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A new sensitive fluorometric method for measurement of vascular permeability
A New Sensitive Fluorometric Vascular Permeability
KAZUYOSHI
WATANABE,
HIDEO
NAKACAWA
Method for Measurement
AND SUSUMU
of
TSURUFUII
A sensitive fluorometric
method has been developed for the measurement of vascular permeability in carrageenin air-pouch inflammation in rats. Fluorescein isothiocyanate-labeled bovine serum albumin (F-BSA) was used as a tracer. This fluorometric method is as simple and reliable as the method using radioiodinelabeled human serum albumin and has the advantages of low cost, no health hazard, and the fact that F-BSA can be stored over a long period. This fluorometric method is probably applicable to other inflammation models such as pleurisy and peritonitis in which inflammatory exudate can be collected. Key Words: Fluorometric measurement; Vascular permeability; bels; Bovine serum albumin; Carrageenin; Inflammation
Fluorescein la-
INTRODUCTION Increase inflammatory Several
in vascular
permeability
processes
methods
and
have been
is one
is frequently developed
of the utilized
most
important
for evaluation
for measurement
phenomena of drug
of vascular
in
action.
permeability.
The method using protein-bound dyes such as Evans Blue or Pontamine Blue is utilized as an alternative method to that of using radioiodine-labeled serum albumin.
In the dye method, vascular permeability is estimated either from the intensity and diameter of the stained area surrounding the injection site or is quantitatively measured by extracting the protein-bound dye of the stained area. However the dye method often lacks sensitivity and precision. The method using radioiodine-labeled serum albumin (Mustard et al., 1965; Di Rosa et al., 1970; Tsurufuji et al., 1977) is the most widely used because of its high sensitivity and the convenience of being able to follow the time course of drug action on vascular permeability. The method, however, has many defects: 1) the use of radioactive isotopes requires a special laboratory; 2) radiation may cause health hazards; 3) isotopes and instruments for their measurement are relatively expensive; and 4) the half-life of isotopes limits the period that radioactive tracers can be stored. We have developed a sensitive and precise fluorometric method for measuring vascular permeability in order to overcome the defects of the method using radioiodine-labeled human serum albumin (*I-HSA) as a tracer. From the Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Sendai 980, Japan. Address reprint requests to Dr. Kazuyoshi Watanabe, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Sendai 980, Japan. Received October 5, 1983; revised and accepted December 5, 1983. 167 Journalof
Pharmacological
Methods
0 1984 Elsevier Science Publishing
11, 167-176
(1984)
Co., Inc., 52 Vanderbilt
0160-5402/84lSO3.00 Avenue,
New York, NY 10017
168
K. Watanabe et al.
MATERIALS AND METHODS Induction
of Inflammation
Carrageenin air-pouch inflammation was induced according to the procedure already reported (Fukuhara and Tsurufuji, 1969). Briefly, male rats (Sprague-Dawley weighing 170-190 g) were subcutaneously injected with 7 ml of air on the dorsum to make an air sac, and 24 hr later 4 ml of 2% (w/v) solution of carrageenin in 0.9% NaCl was injected into the air sac. Preparation
of F-BSA
Conjugation of fluorescein isothiocyanate to bovine serum albumin (BSA) was performed according to the procedure of McKinney et al. (1964), with some modifications. A standard solution of 0.2% fluorescein isothiocyanate (Dozin Chemical institute, Osaka, Japan) in 0.15 M phosphate buffer (pH 9) and 10% BSA solution in 0.15 M phosphate buffer (pH 9.5) were prepared. About 25 ml of the fluorescein isothiocyanate solution was added to 50 ml of the BSA solution. The pH was adjusted to 9.5 by addition of 0.1 M trisodium phosphate solution, and the reaction mixture was stirred for 3 hr at room temperature with occasional pH adjustment (pH 9.5). The solution was fractionated by addition of solid (NH&SO4 to give a 70% saturation. The mixture was stirred for 30 min in an ice bath and centrifuged at 9,000 g for 15 min at 2°C. The precipitate was washed with cold acetone and centrifuged at 3,000 g for 5 min at 2°C. The washing was repeated until the color of fluorescein isothiocyanate was not observed in the supernatant. After the washing, the precipitate was dissolved in approximately 100 ml of cold distilled water and thoroughly dialyzed against distilled water at 4°C. The dialyzed solution was lyophilized. Molecular fluorescein/protein ratio of fluorescein isothiocyanate-labeled bovine serum albumin (F-BSA) obtained was about 3.7. Gel Filtration Gel filtration was performed on a Sephadex G-100 column (12 x 300 mm) at a flow rate of 15-20 ml/hr. The effluent fractions of 3 ml were collected. Preparation
of Radioiodinated
Human Serum Albumin
‘3’l-labeled human serum albumin (13’l-HSA, Daiich Kagaku Co., Tokyo, Japan) and 1251-labeled human serum albumin (lz51-HSA, Kaken Kagaku Co., Tokyo, Japan) were purified before use by subjecting them to Sephadex G-100 column chromatography as described above in order to remove radioactive low molecular impurities (Tsurufuji et al., 1977). Measurement
of Fluorescence
Intensity
of F-BSA
The fluorescence intensity of the solution containing F-BSA was measured with a fluorescence spectrophotometer (Japan Spectroscopic Co., Tokyo, Japan) using 490 nm for excitation and 521 nm for emission.
