Pathophysiological basis of acute inflammatory hyperaemia in the rat knee: roles of cyclo-oxygenase-1 and -2

Abstract

The role of different isoforms of cyclo-oxygenase (COX) in mediating the acute (0-6 h) and late (24 h) phases of inflammation was investigated in the rat knee joint following intra-articular injection of carrageenan. The hyperaemic response was assessed transcutaneously using laser Doppler imaging (LDI). Samples were taken at corresponding time points for detection of synovial COX-1, COX-2 and inducible nitric oxide synthase (iNOS) mRNA, and measurement of urinary prostaglandin (PG) and nitric oxide metabolites (NO(x)). A non-selective COX inhibitor (indomethacin, 15 mg kg(-1) I.P.), a selective COX-2 inhibitor (SC-236, 16.8 mg kg(-1) I.P.) or vehicle were administered 1 h prior to carrageenan in the acute phase study. LDI scans were taken hourly for 4 h post-induction. Inflammatory hyperaemia in the vehicle group was attenuated in the indomethacin- (P < 0.001, two-way ANOVA) and SC-236-treated groups (P < 0.0001), with no difference between these treatments. At 24 h, I.V. infusion of indomethacin (0.1 mg min(-1)), increased vascular resistance (24 +/- 7.1 %; P < 0.05) compared to vehicle infusion, whereas SC-236 (0.11 mg min(-1)) did not. Resistance changes to indomethacin also differed from SC-236 (P < 0.05). Knee joint diameter progressively increased over 24 h (P < 0.0001, one-way ANOVA). Urinary PG levels increased by 6 h (P < 0.05), but returned to baseline by 24 h. COX-1 mRNA was detectable at all time points; COX-2 mRNA only at 3 h. Urinary NO(x) levels increased progressively over 24 h (P < 0.05), paralleled by induction of iNOS in the 3 and 24 h samples. Prostaglandin production via COX-2 appears to mediate the development of acute inflammatory hyperaemia, but nitrergic mechanisms may supervene subsequently. COX-1 but not COX-2 contributes to the maintenance of basal blood flow in the hyperaemic joint at 24 h.

Figure 1. Oedema response in carrageenan-induced joint inflammation

The oedema response was measured as the percentage change in knee joint diameter. ▪, ipsilateral injected knee (P < 0.0001, one-way ANOVA; n = 4–6); □, contralateral non-injected knee.

Figure 2. The role of COX in the development of acute inflammatory hyperaemia in the rat knee

Percentage change in resistance with time (compared to pre-treatment control) is shown for vehicle (•), indomethacin (▪), SC-236 (▴) and untreated control (○) groups. A significant (P < 0.0001, one-way ANOVA) time-dependent hyperaemia was observed in the vehicle (n = 7) group. This was significantly attenuated by indomethacin (n = 7) and SC-236 (n = 7). There was no significant difference between the two COX inhibitors (P = 0.76).

Figure 3. The role of COX in regulating basal perfusion in the 24 h inflamed joint

Percentage change in resistance (compared to pre-treatment control) is shown for vehicle (□), indomethacin (▪) and SC-236 (). Indomethacin (n = 6) produced a significant increase in articular vascular resistance (24 ± 7.1 %) compared to vehicle infusion (* P < 0.05). The change in resistance produced by infusion of an equimolar concentration of the COX-2 inhibitor, SC-236 (n = 8), differed significantly from the indomethacin-treated group (* P < 0.05), but not from the vehicle group (n = 6; P = 0.71).

Figure 4. Urinary levels at control, and 6 and 24 h after inflammation (n = 4–6), corrected for urine osmolality

Compared to control (0 time), PG levels (A) increased significantly (* P < 0.001) only at 6 h, whereas NO_x (B) tended to increase in a time-dependent manner (P = 0.056, one-way ANOVA), reaching significance (* P < 0.05) at the 24 h time point.

Figure 5. COX-1, COX-2, iNOS and β-actin mRNA expression in control (0 h), and 3 and 24 h after inflammation in the joint synovium of the rat analysed by RT-PCR

The PCR products from four rats (lanes 1–4) in each panel from the β-actin, COX-1, COX-2 and iNOS sequences generated after 35 cycles of amplification were analysed by electrophoresis in a 2 % TBE agarose gel. A 123 bp and 100 bp (iNOS) DNA ladder was used as a molecular weight standard. The sizes of the amplified DNA fragments are indicated in base pairs (see Table 1). -, negative control; +, positive control (see Methods).