MICROVASCULAR
RESEARCH
Extravascular
13,225-231(1977)
Protein Measurements by Ultramicrospectrophotometry
in Vivo and in Situ
SIEGFRIED WITTE AND SUSE ZENZES-GEPRAGS Department oJ’Medicine, Diakonissenkrankenhaus, D-7500, Karfsruhe 51, West Germany Received March 22, 1976 By means of ultramicrospectrophotometry the amount of extravascular plasma protein in the perivasal connective tissue was determined in vivo and in situ, using as an object the mesenterial plate of an everted intestinal loop of the rat. As with the circulating blood plasma, the protein content in the perivascular space lies in the range of 7-527:. We calculated the mean protein amount in 1 g of interstitial tissue as 3.4 mg. Outside the wall of venules the protein content was significantly higher than around capillaries or arterioles. Further from the vascular wall, the protein content decreasesin the interstitial tissue. These concentration gradients were significantly higher around the venules than around the arterioles. The extravascular protein in steady state conditions of permeability is, therefore, not equally distributed within the interstitial space.
INTRODUCTION The exchange phenomena between blood and parenchyma take place in the interstitial connective tissue. In these processesproteins are engaged in different ways. Among them there are proteins asconstituents of the connective tissue, mainly collagen, elastin, and other proteins being produced locally by fibrocytes and other cells. Furthermore, plasma proteins pass the connective tissue between blood and parenchyma taking part in permeability processes.Their quantitative determination, therefore, is an important part of permeability studies. Becauseof the locally different permeability of the various segmentsof the terminal vascular bed, it would be most important to measurethe proteins on topographically defined places. For this purpose we used ultramicrospectrophotometry (Caspersson, 1950; Sandritter, 1958)and adapted this method for vital microscopy. The object of examination was the rat’s mesenteric tissue. A loop of the small intestine was exposed in an object chamber made of quartz glass(Witte et al., 1971).Proteins with aromatic amino acids have maximum light absorption at a wavelength of 280 nm. Collagen which is free of aromatic amino acids does not absorb in this wavelength region, contrary to plasma proteins. The molar extinction coefficients of plasma proteins can be accepted as homogeneous. Technical details of this method have been published previously (Witte and Hagel, 1971;Witte and Geprggs, 1974).The appropriatenessof the LanlbertBeerlaw has beenproven. We found significant linear correlations betweenthemeasured absorbancesand the chemically determined quantities of plasma protein, A standard curve as a referencefor quantitative analysis of plasma proteins in the terminal vascular system in vivo and in situ has been established. Copyright 0 1977 by Academic Press, Inc. 225 All rights of reproduction in any form reserved. ISSN 0026-2862 Printed
in Great
Britain
226
WITTE AND ZENZES-GEPRAGS
By this method we measured ilz situ the amount of extravascular and extracellular protein in the perivascular connective tissue and found differenceswith regard to the various types of microcirculatory vessels,as well as to the distance from the vascular wall. METHODS Object
White rats (Wistar, Chbb-Thorn.) weighing 150-220 g were anesthetized by intraperitoneal injection of Thiogenal(0.2 ml/100 g body wt). After attaching the rat to the basis plate of the object chamber the terminal ileum was exposed through a small lateral abdominal incision. An intestinal loop bearing a well-developed microcirculation in the fat free tissue, as checked by stereomicroscopy, was extended and fixed in the object chamber. The chamber which is bordered planoparallel toward the top and the bottom by quartz glass plates has a central depth of 2 mm (Witte et al., 1971). It is continuously perfused with oxygen-saturated Ringer’s solution, added with 5 y0 gelatine, pH 7.4, 37.5”. The chamber was fixed as an object stage on the microscope, so that it could be moved in the directions of the x and y coordinates. Microscope
We used an ultraviolet microscope for transmitting light, from Carl Zeiss (Oberkochen, West Germany). It has two illumination systems which can selectively be switched into the light path. For subjective examination a bulb light was used for bright field or phasecontrast. The phaseannular diaphragm is placed in front of the collector. For ultraviolet microscopy the light comesfrom a xenon high-pressure lamp (XBO 450 with rheostat apparatus for single phase alternating current to stabilize the lamp current), passesa heat protection cuvette, a sectorial diaphragm, rotating with the double current frequency, passesthe monochromator (M 4 Q III) with a symmetrical suspendedaperture and a variable aperture width, and then passesthrough a retractable prism to the microscope. The optical systemsare made of quartz glass. An objective ultrafluar 10: 1, numerical aperture 0.20, is used as a condenser. An ultrafluar 10: 1 is also usedasan objective (Witte, 1969).Passingan intermediate optic in the tube (2.5x), the image is projected either through a prism for subjective observation to a binocular tube (8x) or, after disconnection of this deviation prism, to the photometer head. This photometer head is placed on the microscope tube and consists of a projective (10x) and two exchangeable diaphragms, with either a central perforated mirror (round frame, 3.2-mm diameter) or a front coated mirror. The image projected onto the surface of these mirrors can be observed by a magnifying lens (10x) as eyepiece.For visual observation a releasebutton opens a light-proofed shutter. Measuring Device
The microscope photometer MPM (Carl Zeiss, Oberkochen, West Germany) is equipped with a multiplier RCA 1 P 28, which is centered in the photometer head.