Fluorometric
Measurement
of Radioactivities
of 1251-HSA and 13’1-HSA
The radioactivity was measured in an automatic Aloka JDC-751 (Nihon Musen Co., Mitaka, Japan). The Elimination
Measurement
of F-BSA and ‘*‘I-HSA
well-type
scintillation
counter
from Rat Plasma
F-BSA (20 mg protein in 0.2 ml of 0.9% NaCl solution) or 1251-HSA (1 )*Ci in 0.2 ml of 0.9% NaCl solution) was injected into the tail vein of a rat. At 2, 30, 90, 180, and 300 min after the injection, a blood sample (about 0.2 ml) was drawn from the tail artery, injured with a surgeon’s knife, into a heparinized capillary tube (length: 110 mm, diameter: 2 mm). One end of the capillary tube was closed by heating and then the capillary tube was centrifuged at 2,000 g for 20 min at 4°C. Exactly 25 t~,lof the supernatant was diluted with 4 ml of Ca2+-, Mg2+-free phosphate-buffered saline (PBS( -)). The fluorescence intensity or the radioactivity of the diluted plasma was determined. Vascular Permeability
Assay in the Acute Phase of Inflammation
Rats were injected i.v. with 13’1-HSA (1 t.Li) - F-BSA (20 mg) in 0.2 ml of 0.9% NaCl within 24 hr after carrageenin injection to measure vascular permeability in
24
20 F
-
BSA,
i0 ug/ml
FIGURE 1. The effect of the acute-phase exudate on the fluorescence intensity of F-BSA. Various amounts of F-BSA were dissolved in 5 ml of PBS( -) (O- - - -0) or supernatant of the exudate (AA). The exudate obtained at 6 hr after carrageenin injection was diluted 6fold with PBS(-) and centrifuged at 20,000 g for 20 min at 15°C. Each point represents the mean of three determinations.
169
170
K. Watanabe
et al.
2
1 F
-
BSA,
3
4
5
&ml
FIGURE 2. The effect of the chronic phase exudate on fluorescence intensity of F-BSA. Various amounts of F-BSA were dissolved in 4 ml of PBS( -) (O- - - -0) or supernatant of the exudate (AA). The exudate was obtained 8 days after carrageenin injection and was centrifuged at 300,fMO g for 1 hr at 2°C. The supeinatant was diluted 4-fold with PBS( -) and used for assay. Each point represents the mean of three determinations.
the acute
phase
of inflammation.
Rats were
sacrificed
at 30 min after
the tracer
injection. The entire pouch fluid in the carrageenin air-pouch was collected the weight of the fluid was measured. A portion (I ml) of the fluid was taken
and and
its radioactivity was measured. Another portion (1 ml) of the fluid was mixed with 5 ml of PBS( -) that had been warmed at 37°C in advance to prevent gel formation of the fluid. The mixture was centrifuged at 20,000 g for 20 min at 15°C. Fluorescence intensity of the supernatant was measured. Total radioactivity or fluorescence intensity in the entire pouch fluid was calculated and expressed in terms of percent of the radioactivity or the fluorescence intensity injected and used as an index of the vascular Vascular
permeability Permeability
in the inflammatory
locus.
Assay in the Chronic Phase of Inflammation
Three days after the carrageenin injection, the carrageenin air-pouch inflammation had already entered the chronic proliferative phase in accordance with the development of granulation tissue (Tsurufuji et al., 1978). A small amount of fluorescence is observed in the exudate at this stage. Therefore, a portion (I ml) of the
Fluorometric
Measurement
native exudate (the first sample was used as a blank for the second sample) was withdrawn from each granuloma pouch through a syringe attached to a l/&mm needle. At 2.5 hr after the first sampling of exudate, rats were injected i.v. with 1251HSA (1 t.Ki) - F-BSA (30 mg) in 0.3 ml of 0.9% NaCI. At 30 min after the tracer injection, the second sampling was performed in the same manner. Rats were sacrificed and the weight of the entire exudate of each rat was measured. First and second samples (each 1 ml) were added separately to 3 ml of PBS(-) and then centrifuged at 300,000 g for 1 hr at 2°C. Radioactivity and fluorescence intensity of the supernatant were measured. Calculation of vascular permeability was performed as described above. RESULTS The exudate obtained at 6 hr after carrageenin injection had no effect on the fluorescence intensity of F-BSA; 6-hr exudate had no fluorescence (Figure 1). However, a small amount of fluorescence was found in the g-day exudate, the exudate in the chronic phase of inflammation (Figure 2). Therefore, it is necessary to measure the potent fluorescence intensity of the exudate in advance of the vascular permeability assay in the chronic phase of this inflammation model. There was no significant difference in elimination rates (percent decrease) of FBSA and ‘*‘I-HSA from plasma (Table 1). A decrease of approximately 8% was observed at 30 min after the administration of F-BSA and the half-life of F-BSA in plasma, as well as ‘251-HSA, was over 5 hr.
o----
-0
;
F-BSA
-;Serum F -BSA
0
5
10
Fraction
15
at 3hr after injection ( i.v.1
20
25
30
No.
FIGURE 3. The gel filtration of F-BSA(O- - - -0) and the serum (A -A) obtained at 3 hr after i.v. injection of F-BSA (2B mg) on Sephadex G-100. Each 0.5 ml sample was separately applied to Sephadex G-100 column. Fluorescence intensity of the effluent fractions was de_
.
.
