 0.05 (n = 4). Wound closure rate of morphine-treated MORKO mice were similar to wild-type placebo control. D: Morphine-induced inhibition in wound closure was abolished in MORKO mice. Wild-type and MORKO mice were treated with morphine or placebo pellet and wound beds photographed at day 7. Wound closure in morphine-treated MORKO mice was similar to wild-type placebo-treated animals.

Figure 2

A: Morphine delays leukocyte migration into wound tissue beds. Histological analysis of wound beds taken from the hind limbs was performed using H&E staining techniques. Representative wound sections show significant reduction of infiltrating leukocytes (shown with black arrow) into wound tissues of mice treated with morphine 4 days post-injury. B: Wound sections of morphine-treated animals show significantly lower transmigration of leukocytes out of blood vessels into wound beds. Red arrows indicate blood vessels and black arrows infiltrating leukocytes. C: Representative CD31 staining of wound sections was performed on day seven wound sections. Eight-micron, paraffin-embedded sections were stained with anti-CD 31-PE antibody, an endothelial marker. Cell number was determined using 4,6-diamidino-2-phenylindole staining. CD-31 staining was markedly reduced in morphine-treated wounds (n = 4). Quantitative representation of vessel density (ends), vessel branching (nodes) and vessel length (length) show significant reduction in all parameters tested in morphine-treated animals. *P < 0.01 (n = 4). D: Representative α-SMC staining was performed on day seven wound sections. 8 micron paraffin embedded sections were stained with anti α-SMC-fluorescein isothiocyanate antibody, a myofibroblast marker. Cell number was determined using 4,6-diamidino-2-phenylindole staining. α-SMC-fluorescein isothiocyanate staining was markedly reduced in morphine-treated wounds (n = 4). Quantitative evaluation showed significant reduction in α-SMC expression in morphine-treated wounds. *P < 0.01 (n = 4).

Figure 3

A: FACS analysis of LY6G-labeled neutrophils from PVA sponges. Following injury and removal of PVA sponges, cells were extracted and counted using trypan blue exclusion. 1 × 10⁷ cells were placed into each FACS tubes, labeled with LY6G mouse antibodies, and quantified. In the presence of LPS, a marked increase in cells positive for LY6G (neutrophils) migrated into the PVA sponge 24 hours post-injury. However, significantly fewer LY6G-positive cells were detected when morphine was co-administered and this cell population and did not peak until 72 hours post-injury. Significance of P value is **P ≤ 0.01, *P < 0.05 (n = 4). B: FACS analysis of F480-labeled macrophages from PVA sponges. Following injury and removal of PVA sponges, 1 × 10⁷ cells were placed into each FACS tubes, labeled with F4/80 mouse antibodies, and quantified. A significant decrease (*P ≤ 0.01, n = 4) in F4/80-positive cells (macrophages) was seen following co-treatment of morphine and LPS on days 0.5, 1, 2, 3, and 4. Peak recruitment on day 3 post-injury, was also significantly suppressed (*P ≤ 0.01, n = 4) in the morphine + LPS group. Sustained and significant recruitment was observed in the morphine-treated group on day 5 (*P ≤ 0.01, n = 4).

Figure 4

A: MIP-2 expression profile from PVA sponge extracts. The potent neutrophil chemoattractant MIP-2 from PVA sponge supernatants were analyzed using a MIP-2 protein detection ELISA kit. Interestingly, no significant modulation of protein expression was seen between placebo- and morphine-treated groups on days 1 and 3 post-injury. However, a significant (**P < 0.01, n = 4) elevation of MIP-2 protein levels were seen in response to the endotoxin LPS (placebo and morphine in the presence of LPS). B: KC expression profiles from PVA sponge extracts. The potent neutrophil chemoattractant KC from PVA sponge supernatants were analyzed using the KC protein detection ELISA kit. Interestingly, KC levels in the placebo group were higher at day 1 compared with day 3 in both LPS treated and non-treated animals. Morphine treatment resulted in a significant (*P < 0.01, n = 4) reduction in KC protein levels, when compared with placebo-treated groups on both days 1 and 3 post-injury. A significant (*P < 0.01, n = 4) elevation of KC protein levels were seen in response to the endotoxin LPS. C: MCP-1 expression profiles from PVA sponge extracts. The macrophage chemokine MCP-1 from PVA sponge supernatants was analyzed using ELISA. By day 3 post-injury, significant suppression (**P ≤ 0.01, n = 4) of MCP-1 was seen following morphine administration when compared with its control (placebo). In response to the LPS stimuli, MCP-1 protein levels were markedly increased (placebo + LPS). However, a significant (**P ≤ 0.01, n = 4) decline in protein expression was seen when morphine was co-administered (morphine + LPS).

