1.Prepare Evans Blue
Prepare the EB solution for injection in 0.9% saline at a concentration of 50 mg/ml. Sonicate. Once prepared, the solution can be stored at 4ºC. TIP: It is advisable to prepare in advance.
2.Prepare capsaicin cream
Mix 22.6% of Base L-200, 3.7% of Propylene glycol and 73.7% of distilled water and then add 1.6 % of capsaicin powder. Store at room temperature.
3. Preparation and collection of the tissue
a. Cut aluminium foil into 7-8 cm square pieces and label them with sample numbers.
b. Prepare Pentobarbital working solution for anesthetizing the rats.
c. Inject the anaesthesia intraperitoneally \(50 mg/Kg) depending on the weight of the rat and wait until it is deeply anesthetized.
d. Cut carefully the vibrissae on both sides of the snout with curved scissors.
e. Apply the depilatory cream to the whisker pad area and leave for 7 or 8 minutes.
f. Remove the cream with a flat spatula, taking care not to injure or irritate the area.
g. Rinse the area with a gauze wet in distilled water
h. Prior to the injection of EB, vasodilatation should be induced in the tail vein for easier cannulation. Immerse the rat tail in warm water to induce vein vasodilatation and improve their visualization.
TIP: Be careful not to overheat the water to prevent flaking of the skin. Temperature should not exceed 43ºC. Infrared lamps can be used, as well as vasodilating agents such as Xylazine and Acepromazine.
i. Inject the prepared EB solution \(50 mg/kg of 50 mg/ml solution) intravenously in the lateral vein of the tail with an insulin needle at a rate of 0.01 ml/g of animal weight \(Figure 1A). The lateral veins are located on both sides of the centerline of the tail and are very shallow so that the injection should be almost parallel to the surface. Once injected, remove the needle and press the injection site with cotton to stop bleeding. Time should be measured from the moment the skin turns blue.
TIP: Make sure that the vein is not broken during the injection and all the EB is injected properly. It is best to begin the injection into the distal part of the tail. If a lump is formed at the injection site, it means that the vein is broken, and a subsequent injection should be performed more proximally. In this case the syringe should be refilled to replace any lost volume of EB.
j. Five minutes after the injection, apply capsaicin or vehicle cream \(according the group it belongs to) in the whisker pad area on both sides of the snout.
k. Wait 10 minutes after application of the cream and remove the cream with the aid of a gauze \(Figure 1B).
l. Perfuse the animal through the ascending aorta with saline.
m. Quickly remove the whisker pads. Dissect the area between the nose and the inferior lip. Remove the top layers \(about 5 mm in depth) making sure not to include the intraoral epithelium.
n. Wrap each pad separately in a piece of aluminium foil, leaving the upper side of the package open to allow better drying.
4. Tissue processing
a. Place the tissue in aluminium foil in an oven at 56ºC for two days to dry.
b. After two days, take the tissue out of the oven and immerse each one of the samples in a plastic tube with formamide to extract the EB out of the tissue. Add 8 ml of formamide per 1 g of dry tissue.
TIP: Cover the tubes with aluminum foil.
c. Leave the plastic tubes containing the sample embedded in formamide in an oven at 56ºC over two days.
d. After two days in the oven, extract the formamide with a pipette \(it should be blue) to a covered, labelled eppendorf tube and discard the tissue.
e. Prepare the dilutions for the standard curve: dilute EB in the formamide solution in exponential concentrations. The concentrations used in our study, adjusted to the level required for the absorbance of the samples obtained, were as follows:
0 ; 1 ; 2 ; 4 ; 8 ; 16 ; 32 ; 64 ; 128 μg/ml
TIP: Prepare just before measuring the absorbance of the samples. A standard curve is necessary for each experimental session
f. Take 200 μl of the formamide extracted from each sample and put each one in a different well of a P96 multiwell plate. In the same plate put 200 μl of the corresponding dilutions for the standard curve.
h. Measure the absorbance with the help of a spectrophotometer at a wavelenght of 620 nm. We use the program Shortcut to Spf.
5. Data fit
a. Obtain absorbance values from the solutions forming the standard curve. These values should grow exponentially \(Table 1).
b. Adjust the standard curve to a theoretical sigmoidal curve. The regression parameters \(a, b, x0, y0) can be estimated from the experimental absorbance data using any statistical program \(such as Sigma Plot 8.0). Then, the non-linear regression formula is applied:
where the independent variable x indicates the concentration, the dependent variable y refers to the absorbance and a theoretical standard curve is obtained \(Figure 2). This allows us to calculate any point with respect to the formula, thus obtaining a set value.
c. Extrapolate the concentration of extravasated EB obtained in the study from the known concentrations of EB and formamide measured in the standard curve obtained in step 5b. The values can be calculated by isolating the variable x from the previous equation:
x=x0-ln\(\(-y+y0+a)/\(y-y0)) ** b
d. TIP:_this step is necessary if the tissue collection and spectrophotometric analysis was performed on various days. If all spectrophotometry measurements were obtained in one session, this step can be skipped._
After obtaining the x value, all data should be normalized for comparison with each of those obtained in different experimental sessions. The data are normalized with respect to the cut off point xc that is given by assuming that the derivative of the sigmoid curve at x=xc is equal to 0.0005,
dy/dx \(x=xc) = 0.0005
This very low arbitrary value establishes that at the cut off point, the absorbance curve is saturated \(practically unchanged, as indicated by a derivative close to zero).
Finally, x concentrations are converted into dimensionless magnitudes by transforming them into percentages with respect to xc
% = x ** 100/xc
This procedure allows for normalizing the data from different experimental sessions on separate days, which otherwise would lead to non-comparable data.