171
172
K. Watanabe et al.
The elution profiles of F-BSA and the serum obtained at 3 hr after i.v. injection of F-BSA on the Sephadex G-100 column are shown in Figure 3. The elution peak of the serum was in good agreement with that of F-BSA, and no fluorescence was found except on the elution position of BSA. Therefore, F-BSA was not degraded into small molecules in the bloodstream until at least 3 hr after the i.v. injection. The inhibitory effect of indomethacin on acute phase vascular permeability was evaluated by two methods using F-BSA or 13’1-HSA. As shown in Figure 4, the results obtained with both tracers were very similar in the dose-dependent suppression of vascular permeability and in the magnitude of standard error. Furthermore, there was a close significant relation (r = 0.973, p < 0.001) between the exudation of FBSA and that of 1371-HSA into the inflammato~ locus (Figure 5).
.
^
I
( Acute
8 IM(mg/kg) Tracer
~
-
Phase
= 7=
)
8 0
0 F -
BSA
1
10
1311 - WSA
FIGURE 4 A comparative evaluation of the effect of indomethacin on vascular permeability in the acute phase of carrageenin air-pouch inflammation using F-BSA or ‘311-HSA. Indomethacin (1 or 10 mg/kg) suspended in 0.5% (w/v) carboxymethyl cellulose solution (1 ml/kg) was administered orally 1 hr before the carrageenin injection. A mixture of F-BSA(20 mg) ‘“I-HSA(l +i) in 0.2 ml of 0.9% NaCl was injected i.v. 5 hr after the carrageenin injection; 30 min later, the entire exudate in the pouch was collected, The total amount of each tracer in the exudate was measured and was expressed in terms of percent of the amount injected. The vertical bars indicate S.E.M. Numbers.in open columns represent the number of rats used for assay. Statistically significant differences with respect to the corresponding control are shown by the following symbols: *p < 0.001; **p < 0.005.
173 l
( Acute-Phase
)
l
lO
0
r
l
u
i 0.5 .u 2
=
0.973
l
1
J *e
0.5
1.0 intensity
F 1 uorescence
FIGURE 5. The relationship between fluorescence intensity and radioactivi~ in the exudate in the acute phase of inflammation. These data were obtained from the experiments shown in Figure 4. A positive relation between the fluorescence intensity and the radioactivity in 1 ml of the exudate exists (r = 0.973; p < 0.001).
TABLE 1
Elimination of F-BSA and ‘%HSA
from Plasma in Rats
F-BSA (6)
‘=I-HSA
~~~
(5)
~--~
FLUORESCENCE INTENSITY TIME
AFTER ~NIECXON
2 30 1.5 3.0 5.0
min min hr hr hr
OF 25
PCASMA
1.26 1.16 0.91 0.85 0.68
k r k ” t
0.05 0.03 0.02 0.03 0.03
FL
RADIOACTIVITY PERCENT DECREASE
7.9 27.8 32.5 46.4
0 k ” L 2
2.4 1.6 2.4 2.4
OF 25
(CPM)
FL PLASMA
8145 7231 6390 5568 4467
5 + -+ 1: -c
356 272 434 214 194
PERCENT DECREASE
11.2 21.5 31.6 45.2
0 + 3.3 r 5.3 +- 2.6 ‘-c 2.4
F-BSA (20 mg in 0.2 ml of 0.9% NaCI solution) or ?-HSA (1 FCi in 0.2 ml of 0.9% NaCl solution) was injected into the tail vein of the rat. At various times after the injection, blood samples were drawn from the tail artery. After centrifugation of the sample, 25 PI of plasma was dituted with 4 ml of PBS{ - ). The fluorescence intensity or the radioactivity of the diluted plasma was determined. The number of rats is in parentheses. Data are shown as mean 2 S.E.M.
174
IL Watanabe
et al. ( Chronic
Dex (mg/kg) Tracer
-
Phase
)
8
8
0
0 F -
BSA
125, - HSA
FIGURE 6. A comparative evaluation of the effect of dexamethasone on vascular permea. bility in the chronic phase of carrageenin air-pouch inflammation by the methods using FBSA and 1251-HSA. Dexamethasone (0.1 or 1 mgkg) dissolved in 25% ethanol - 0.9% NaCl (1 ml/kg) was injected subcutaneously in the rats 8 days after carrageenin injection. The first sampling (1 ml of exudate) from the pouch was performed immediately after the dexamethasone injection. The mixture of F-BSA (30 mg) - ‘251-HSA (1 pCi) in 0.3 ml of 0.9% NaCl was injected iv. 2.5 hr after the dexamethasone injection; 30 min later, a second sample (1 ml of exudate) was withdrawn from the pouch and then the whole exudate was collected. The total amount of each tracer in the entire exudate was measured and expressed in terms of percent of the amount injected. Numbers in open columns represent the number of rats used for assay. The vertical bars indicate S.E.M. Statistically significant differences with respect to the corresponding control are shown by the following symbols: *p < 0.805; **p < 0.01.
The inhibitory effect of dexamethasone on the chronic phase vascular permeability was also evaluated with F-BSA and 1251-HSA (Figure 6). The results obtained with both tracers were very similar in the dose-dependent suppression of vascular permeability and in the magnitude of standard error. Furthermore, there was a close significant relation (r = 0,947, p < 0.001) between the exudation of F-BSA and “‘IHSA into the inflammatory locus (Figure 7). DISCUSSION
F-BSA is suitable as a tracer for measurement of vascular permeability because degradation of F-BSA in plasma was not observed until at least 3 hr after its administration to the blood stream (Figure 3) and the half-life of F-BSA, as well as that of lz51-HSA, was over 5 hr (Table 1). The method using F-BSA is not inferior to that using *I-HSA in simplicity, precision, and reliability based on the results from the double tracer experiments using F-BSA and *I-HSA (Figures 4-7).