The measuring device consists of a high voltage unit for the multiplier, photo cell amplifier, and light scale galvanometer. This apparatus is remarkably stabilized by synchronization to the light modulator (rotating sectorial diaphragm). The measuring
EXTRAVASCULAR
PROTEIN
MEASUREMENT
227
range is linear up to a factor ~10~. The time constant of the galvanometer is variable from l-20 sec.Mostly, an adjusting time of 1 set has beenchosen.The measuring field is limited by diaphragms which were inserted into the microscope stage. For the chosen optical conditions we used a diaphragm 0.2 mm in diameter. Process of Measurement
Fat free connective tissue was chosen.A field with a well-developed terminal vascular net was focused. In the samemicroscopic field should be an empty spot that is free of structure. Little round holes which often occur spontaneously in the mesenteric plate are suited for this. Otherwise, we cut an appropriate hole under microscopic vision (diameter approximately 30-60 pm) in a vascular free part of the tissue. In this ho1ethe instrument will be adjusted for a transmission standard of 100‘A. For this, the edge of the empty spot will be focused in visible light with the eyepieceof the photometer head. Then the center of the empty spot will be moved into the measuring field, the bulb light will be switched to monochromatic ultraviolet (wavelength 1= 280 nrn), and the scale on the galvanometer is then setto full deflection (transmission 100).To this purpose the monochromator aperture will be closed till the galvanometer is in a medium range. After reswitching to bright field illumination, the object stage will be moved to the chosen measuring point of the object. Now the ultraviolet absorbance is measured. After this we recheck the standard to confirm whether the reference value remained constant, i.e., full transmission is indicated again in the empty spot. We only considered measurementsfor which the referencevalue remained constant. The optical adjustment of the microscope should not be changed during the whole procedure, which consists of the three consecutive measurements:the referenceobject, the measuring object, and again the referenceobject. The measurementprocedure was repeatedtwo to three times on each object spot, and the mean value of absorbance was calculated. The standard deviation of this method in repeatedmeasurementsof the sameobject field was found as f5 % (Witte and Hagel, 1971). For the conversion of the measured absorbance values into absolute amounts of plasma protein, standard curves we had set up before were used (Witte and Geprags, 1974). We made measurements on the different types of vesselsin the terminal vascular bed and established three groups: (I) arterioles, (2) capillaries, and (3) venules. The classification was made according to the topographical arrangement of the vascular system concerned, the direction of the blood flow, and the composition of the vessel wall. The radial distance between the measuredvesselswas greater than 150 pm. For statistical evaluation the mean values have been compared using a paired t test (Cavalli-Sforza, 1969).
RESULTS 1. The Perivascular Plasma Protein Absorbance a. Findings on both sides outside the vascular wall. First we measuredthe absorbance closely outside the microcirculatory vessels.The distance of the edge of the measuring field from the outer vascular wall was approximately 10 pm (Fig. 1). We checked
228
WI’ITE AND ZENZES-GEPR;iGS
FIG. 1. Typical object field for ultramicrospectrophotometry outside the vessel wall (venule) in the perivascular connective tissue. Radiant field stop (diameter, 0.2 mm) under the optical conditions of the microscope seenwith a diameter of 32 pm. Everted mesenterial plate of the rat, bright field illumination, objective lO:l, ocular 8x.
