Aspirin partially protects Swiss albino mice from PAF-induced lethality and amplifies the cross-tolerance exerted by hyperactivated TLR-4.

<p>Mice were divided into 11 groups. Three groups were intraperitoneally injected with a 10 mg/kg dose of aspirin 30 min before being injected with the indicated doses of PAF, LPS, or both. Three other groups were intraperitoneally injected with a 20 mg/kg dose of aspirin 30 min before receiving PAF, LPS, or both. The 20 mg/kg dose of aspirin partially protected the animals from PAF-induced sudden death, whereas it completely protected the animals from delayed death due to PAF (5 μg/mouse) plus a high dose of LPS (20 mg/kg). The animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

Specific COX-2 inhibitor confers complete protection against both PAF-induced lethality and delayed death due to PAF and hyperactivated TLR-4.

<p>Mice were divided into 7 groups containing 6 animals each. Three groups received an intraperitoneal injection of NS-398 (20 mg/kg body wt) 30 min before receiving PAF, LPS, or a combination of both. NS-398 (20 mg/kg body wt) completely abolished PAF-induced sudden death and enhanced the cross-tolerance effect exerted by the high dose of LPS (20 mg/kg). The animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

LPS cross-tolerance to PAF-induced lethality in Swiss albino mice is temporal.

<p>Mice were divided into 6 groups containing 6 animals per group. PAF (5 μg/mouse) was intraperitoneally injected together with LPS (20 mg/kg body wt) or at 30 min, 3 h, or 6 h after LPS was injected. All the animals injected with PAF alone (5 μg/mouse) or at 3 h or 6 h after receiving LPS (20 mg/kg) died in 15–20 min. In the group that received PAF concomitantly with LPS (20 mg/kg body wt), 33.4% of the animals died within 24 h, and 66.6% survived for 6 days. In the group that received PAF (5 μg/mouse) 30 min after LPS (20 mg/kg) was administered, the time until death was delayed to 12–24 h. All the animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

LPS cross-tolerance is not transferable.

<p>Mice were divided into 4 groups containing 4 animals each. In the initial phase of this experiment, animals were intraperitoneally injected with 0.5 mL of PBS or with LPS (20 mg/kg) + PAF (5 μg/mouse). These animals were then anesthetized, and their blood was collected 1 h after treatment. In the second phase of this experiment, 100 μL of the serum obtained from the mice treated with PBS or LPS (20 mg/kg body wt) + PAF (5 μg/mouse) was intraperitoneally injected into the other 2 groups of mice 30 min before they were injected with PAF (5 μg/mouse). Half of the mice in the group that originally received PAF (5 μg/mouse) + LPS (20 mg/kg) survived, but the serum from these animals failed to protect the naive animals injected with PAF (5 μg/mouse) 30 min after receiving the serum. The results are representative of 3 individual trials.</p

PAF induces sudden death in Swiss albino mice.

<p>Swiss albino mice were divided into 9 groups containing 6 animals each. The indicated amounts of WEB-2086 or BN-52021 were aliquoted from a stock in DMSO, brought up to a total volume of 0.5 mL in PBS, and injected intraperitoneally into animals 30 min before PAF (5 μg/mouse) was injected. Animals that received rPAF-AH (25 μg/mouse) were intraperitoneally injected with it 30 min before being injected with PAF (5 μg/mouse), as were the animals that received the vehicle for rPAF-AH (sodium citrate, sucrose, pluronic, and Tween-80). All the animals injected with PAF alone (5 μg/mouse) died within 15–20 min. In contrast, all the animals that received either a PAF-R antagonist or rPAF-AH 30 min before being injected with a lethal dose of PAF survived. All animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

Dose-dependent effect of PAF on mortality in Swiss albino mice.

<p>The indicated amounts of PAF were aliquoted from a stock of 5 mg/mL in methanol, evaporated under a stream of nitrogen, reconstituted in phosphate buffered saline (PBS) containing 0.1% albumin, and sonicated. The animals were divided into 6 groups, each containing 6 animals. Each mouse was intraperitoneally injected with the designated dose of PAF in a total volume of 0.5 mL of PBS containing 0.1% albumin. Control animals received 0.5 mL PBS containing 0.1% albumin. The animals were monitored for survival for up to 6 days. The results are representative of 3 independent trials.</p

Biologically inactive structural analogs of PAF are not lethal to Swiss albino mice.