Fluorometric
Measurement
Similar studies on the fluorometric method for vascular permeability were also performed by using rhodamine B isothiocyanate-labeled BSA. This conjugate was considerably inferior to F-BSA in sensitivity. Furthermore, under the wavelength conditions that enable the conjugate to be measured, fluorescence was detected in the exudates not only from the chronic phase but also from the acute phase of inflammation (data not shown). Therefore, rhodamine B isothiocyanate-labeled BSA is not a suitable tracer for measurement of vascular permeability. The fluorescence intensity in the 6-hr exudate was negligible, but that in the 8day exudate was detectable and was equivalent to about 5% of the total fluorescence intensity of the exudate obtained at 30 min after F-BSA injection in the chronic phase of inflammation. Although the fluorescence intensity of chronic phase exudate was not so high as to critically influence the measurement of vascular permeability, the sampling of native exudate before F-BSA injection is necessary to obtain more precise results in the carrageenin air-pouch inflammation. The exuded F-BSA was highly diluted with a large quantity of exudate (usually 40-60 ml) already accumulated in
2.4
l
L v W-l I
2.0-
x
1.6’
( Chronic-Phase
>
0
V
.-
0 0
+J
0
;
.z 0.8-u 2 0.4
5
+ 0
l
0.1
*’
0
r = 0.947
l.
0.2
0.3
Fluorescence
0.4
0.5
0.6
0.7
intensity
FIGURE 7. The relationship between fluorescence intensity and radioactivity in the exudate in the chronic phase of inflammation. These data were obtained from the experiments shown in Figure 6. A positive relation between the fluorescence intensity and the radioactivity in 1 ml of the exudate exists (r = 0.95; p < 0.001).
175
176
K. Watanabe
et al.
the pouch in the chronic phase of the carrageenin air-pouch inflammation, resulting in a relative increase in fluorescence intensity of native exudate in the total fluorescence intensity. Therefore, in other inflammation models having less quantity of exudate, the sampling of native exudate before F-BSA injection is probably not necessary. For example, experimental inflammation models such as pleurisy (Di Rosa et al., 1970,1972; Rothkopf, 1978) and peritonitis (Hancocket et al., 1978; Giri et al., 1979) induced by various phlogogens have a small quantity of exudate compared with the carrageenin air-pouch inflammation, and vascular permeability in such models can probably be measured by the present fluorometric method without the sampling of native exudate. This fluorometric method using F-BSA overcame all the defects of the method using *I-HSA; the method using F-BSA is low-cost and does not create a health hazard or require a special laboratory. Furthermore, F-BSA lyophylized can be stored for a long period at 4°C. These advantages and the application of this fluorometric method to other inflammation models in which inflammatory exudate can be collected promise us the convenient and precise measurement of vascular permeability not only in rats, but also in mice (Yamashita et al., 1982) and other animals.
REFERENCES Caster WO, Simon AB, Armstrong WD (1954) An evans blue method for the determination of plasmavolume in the soft tissues in the rat./Appl Physiol6: 724-726 Di Rosa M, Ciroud JP, Willoughby DA (1970) Studies of the mediators of the acute inflammatory response induced in rats in different sites by carrageenin and turpentine. / Path 104:15-29. Di Rosa M, Sorrentino L, Parente L (1972) Non-steroidal antiinflammatory drugs and leucocyte emigration. / Pharm Pharmac 241575-577. Fukuhara M, Tsurufuji S (1969) The effect of locally injected antiinflammatory drugs on the carrageenin granuloma in rats. Biochem Pharmac 18:475-484.
Ciri SN, Jain NC, Hollinger MA (1979) Biochemical and pathological feature of cadmium induced peritonitis in rats. Exp MO/ Patho/30:394-408. Hancock PM, Hill MW, Johnson NW (1978) The inflammatory response to paraffin in the peritoneal cavity of the rat. Br / exp Path 59:128-136. McKinney RM, Spillane JT, Pearce GW (1964) Factors affecting the rate of reaction of fluorescein isothiocyanatewith serum proteins. / lmmunol 931232-242.
Menkin V, Menkin MF (1930) Studies on inflammation. A measure of the permeability of capillaries in an inflamed area. / Exp Med51:285-293. Mustard JF, Movat HJ, Macmorine DRL, Senyi A (1965) Release of permeability factors from the blood platelet. Proc Sot Exp Biol Med 19:988991.
Rothkopf IM (1978) Phenyl isothiocyanate pleurisy in rats: A model for the evaluation of anti-exudative substances. Agents Actions 8:sh6:610617. Tsurufuji S, Sato H, Min KR, Ohuchi K (1978) Difference in the antiinflammatory effect of indomethacin between acute and chronic stage of carrageenin-induced inflammation. / Pharm Dyn I:&14.
Tsurufuji S, Sugio K, Endo Y (1977) Inhibition of vascular permeability by cycloheximide in granulomatous inflammation. Biochem Pharmacol 26:1131-1136.
Yamashita T, lshibashi Y, Nagaoka I, Kasuya K, Masuda K, Warabi H, Shiokawa Y (1982) Studies on glycogen-induced inflammation of mice: Dynamics of inflammatory response and influence of antiinflammatory drugs and protease inhibitors. inflammation 6:87-101.