whether any differencesin the absorbance (A,,,,) on either side of the vesselexist. For this, object fields which were located on exactly opposite sides of a certain vesselspot and which did not embody tissue cells were measured.The mean values of thesecontrol pairs (Table 1) did not differ significantly. Therefore, in further measurements we calculated the mean values from both sides. TABLE I THE ABSORBANCE AT
1280 nm OF THE
PERIVASCULAR TISSUE OUTSIDE THE VASCULAR BOTH SIDES OF THE VESSEL
Vessel
Arterioles Capillaries Venules
One side Opposite One side Opposite One side Opposite
n
m
SD
17 17 4 4 15 15
0.22 0.22 0.20 0.20 0.26 0.26
0.06 0.04 0.05 0.05 0.07 0.1
WALL,
COMPARING
1
P
0.5
>O.l
0.14
>O.l
0.36
>O.l
b. Findings on d$erent types oj’vessels. In a second serieswe measured the protein absorbance in the perivascular tissue surrounding different microcirculatory vessels. On the averagewe found the highest absorbance values outside the venules (Table 2). These values were significantly higher than those measured around capillaries and
EXTRAVASCULAR
229
PROTEIN MEASUREMENT
arterioles. The perivascular absorbances of arterioles and capillaries did not differ significantly from each other. TABLE
2
THE ABSORBANCE AT 1280 nm OF THE PERIVASCLJLARTISSUEOUTSIDE THE VASCULAR WALL, COMPARING DIFFERENT KINDS OF VESSELS
n
m
30 9 34
0.22 0.20 0.26
Vessel Arterioles Capillaries Venules
t
SD ;;]
1.26 2.17
0:07 ]
P
1 z-o.1 2.45 < 0.01 0.025-0.0125
2. The Plasma Protein Absorbancein the Interstitial Space Next the perivascular protein absorbances at various topographical distances from the vessel wall were investigated. To accomplish this we measured object points, which were in the interstitial space perpendicular to the vessel wall at a distance of 20-30 /trn from the perivascular measuring points near the wall. We always found differences, in the mean, with lower protein absorbances being measured more distant to the vascular wall (Table 3). This decrease of absorbance to the peripheral interstitial space was TABLE
3
THE ABSORBANCEAT 1280 nm OF THE INIERSTITIAL TISSUE WITH THE CALCULATED PROTEIN QUANTITY IN pg AND STATISTICAL DATA, COMPARING DIFFERENT DISTANCES FROM THE VESSELWALL
Vessel Arterioles Capillaries
Distance from the vascular wall (id
t
n
10
23 23
0.23 0.20
41 21
0.05 0.05
10
7 7 23 23
0.22
35 9 68 35
0.04 0.03 0.08 0.07
30
30 10 30
0.18 0.27 0.22
P
2.55
RESEARCH
Extravascular
13,225-231(1977)
Protein Measurements by Ultramicrospectrophotometry
in Vivo and in Situ
SIEGFRIED WITTE AND SUSE ZENZES-GEPRAGS Department oJ’Medicine, Diakonissenkrankenhaus, D-7500, Karfsruhe 51, West Germany Received March 22, 1976 By means of ultramicrospectrophotometry the amount of extravascular plasma protein in the perivasal connective tissue was determined in vivo and in situ, using as an object the mesenterial plate of an everted intestinal loop of the rat. As with the circulating blood plasma, the protein content in the perivascular space lies in the range of 7-527:. We calculated the mean protein amount in 1 g of interstitial tissue as 3.4 mg. Outside the wall of venules the protein content was significantly higher than around capillaries or arterioles. Further from the vascular wall, the protein content decreasesin the interstitial tissue. These concentration gradients were significantly higher around the venules than around the arterioles. The extravascular protein in steady state conditions of permeability is, therefore, not equally distributed within the interstitial space.