Figure 5

A: Chemotaxis assay of naïve neutrophil migration toward supernatants of PVA sponge extracts. Bone marrow was harvested from GFP-transgenic mice and treated with morphine (1 μmol/L) or saline for 24 hours. 5 × 10⁶ neutrophils were added to top chambers of chemotaxis plates. Supernatants from day 1 PVA sponge extracts were placed in the bottom chambers. Following 1-hour incubation, top chambers were removed, and cells in the bottom chambers were quantified by plate reader using GFP excitation and emission wavelengths (GFP was detected at a 485:520 excitation/emission wavelength). Significantly fewer neutrophils migrated toward the supernatants containing morphine, as compared with the placebo treated group (P ≤ 0.001, n = 4). In parallel studies, day 1 PVA supernatant was pretreated with either MIP-2 or KC antibody for 12 hours before being placed on the top chamber of the chemotaxis plate. Pretreatment with MIP-2 antibody did not significantly decrease chemotaxis of cells toward the bottom chamber. B: In contrast, pretreatment with KC antibody resulted in a significant decrease in migration of naïve neutrophil toward placeco+LPS supernatant. C and D: Morphine inhibits LPS-induced CXCR2 expression in neutrophils. Bone marrow-derived neutrophils were pretreated with morphine (1 μmol/L) for 24 hours and treated with LPS (10 ng and 100 ng) for 2 hours and CXCR2 expression determined using reverse transcription PCR. Results show that morphine treatment resulted in a dramatic decrease in LPS-induced CXCR2 expression levels (D). β-actin was used as a loading control (*P ≤ 0.01, **P ≤ 0.001, n = 4). D: Densitometric analysis of four representative PCR gel blots. (*P < 0.01).

Figure 6

A: Morphine inhibits LPS-induced MCP-1 expression and had no effect on CCR2 expression in macrophages. Naïve peritoneal macrophages were pretreated with either saline or morphine (1 μmol/L) for 24 hours and then treated with LPS for 12 and 24 hours. MCP-1 and CCR2 receptor expression was determined using reverse transcription PCR. LPS treatment resulted in a time-dependent increase in MCP-1 expression. However, morphine treatment of macrophages significantly inhibited LPS-induced MCP-1 induction. Morphine treatment did not result in a decrease in CCR2 expression. B: Densitometric analysis of 4 representative PCR gel blots. (*P < 0.01 and **P < 001).

Figure 7

Dissemination of E. coli out of wound tissues into blood and organs. PVA sponges containing live bacteria E. coli at 18 colony forming units were implanted into hind limbs of mice administered placebo or morphine. Homogenized spleens, livers, and blood samples were incubated on blood agar plates. Averages of plate colony counts (n = 4) were taken. A significant increase in GFP-tagged E. coli was seen in the spleen, liver, and blood of morphine-treated groups 7 days following injury (*P < 0.01, n = 4). Adequate clearance of bacteria was found to occur in the blood, spleen, and liver tissues of placebo-treated mice. Horizontal lines represent mean bacterial load. Mean ± SE is shown in the table below.

Figure 8

Sponge supernatant from morphine-treated mice attenuated new blood vessel formation in Matrigel plugs. Placebo and morphine PVA sponge supernatants containing Matrigels were injected into the hind limb of placebo or morphine pelleted mice. Matrigel plug was removed at day 7 following euthanasia. Cryostat sections from liquid nitrogen–snap-frozen Matrigel samples were stained with PECAM (CD31) endothelial marker. In the placebo-treated animals that received placebo supernatant (Pp + Ps) (A) angiogenesis was apparent and readily visible throughout the day 7 Matrigel plugs. Substantial decrease in angiogenesis was observed in the placebo-treated animals that received morphine supernatant (Pp + Ms) (C). Chronic morphine treatment significantly decreased the formation of blood vessels in the placebo supernatant (Mp + Ps) (E)-containing plugs. The most dramatic decrease in angiogenesis was observed in morphine-treated animals that received morphine sponge supernatant (Mp + Ms) (G). Panels B, D, F, and H are skeletonized images of panels A, C, E, and G, respectively. B: Areas of vessel density in Matrigel plugs were then examined under higher magnification (×20) and counted after fluorescent images were binearized for PE-positive pixels, and skeletonized (gray images B, D, F, and H) using Reindeer Image Analysis tool kit and Adobe PhotoShop. I, J, K: Morphometric analysis of angiogenic events. Quantification of the skeletonized images, shown in Figure 8, B, D, F, and H was evaluated for angiogenic events [vessel density-ends (I), vessel branch points-nodes (J), and vessel length (K) of new vessels]. Plugs extracted from mice treated with morphine pellets showed a significant reduction in blood vessel density (ends), vessel branching (nodes), and vessel length, when compared with placebo treated groups (*P < 0.05, n = 4).