KAZUYOSHI
WATANABE,
HIDEO
NAKACAWA
Method for Measurement
AND SUSUMU
of
TSURUFUII
A sensitive fluorometric
method has been developed for the measurement of vascular permeability in carrageenin air-pouch inflammation in rats. Fluorescein isothiocyanate-labeled bovine serum albumin (F-BSA) was used as a tracer. This fluorometric method is as simple and reliable as the method using radioiodinelabeled human serum albumin and has the advantages of low cost, no health hazard, and the fact that F-BSA can be stored over a long period. This fluorometric method is probably applicable to other inflammation models such as pleurisy and peritonitis in which inflammatory exudate can be collected. Key Words: Fluorometric measurement; Vascular permeability; bels; Bovine serum albumin; Carrageenin; Inflammation
Fluorescein la-
INTRODUCTION Increase inflammatory Several
in vascular
permeability
processes
methods
and
have been
is one
is frequently developed
of the utilized
most
important
for evaluation
for measurement
phenomena of drug
of vascular
in
action.
permeability.
The method using protein-bound dyes such as Evans Blue or Pontamine Blue is utilized as an alternative method to that of using radioiodine-labeled serum albumin.
In the dye method, vascular permeability is estimated either from the intensity and diameter of the stained area surrounding the injection site or is quantitatively measured by extracting the protein-bound dye of the stained area. However the dye method often lacks sensitivity and precision. The method using radioiodine-labeled serum albumin (Mustard et al., 1965; Di Rosa et al., 1970; Tsurufuji et al., 1977) is the most widely used because of its high sensitivity and the convenience of being able to follow the time course of drug action on vascular permeability. The method, however, has many defects: 1) the use of radioactive isotopes requires a special laboratory; 2) radiation may cause health hazards; 3) isotopes and instruments for their measurement are relatively expensive; and 4) the half-life of isotopes limits the period that radioactive tracers can be stored. We have developed a sensitive and precise fluorometric method for measuring vascular permeability in order to overcome the defects of the method using radioiodine-labeled human serum albumin (*I-HSA) as a tracer. From the Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Sendai 980, Japan. Address reprint requests to Dr. Kazuyoshi Watanabe, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Sendai 980, Japan. Received October 5, 1983; revised and accepted December 5, 1983. 167 Journalof
Pharmacological
Methods
0 1984 Elsevier Science Publishing
11, 167-176
(1984)
Co., Inc., 52 Vanderbilt
0160-5402/84lSO3.00 Avenue,
New York, NY 10017
168
K. Watanabe et al.
MATERIALS AND METHODS Induction
of Inflammation
Carrageenin air-pouch inflammation was induced according to the procedure already reported (Fukuhara and Tsurufuji, 1969). Briefly, male rats (Sprague-Dawley weighing 170-190 g) were subcutaneously injected with 7 ml of air on the dorsum to make an air sac, and 24 hr later 4 ml of 2% (w/v) solution of carrageenin in 0.9% NaCl was injected into the air sac. Preparation
of F-BSA
Conjugation of fluorescein isothiocyanate to bovine serum albumin (BSA) was performed according to the procedure of McKinney et al. (1964), with some modifications. A standard solution of 0.2% fluorescein isothiocyanate (Dozin Chemical institute, Osaka, Japan) in 0.15 M phosphate buffer (pH 9) and 10% BSA solution in 0.15 M phosphate buffer (pH 9.5) were prepared. About 25 ml of the fluorescein isothiocyanate solution was added to 50 ml of the BSA solution. The pH was adjusted to 9.5 by addition of 0.1 M trisodium phosphate solution, and the reaction mixture was stirred for 3 hr at room temperature with occasional pH adjustment (pH 9.5). The solution was fractionated by addition of solid (NH&SO4 to give a 70% saturation. The mixture was stirred for 30 min in an ice bath and centrifuged at 9,000 g for 15 min at 2°C. The precipitate was washed with cold acetone and centrifuged at 3,000 g for 5 min at 2°C. The washing was repeated until the color of fluorescein isothiocyanate was not observed in the supernatant. After the washing, the precipitate was dissolved in approximately 100 ml of cold distilled water and thoroughly dialyzed against distilled water at 4°C. The dialyzed solution was lyophilized. Molecular fluorescein/protein ratio of fluorescein isothiocyanate-labeled bovine serum albumin (F-BSA) obtained was about 3.7. Gel Filtration Gel filtration was performed on a Sephadex G-100 column (12 x 300 mm) at a flow rate of 15-20 ml/hr. The effluent fractions of 3 ml were collected. Preparation
of Radioiodinated
Human Serum Albumin
‘3’l-labeled human serum albumin (13’l-HSA, Daiich Kagaku Co., Tokyo, Japan) and 1251-labeled human serum albumin (lz51-HSA, Kaken Kagaku Co., Tokyo, Japan) were purified before use by subjecting them to Sephadex G-100 column chromatography as described above in order to remove radioactive low molecular impurities (Tsurufuji et al., 1977). Measurement
of Fluorescence
Intensity
of F-BSA
The fluorescence intensity of the solution containing F-BSA was measured with a fluorescence spectrophotometer (Japan Spectroscopic Co., Tokyo, Japan) using 490 nm for excitation and 521 nm for emission.