INTRODUCTION The exchange phenomena between blood and parenchyma take place in the interstitial connective tissue. In these processesproteins are engaged in different ways. Among them there are proteins asconstituents of the connective tissue, mainly collagen, elastin, and other proteins being produced locally by fibrocytes and other cells. Furthermore, plasma proteins pass the connective tissue between blood and parenchyma taking part in permeability processes.Their quantitative determination, therefore, is an important part of permeability studies. Becauseof the locally different permeability of the various segmentsof the terminal vascular bed, it would be most important to measurethe proteins on topographically defined places. For this purpose we used ultramicrospectrophotometry (Caspersson, 1950; Sandritter, 1958)and adapted this method for vital microscopy. The object of examination was the rat’s mesenteric tissue. A loop of the small intestine was exposed in an object chamber made of quartz glass(Witte et al., 1971).Proteins with aromatic amino acids have maximum light absorption at a wavelength of 280 nm. Collagen which is free of aromatic amino acids does not absorb in this wavelength region, contrary to plasma proteins. The molar extinction coefficients of plasma proteins can be accepted as homogeneous. Technical details of this method have been published previously (Witte and Hagel, 1971;Witte and Geprggs, 1974).The appropriatenessof the LanlbertBeerlaw has beenproven. We found significant linear correlations betweenthemeasured absorbancesand the chemically determined quantities of plasma protein, A standard curve as a referencefor quantitative analysis of plasma proteins in the terminal vascular system in vivo and in situ has been established. Copyright 0 1977 by Academic Press, Inc. 225 All rights of reproduction in any form reserved. ISSN 0026-2862 Printed
in Great
Britain
226
WITTE AND ZENZES-GEPRAGS
By this method we measured ilz situ the amount of extravascular and extracellular protein in the perivascular connective tissue and found differenceswith regard to the various types of microcirculatory vessels,as well as to the distance from the vascular wall. METHODS Object
White rats (Wistar, Chbb-Thorn.) weighing 150-220 g were anesthetized by intraperitoneal injection of Thiogenal(0.2 ml/100 g body wt). After attaching the rat to the basis plate of the object chamber the terminal ileum was exposed through a small lateral abdominal incision. An intestinal loop bearing a well-developed microcirculation in the fat free tissue, as checked by stereomicroscopy, was extended and fixed in the object chamber. The chamber which is bordered planoparallel toward the top and the bottom by quartz glass plates has a central depth of 2 mm (Witte et al., 1971). It is continuously perfused with oxygen-saturated Ringer’s solution, added with 5 y0 gelatine, pH 7.4, 37.5”. The chamber was fixed as an object stage on the microscope, so that it could be moved in the directions of the x and y coordinates. Microscope
We used an ultraviolet microscope for transmitting light, from Carl Zeiss (Oberkochen, West Germany). It has two illumination systems which can selectively be switched into the light path. For subjective examination a bulb light was used for bright field or phasecontrast. The phaseannular diaphragm is placed in front of the collector. For ultraviolet microscopy the light comesfrom a xenon high-pressure lamp (XBO 450 with rheostat apparatus for single phase alternating current to stabilize the lamp current), passesa heat protection cuvette, a sectorial diaphragm, rotating with the double current frequency, passesthe monochromator (M 4 Q III) with a symmetrical suspendedaperture and a variable aperture width, and then passesthrough a retractable prism to the microscope. The optical systemsare made of quartz glass. An objective ultrafluar 10: 1, numerical aperture 0.20, is used as a condenser. An ultrafluar 10: 1 is also usedasan objective (Witte, 1969).Passingan intermediate optic in the tube (2.5x), the image is projected either through a prism for subjective observation to a binocular tube (8x) or, after disconnection of this deviation prism, to the photometer head. This photometer head is placed on the microscope tube and consists of a projective (10x) and two exchangeable diaphragms, with either a central perforated mirror (round frame, 3.2-mm diameter) or a front coated mirror. The image projected onto the surface of these mirrors can be observed by a magnifying lens (10x) as eyepiece.For visual observation a releasebutton opens a light-proofed shutter. Measuring Device
The microscope photometer MPM (Carl Zeiss, Oberkochen, West Germany) is equipped with a multiplier RCA 1 P 28, which is centered in the photometer head.
The measuring device consists of a high voltage unit for the multiplier, photo cell amplifier, and light scale galvanometer. This apparatus is remarkably stabilized by synchronization to the light modulator (rotating sectorial diaphragm). The measuring
EXTRAVASCULAR
PROTEIN
MEASUREMENT
227
range is linear up to a factor ~10~. The time constant of the galvanometer is variable from l-20 sec.Mostly, an adjusting time of 1 set has beenchosen.The measuring field is limited by diaphragms which were inserted into the microscope stage. For the chosen optical conditions we used a diaphragm 0.2 mm in diameter. Process of Measurement
Fat free connective tissue was chosen.A field with a well-developed terminal vascular net was focused. In the samemicroscopic field should be an empty spot that is free of structure. Little round holes which often occur spontaneously in the mesenteric plate are suited for this. Otherwise, we cut an appropriate hole under microscopic vision (diameter approximately 30-60 pm) in a vascular free part of the tissue. In this ho1ethe instrument will be adjusted for a transmission standard of 100‘A. For this, the edge of the empty spot will be focused in visible light with the eyepieceof the photometer head. Then the center of the empty spot will be moved into the measuring field, the bulb light will be switched to monochromatic ultraviolet (wavelength 1= 280 nrn), and the scale on the galvanometer is then setto full deflection (transmission 100).To this purpose the monochromator aperture will be closed till the galvanometer is in a medium range. After reswitching to bright field illumination, the object stage will be moved to the chosen measuring point of the object. Now the ultraviolet absorbance is measured. After this we recheck the standard to confirm whether the reference value remained constant, i.e., full transmission is indicated again in the empty spot. We only considered measurementsfor which the referencevalue remained constant. The optical adjustment of the microscope should not be changed during the whole procedure, which consists of the three consecutive measurements:the referenceobject, the measuring object, and again the referenceobject. The measurementprocedure was repeatedtwo to three times on each object spot, and the mean value of absorbance was calculated. The standard deviation of this method in repeatedmeasurementsof the sameobject field was found as f5 % (Witte and Hagel, 1971). For the conversion of the measured absorbance values into absolute amounts of plasma protein, standard curves we had set up before were used (Witte and Geprags, 1974). We made measurements on the different types of vesselsin the terminal vascular bed and established three groups: (I) arterioles, (2) capillaries, and (3) venules. The classification was made according to the topographical arrangement of the vascular system concerned, the direction of the blood flow, and the composition of the vessel wall. The radial distance between the measuredvesselswas greater than 150 pm. For statistical evaluation the mean values have been compared using a paired t test (Cavalli-Sforza, 1969).