<p>Aliquots of lysoPAF and lysoPC were taken from a stock of 5 mg/mL in methanol, dried under nitrogen, reconstituted in PBS containing 0.1% albumin, and sonicated. The indicated doses of each were brought up to a total volume of 0.5 mL with PBS containing 0.1% albumin and intraperitoneally injected into the mice. Animals injected with the vehicle (PBS containing 0.1% albumin) received the same volume. PAF (5 μg/mouse) was used as a positive control; all the animals injected with PAF died within 15–20 min. No mortality was observed for the animals intraperitoneally injected with the lyso analogs at doses equivalent to that used for PAF or 10-fold higher (50 μg/mouse). All the animals were monitored for survival for up to 6 days. The results are representative of 3 individual trials.</p

Strain-specific effects of PAF in murine species.

<p>Five-microgram aliquots of PAF were taken from a stock of 5 mg/mL in methanol, evaporated under a stream of nitrogen, reconstituted to a final volume of 0.5 mL in phosphate buffered saline containing 0.1% albumin, and sonicated. Then, the 5 μg doses of PAF were intraperitoneally injected into 3 strains of mice: Swiss albino, C57BL/6J, and BALB/c (n = 6 per strain). The mice were monitored for survival for up to 6 days. All of the Swiss albino mice that received PAF died within 15–20 min, whereas none of the C57BL/6J or BALB/c mice injected with PAF died. The results are representative of 3 individual trials.</p

Hyperactivation of TLR-4 delays PAF-induced sudden death in Swiss albino mice.

<p>Animals were divided into 8 groups, each containing 6 animals. Three groups were intraperitoneally injected with LPS alone (5, 10, or 20 mg/kg body wt) dissolved in 0.5 mL PBS. Three other groups underwent intraperitoneal injections of LPS (5, 10, or 20 mg/kg body wt) immediately followed by PAF (5 μg/mouse) in 0.5 mL of PBS containing 0.1% albumin. No mortality was observed in the group injected with 10 mg/kg LPS, whereas 30% of the animals injected with 20 mg/kg LPS died in 5–24 h. All the animals injected with PAF alone (5 μg/mouse) and in conjunction with 5 mg/kg LPS died in 15–20 min, whereas a delay in PAF-induced death (2–48 h) was observed when higher doses of LPS (10 and 20 mg/kg) were used. Sixty-six percent of the animals that received PAF (5 μg/mouse) + 10 mg/kg LPS and 50% that received 20 mg/kg LPS + PAF (5 μg/mouse) survived for 6 days. The results are representative of 3 individual trials. *<i>P</i><0.05 and **<i>P</i><0.001, as compared with PAF-challenged mice. The <i>inset</i> graph represents survival rate of animals in minutes for the first 30 min after injection.</p

Serum levels of TNF-α and IL-10 in mice challenged with PAF in the presence and absence of LPS.

<p>(<b>a</b>, <b>b</b>) Blood was collected from the animals injected with PAF alone (5 μg/mouse) or with 5 mg/kg LPS + PAF (5 μg/mouse) 7 min after challenge, whereas blood was collected 4 h after challenge for the other animals. Serum was then separated from the blood for analysis. The levels of TNF-α (a) and IL-10 (b) were measured using ELISA kits, as per the manufacturer’s instructions. The insets show the TNF-α and IL-10 levels for the mice injected with vehicle or PAF (5 μg/mouse) or 5 mg/kg LPS + PAF. All values are expressed as pg/mL and presented as mean ± SD (n = 3). <i>*P</i><0.05; **<i>P</i><0.01, as compared with mice injected with vehicle. LPS cross-tolerance to PAF-induced sudden death neither increased the level of circulating anti-inflammatory IL-10 nor decreased the level of pro-inflammatory TNF-α.</p