Fluorometric
Measurement
of Radioactivities
of 1251-HSA and 13’1-HSA
The radioactivity was measured in an automatic Aloka JDC-751 (Nihon Musen Co., Mitaka, Japan). The Elimination
Measurement
of F-BSA and ‘*‘I-HSA
well-type
scintillation
counter
from Rat Plasma
F-BSA (20 mg protein in 0.2 ml of 0.9% NaCl solution) or 1251-HSA (1 )*Ci in 0.2 ml of 0.9% NaCl solution) was injected into the tail vein of a rat. At 2, 30, 90, 180, and 300 min after the injection, a blood sample (about 0.2 ml) was drawn from the tail artery, injured with a surgeon’s knife, into a heparinized capillary tube (length: 110 mm, diameter: 2 mm). One end of the capillary tube was closed by heating and then the capillary tube was centrifuged at 2,000 g for 20 min at 4°C. Exactly 25 t~,lof the supernatant was diluted with 4 ml of Ca2+-, Mg2+-free phosphate-buffered saline (PBS( -)). The fluorescence intensity or the radioactivity of the diluted plasma was determined. Vascular Permeability
Assay in the Acute Phase of Inflammation
Rats were injected i.v. with 13’1-HSA (1 t.Li) - F-BSA (20 mg) in 0.2 ml of 0.9% NaCl within 24 hr after carrageenin injection to measure vascular permeability in
24
20 F
-
BSA,
i0 ug/ml
FIGURE 1. The effect of the acute-phase exudate on the fluorescence intensity of F-BSA. Various amounts of F-BSA were dissolved in 5 ml of PBS( -) (O- - - -0) or supernatant of the exudate (AA). The exudate obtained at 6 hr after carrageenin injection was diluted 6fold with PBS(-) and centrifuged at 20,000 g for 20 min at 15°C. Each point represents the mean of three determinations.
169
170
K. Watanabe
et al.
2
1 F
-
BSA,
3
4
5
&ml
FIGURE 2. The effect of the chronic phase exudate on fluorescence intensity of F-BSA. Various amounts of F-BSA were dissolved in 4 ml of PBS( -) (O- - - -0) or supernatant of the exudate (AA). The exudate was obtained 8 days after carrageenin injection and was centrifuged at 300,fMO g for 1 hr at 2°C. The supeinatant was diluted 4-fold with PBS( -) and used for assay. Each point represents the mean of three determinations.
the acute
phase
of inflammation.
Rats were
sacrificed
at 30 min after
the tracer
injection. The entire pouch fluid in the carrageenin air-pouch was collected the weight of the fluid was measured. A portion (I ml) of the fluid was taken
and and
its radioactivity was measured. Another portion (1 ml) of the fluid was mixed with 5 ml of PBS( -) that had been warmed at 37°C in advance to prevent gel formation of the fluid. The mixture was centrifuged at 20,000 g for 20 min at 15°C. Fluorescence intensity of the supernatant was measured. Total radioactivity or fluorescence intensity in the entire pouch fluid was calculated and expressed in terms of percent of the radioactivity or the fluorescence intensity injected and used as an index of the vascular Vascular
permeability Permeability
in the inflammatory
locus.
Assay in the Chronic Phase of Inflammation
Three days after the carrageenin injection, the carrageenin air-pouch inflammation had already entered the chronic proliferative phase in accordance with the development of granulation tissue (Tsurufuji et al., 1978). A small amount of fluorescence is observed in the exudate at this stage. Therefore, a portion (I ml) of the
Fluorometric
Measurement
native exudate (the first sample was used as a blank for the second sample) was withdrawn from each granuloma pouch through a syringe attached to a l/&mm needle. At 2.5 hr after the first sampling of exudate, rats were injected i.v. with 1251HSA (1 t.Ki) - F-BSA (30 mg) in 0.3 ml of 0.9% NaCI. At 30 min after the tracer injection, the second sampling was performed in the same manner. Rats were sacrificed and the weight of the entire exudate of each rat was measured. First and second samples (each 1 ml) were added separately to 3 ml of PBS(-) and then centrifuged at 300,000 g for 1 hr at 2°C. Radioactivity and fluorescence intensity of the supernatant were measured. Calculation of vascular permeability was performed as described above. RESULTS The exudate obtained at 6 hr after carrageenin injection had no effect on the fluorescence intensity of F-BSA; 6-hr exudate had no fluorescence (Figure 1). However, a small amount of fluorescence was found in the g-day exudate, the exudate in the chronic phase of inflammation (Figure 2). Therefore, it is necessary to measure the potent fluorescence intensity of the exudate in advance of the vascular permeability assay in the chronic phase of this inflammation model. There was no significant difference in elimination rates (percent decrease) of FBSA and ‘*‘I-HSA from plasma (Table 1). A decrease of approximately 8% was observed at 30 min after the administration of F-BSA and the half-life of F-BSA in plasma, as well as ‘251-HSA, was over 5 hr.
o----
-0
;
F-BSA
-;Serum F -BSA
0
5
10
Fraction
15
at 3hr after injection ( i.v.1
20
25
30
No.
FIGURE 3. The gel filtration of F-BSA(O- - - -0) and the serum (A -A) obtained at 3 hr after i.v. injection of F-BSA (2B mg) on Sephadex G-100. Each 0.5 ml sample was separately applied to Sephadex G-100 column. Fluorescence intensity of the effluent fractions was de_
.
.