RESULTS 1. The Perivascular Plasma Protein Absorbance a. Findings on both sides outside the vascular wall. First we measuredthe absorbance closely outside the microcirculatory vessels.The distance of the edge of the measuring field from the outer vascular wall was approximately 10 pm (Fig. 1). We checked
228
WI’ITE AND ZENZES-GEPR;iGS
FIG. 1. Typical object field for ultramicrospectrophotometry outside the vessel wall (venule) in the perivascular connective tissue. Radiant field stop (diameter, 0.2 mm) under the optical conditions of the microscope seenwith a diameter of 32 pm. Everted mesenterial plate of the rat, bright field illumination, objective lO:l, ocular 8x.
whether any differencesin the absorbance (A,,,,) on either side of the vesselexist. For this, object fields which were located on exactly opposite sides of a certain vesselspot and which did not embody tissue cells were measured.The mean values of thesecontrol pairs (Table 1) did not differ significantly. Therefore, in further measurements we calculated the mean values from both sides. TABLE I THE ABSORBANCE AT
1280 nm OF THE
PERIVASCULAR TISSUE OUTSIDE THE VASCULAR BOTH SIDES OF THE VESSEL
Vessel
Arterioles Capillaries Venules
One side Opposite One side Opposite One side Opposite
n
m
SD
17 17 4 4 15 15
0.22 0.22 0.20 0.20 0.26 0.26
0.06 0.04 0.05 0.05 0.07 0.1
WALL,
COMPARING
1
P
0.5
>O.l
0.14
>O.l
0.36
>O.l
b. Findings on d$erent types oj’vessels. In a second serieswe measured the protein absorbance in the perivascular tissue surrounding different microcirculatory vessels. On the averagewe found the highest absorbance values outside the venules (Table 2). These values were significantly higher than those measured around capillaries and
EXTRAVASCULAR
229
PROTEIN MEASUREMENT
arterioles. The perivascular absorbances of arterioles and capillaries did not differ significantly from each other. TABLE
2
THE ABSORBANCE AT 1280 nm OF THE PERIVASCLJLARTISSUEOUTSIDE THE VASCULAR WALL, COMPARING DIFFERENT KINDS OF VESSELS
n
m
30 9 34
0.22 0.20 0.26
Vessel Arterioles Capillaries Venules
t
SD ;;]
1.26 2.17
0:07 ]
P
1 z-o.1 2.45 < 0.01 0.025-0.0125
2. The Plasma Protein Absorbancein the Interstitial Space Next the perivascular protein absorbances at various topographical distances from the vessel wall were investigated. To accomplish this we measured object points, which were in the interstitial space perpendicular to the vessel wall at a distance of 20-30 /trn from the perivascular measuring points near the wall. We always found differences, in the mean, with lower protein absorbances being measured more distant to the vascular wall (Table 3). This decrease of absorbance to the peripheral interstitial space was TABLE
3
THE ABSORBANCEAT 1280 nm OF THE INIERSTITIAL TISSUE WITH THE CALCULATED PROTEIN QUANTITY IN pg AND STATISTICAL DATA, COMPARING DIFFERENT DISTANCES FROM THE VESSELWALL
Vessel Arterioles Capillaries
Distance from the vascular wall (id
t
n
10
23 23
0.23 0.20
41 21
0.05 0.05
10
7 7 23 23
0.22
35 9 68 35
0.04 0.03 0.08 0.07
30
30 10 30
0.18 0.27 0.22
P
2.55
