Pyrilamine maleate

Both flanks of adult anaesthetized mice were shaven. The next day, 100 µl of 1% Evans Blue (Sigma-Aldrich) in sterile saline (wt/vol) was injected intravenously through the lateral tail vein. In some experiments, mice received an intraperitoneal injection of pyrilamine maleate (4 mg/kg body weight in 0.9% saline; Sigma-Aldrich) before Evans Blue injection to inhibit release of endogenous histamine. 30 min after Evans Blue injection, 20 µl of PBS or PBS containing 50 ng VEGF164 (PeproTech), 300 ng SEMA3A (R&D Systems), or 50 ng histamine (Sigma-Aldrich) were injected intradermally each at three sites into the flank skin of anaesthetized mice. After 20 min, mice were culled, the skin was separated from the underlying muscle, and the tissue surrounding the injection sites excised (circled in Fig. 1 A) and dried overnight at 55°C. Evans Blue was extracted by incubation in formamide at 55°C overnight and quantified by spectrophotometry at 620 nm after subtraction of background absorbance at 740 nm. Data from the three sites injected with the same agent (ligand or PBS) were averaged and expressed as fold change relative to PBS control per each mouse. In some experiments, the inner side of the skin was imaged on an MZ16 stereomicroscope (Leica) equipped with a Micropublisher camera (Perkin-Elmer). In other experiments, a sample of liver or skin tissue was retained for immunoblotting.

Analytical grade chemicals were used in experiments. Acetylcholine chloride, atropine sulphate, potassium chloride (KCl), magnesium chloride (MgCl₂), verapamil hydrochloride, pyrilamine maleate, ethylenediaminetetracetic acid (EDTA), carboxymethylcellulose sodium, castor oil, gallic acid, quercetin and loperamide hydrochloride were of Sigma Chemicals (St. Louis, USA) origin. Sodium chloride (NaCl), sulphuric acid (H₂SO₄), hydrochloric acid (HCl), charcoal and sodium nitrite (NaNO₂) were procured from BDH Laboratory Supplies, Poole, UK. Glucose, ethanol, methanol, calcium chloride (CaCl₂), magnesium sulphate (MgSO₄), monopotassium phosphate (KH₂PO₄), monosodium phosphate (NaH₂PO₄), sodium carbonate (Na₂CO₃), sodium bicarbonate (NaHCO₃) and dimethyl sulfoxide (DMSO) were procured from Merck, Darmstadt, Germany. VWR International Ltd. Poole, UK was supplier of ethylacetate.

To assess histamine-induced vascular leak in mice, we used the Miles assay as described previously²⁶ . Briefly, mice were injected intraperitoneally with pyrilamine maleate (4 mg per kg body weight, Sigma) 30 min prior to injection with EB dye to reduce background permeability during handling. Mice were then injected with 100 µl of 0.5% EB dye in PBS (Sigma) via retro-orbital injection, followed by intradermal injections of histamine or saline (50 μl total volume). 30 min after the intradermal injection, the dorsal skin was collected with a 12-mm biopsy punch, and EB dye was extracted with formamide (Sigma; 56 °C for 48 h). The amount of EB in each sample was determined by measuring the absorbance at 620 nm, and results were expressed as EB dye amount (ng) per 100 mm² of the skin, with quantification against a standard curve.