171
172
K. Watanabe et al.
The elution profiles of F-BSA and the serum obtained at 3 hr after i.v. injection of F-BSA on the Sephadex G-100 column are shown in Figure 3. The elution peak of the serum was in good agreement with that of F-BSA, and no fluorescence was found except on the elution position of BSA. Therefore, F-BSA was not degraded into small molecules in the bloodstream until at least 3 hr after the i.v. injection. The inhibitory effect of indomethacin on acute phase vascular permeability was evaluated by two methods using F-BSA or 13’1-HSA. As shown in Figure 4, the results obtained with both tracers were very similar in the dose-dependent suppression of vascular permeability and in the magnitude of standard error. Furthermore, there was a close significant relation (r = 0.973, p < 0.001) between the exudation of FBSA and that of 1371-HSA into the inflammato~ locus (Figure 5).
.
^
I
( Acute
8 IM(mg/kg) Tracer
~
-
Phase
= 7=
)
8 0
0 F -
BSA
1
10
1311 - WSA
FIGURE 4 A comparative evaluation of the effect of indomethacin on vascular permeability in the acute phase of carrageenin air-pouch inflammation using F-BSA or ‘311-HSA. Indomethacin (1 or 10 mg/kg) suspended in 0.5% (w/v) carboxymethyl cellulose solution (1 ml/kg) was administered orally 1 hr before the carrageenin injection. A mixture of F-BSA(20 mg) ‘“I-HSA(l +i) in 0.2 ml of 0.9% NaCl was injected i.v. 5 hr after the carrageenin injection; 30 min later, the entire exudate in the pouch was collected, The total amount of each tracer in the exudate was measured and was expressed in terms of percent of the amount injected. The vertical bars indicate S.E.M. Numbers.in open columns represent the number of rats used for assay. Statistically significant differences with respect to the corresponding control are shown by the following symbols: *p < 0.001; **p < 0.005.
173 l
( Acute-Phase
)
l
lO
0
r
l
u
i 0.5 .u 2
=
0.973
l
1
J *e
0.5
1.0 intensity
F 1 uorescence
FIGURE 5. The relationship between fluorescence intensity and radioactivi~ in the exudate in the acute phase of inflammation. These data were obtained from the experiments shown in Figure 4. A positive relation between the fluorescence intensity and the radioactivity in 1 ml of the exudate exists (r = 0.973; p < 0.001).
TABLE 1
Elimination of F-BSA and ‘%HSA
from Plasma in Rats
F-BSA (6)
‘=I-HSA
~~~
(5)
~--~
FLUORESCENCE INTENSITY TIME
AFTER ~NIECXON
2 30 1.5 3.0 5.0
min min hr hr hr
OF 25
PCASMA
1.26 1.16 0.91 0.85 0.68
k r k ” t
0.05 0.03 0.02 0.03 0.03
FL
RADIOACTIVITY PERCENT DECREASE
7.9 27.8 32.5 46.4
0 k ” L 2
2.4 1.6 2.4 2.4
OF 25
(CPM)
FL PLASMA
8145 7231 6390 5568 4467
5 + -+ 1: -c
356 272 434 214 194
PERCENT DECREASE
11.2 21.5 31.6 45.2
0 + 3.3 r 5.3 +- 2.6 ‘-c 2.4
F-BSA (20 mg in 0.2 ml of 0.9% NaCI solution) or ?-HSA (1 FCi in 0.2 ml of 0.9% NaCl solution) was injected into the tail vein of the rat. At various times after the injection, blood samples were drawn from the tail artery. After centrifugation of the sample, 25 PI of plasma was dituted with 4 ml of PBS{ - ). The fluorescence intensity or the radioactivity of the diluted plasma was determined. The number of rats is in parentheses. Data are shown as mean 2 S.E.M.
174
IL Watanabe
et al. ( Chronic
Dex (mg/kg) Tracer
-
Phase
)
8
8
0
0 F -
BSA
125, - HSA
FIGURE 6. A comparative evaluation of the effect of dexamethasone on vascular permea. bility in the chronic phase of carrageenin air-pouch inflammation by the methods using FBSA and 1251-HSA. Dexamethasone (0.1 or 1 mgkg) dissolved in 25% ethanol - 0.9% NaCl (1 ml/kg) was injected subcutaneously in the rats 8 days after carrageenin injection. The first sampling (1 ml of exudate) from the pouch was performed immediately after the dexamethasone injection. The mixture of F-BSA (30 mg) - ‘251-HSA (1 pCi) in 0.3 ml of 0.9% NaCl was injected iv. 2.5 hr after the dexamethasone injection; 30 min later, a second sample (1 ml of exudate) was withdrawn from the pouch and then the whole exudate was collected. The total amount of each tracer in the entire exudate was measured and expressed in terms of percent of the amount injected. Numbers in open columns represent the number of rats used for assay. The vertical bars indicate S.E.M. Statistically significant differences with respect to the corresponding control are shown by the following symbols: *p < 0.805; **p < 0.01.
The inhibitory effect of dexamethasone on the chronic phase vascular permeability was also evaluated with F-BSA and 1251-HSA (Figure 6). The results obtained with both tracers were very similar in the dose-dependent suppression of vascular permeability and in the magnitude of standard error. Furthermore, there was a close significant relation (r = 0,947, p < 0.001) between the exudation of F-BSA and “‘IHSA into the inflammatory locus (Figure 7). DISCUSSION
F-BSA is suitable as a tracer for measurement of vascular permeability because degradation of F-BSA in plasma was not observed until at least 3 hr after its administration to the blood stream (Figure 3) and the half-life of F-BSA, as well as that of lz51-HSA, was over 5 hr (Table 1). The method using F-BSA is not inferior to that using *I-HSA in simplicity, precision, and reliability based on the results from the double tracer experiments using F-BSA and *I-HSA (Figures 4-7).
Fluorometric
Measurement
Similar studies on the fluorometric method for vascular permeability were also performed by using rhodamine B isothiocyanate-labeled BSA. This conjugate was considerably inferior to F-BSA in sensitivity. Furthermore, under the wavelength conditions that enable the conjugate to be measured, fluorescence was detected in the exudates not only from the chronic phase but also from the acute phase of inflammation (data not shown). Therefore, rhodamine B isothiocyanate-labeled BSA is not a suitable tracer for measurement of vascular permeability. The fluorescence intensity in the 6-hr exudate was negligible, but that in the 8day exudate was detectable and was equivalent to about 5% of the total fluorescence intensity of the exudate obtained at 30 min after F-BSA injection in the chronic phase of inflammation. Although the fluorescence intensity of chronic phase exudate was not so high as to critically influence the measurement of vascular permeability, the sampling of native exudate before F-BSA injection is necessary to obtain more precise results in the carrageenin air-pouch inflammation. The exuded F-BSA was highly diluted with a large quantity of exudate (usually 40-60 ml) already accumulated in
2.4
l
L v W-l I
2.0-
x
1.6’
( Chronic-Phase
>
0
V
.-
0 0
+J
0
;
.z 0.8-u 2 0.4
5
+ 0
l
0.1
*’
0
r = 0.947
l.
0.2
0.3
Fluorescence
0.4
0.5
0.6
0.7
intensity
FIGURE 7. The relationship between fluorescence intensity and radioactivity in the exudate in the chronic phase of inflammation. These data were obtained from the experiments shown in Figure 6. A positive relation between the fluorescence intensity and the radioactivity in 1 ml of the exudate exists (r = 0.95; p < 0.001).
175
176
K. Watanabe
et al.
the pouch in the chronic phase of the carrageenin air-pouch inflammation, resulting in a relative increase in fluorescence intensity of native exudate in the total fluorescence intensity. Therefore, in other inflammation models having less quantity of exudate, the sampling of native exudate before F-BSA injection is probably not necessary. For example, experimental inflammation models such as pleurisy (Di Rosa et al., 1970,1972; Rothkopf, 1978) and peritonitis (Hancocket et al., 1978; Giri et al., 1979) induced by various phlogogens have a small quantity of exudate compared with the carrageenin air-pouch inflammation, and vascular permeability in such models can probably be measured by the present fluorometric method without the sampling of native exudate. This fluorometric method using F-BSA overcame all the defects of the method using *I-HSA; the method using F-BSA is low-cost and does not create a health hazard or require a special laboratory. Furthermore, F-BSA lyophylized can be stored for a long period at 4°C. These advantages and the application of this fluorometric method to other inflammation models in which inflammatory exudate can be collected promise us the convenient and precise measurement of vascular permeability not only in rats, but also in mice (Yamashita et al., 1982) and other animals.
REFERENCES Caster WO, Simon AB, Armstrong WD (1954) An evans blue method for the determination of plasmavolume in the soft tissues in the rat./Appl Physiol6: 724-726 Di Rosa M, Ciroud JP, Willoughby DA (1970) Studies of the mediators of the acute inflammatory response induced in rats in different sites by carrageenin and turpentine. / Path 104:15-29. Di Rosa M, Sorrentino L, Parente L (1972) Non-steroidal antiinflammatory drugs and leucocyte emigration. / Pharm Pharmac 241575-577. Fukuhara M, Tsurufuji S (1969) The effect of locally injected antiinflammatory drugs on the carrageenin granuloma in rats. Biochem Pharmac 18:475-484.
Ciri SN, Jain NC, Hollinger MA (1979) Biochemical and pathological feature of cadmium induced peritonitis in rats. Exp MO/ Patho/30:394-408. Hancock PM, Hill MW, Johnson NW (1978) The inflammatory response to paraffin in the peritoneal cavity of the rat. Br / exp Path 59:128-136. McKinney RM, Spillane JT, Pearce GW (1964) Factors affecting the rate of reaction of fluorescein isothiocyanatewith serum proteins. / lmmunol 931232-242.
Menkin V, Menkin MF (1930) Studies on inflammation. A measure of the permeability of capillaries in an inflamed area. / Exp Med51:285-293. Mustard JF, Movat HJ, Macmorine DRL, Senyi A (1965) Release of permeability factors from the blood platelet. Proc Sot Exp Biol Med 19:988991.
Rothkopf IM (1978) Phenyl isothiocyanate pleurisy in rats: A model for the evaluation of anti-exudative substances. Agents Actions 8:sh6:610617. Tsurufuji S, Sato H, Min KR, Ohuchi K (1978) Difference in the antiinflammatory effect of indomethacin between acute and chronic stage of carrageenin-induced inflammation. / Pharm Dyn I:&14.
Tsurufuji S, Sugio K, Endo Y (1977) Inhibition of vascular permeability by cycloheximide in granulomatous inflammation. Biochem Pharmacol 26:1131-1136.
Yamashita T, lshibashi Y, Nagaoka I, Kasuya K, Masuda K, Warabi H, Shiokawa Y (1982) Studies on glycogen-induced inflammation of mice: Dynamics of inflammatory response and influence of antiinflammatory drugs and protease inhibitors. inflammation 6:87-101.