AMERICAN JOURNAL OF PHYSIOLOGY 1’01. 228, No. 1, January 1975. Printed
in U.S.A.
Effect of age on transvascular
fluid
movement
SANFORD M. ROSENTHAL AND LAWRENCE A. LAJOHN Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014
SANFORD transuascular@d
M., AND movement.
ROSENTHAL,
of age on
LAWRENCE A. Am. J Physiol.
LAJOHN. Effect 228(X > : l34-
140. 1975.-Swellings of the mouse tail and ear were produced by subjecting them to subatmospheric pressures of -40 to - 80 mmHg for 15-60 min. Increase in volume was measured volumetrically in the tail and gravimetrically in the ear. Blood volume increases in the tail, as measured with 51Cr erythrocytes, contributed a minor part of the fluid increase. Comparison of mice
from
3 to 36 wk
in
age
showed
a large
decrease
of fluid
movement with age, with major changes during the growth period. Study of permeability of the ear under decreased pressure, to intravenously administered Evans blue, showed no influence of age on permeability to the protein-bound dye. Measurement of transmission of the applied negative pressure through the skin, and of compliance of the tissues of the ear and tail in mice of different age groups, indicated that these factors were not responsible for the observed changes with age. hydrostatic pressure and edema; influence interstitial fluids; capillary permeability
of age on swelling;
THE LARGE LITERATURE on capillary permeability and transvascular fluid movement contains little reference to the influence of age (9, 12). A decreasing local edema with age, in response to trauma, was demonstrated by Little (10) in the hindlimbs of rabbits, under conditions where damage to the capillary wall had destroyed its selective permeability. In the present study local edema has been produced by exposure of the mouse tail or ear to relatively mild degrees of subatmospheric pressure. The influence of age on the resulting edema has been investigated. While most previous studies have employed increased arteriolar or venous pressure to effect transcapillary fluid movement, the vascular reactions and fluid movement in human extremities subjected to subatmospheric pressure have been reported by Greenfield and Patterson (4) and in subsequent reports from this laboratory (1, 2, 3), by Yamada and Burton (17), and by Guyton et al. (5). The local vascular reactions to hydrodynamic changes have been recently reviewed (12). METHODS
The apparatus for obtaining graded subatmospheric pressures to the mouse tail or ear is shown in Fig. 1. It consists of a column of mercury in a 100-ml stoppered cylinder, through which air is drawn. It is connected to a vacuum source through a T tube inserted into the stopper (B). The intake of air is through a second glass tube (A)
and
in the stopper inserted to any desired depth in the mercury column; the depth of the insertion determines the pressure at which air will be drawn in through the mercury. The second tube (A) is connected to another loo-ml cylinder with a similar arrangement, but filled with water; this serves as a fine adjustment for pressure regulation (Fig. 1). The T tube from the mercury cylinder (B) is connected to a series of six T tubes (C) joined together with rubber tubing, which are fastened to a board of cork or insulating material of such consistency that small nails can be pushed into it by hand. The outlets of the six T tubes are connected to glass tubes (D) (0.5 cm OD) 10 cm in length by strips of thick-walled rubber tubing, each fitted with a metal clamp. The open ends of the six tubes (D) are fitted with a segment of thin-walled flexible rubber tubing (G) (ss ID X 552 inch wall thickness), about 3 cm in length, protruding about 1.5 cm. The open end of the last T tube (E) is connected to a mercury manometer. For applying subatmospheric pressure to the tail, the mouse is placed head first in a containing cylinder (F) closed at one end with aluminum wire screening; the animal is held within by three no. 30 rubber bands enclosing the length of the cylinder, The cylinder should be of such size that the mouse cannot turn around, but not too tight to restrict respiration. An assortment of sizes should be available to fit individual mice. The protruding tail is inserted to the base into the rubbertipped tube (G), and sealing is effected by applying petrolatum jelly to the hair around the base of the tail. The animal is positioned with slight pressure against the rubbertipped tube, and the cylinder is held in place by three small nails around the containing cylinder, pushed into the board. With the apparatus adjusted to a given pressure, a continuous bubbling of air through the mercury occurs, and any leakage is indicated by a decrease or cessation of bubbling, as well as by a fall in the level of the manometer. With a vigorous flow of air, minute leakages do not affect the pressure values No anesthesia is required. Swelling of the tail is measured by a previouslv published procedure for measuring tail volume (13); it iS a plethysmographic method based on fluid displacement* The accuracy with groups of 10 mice is & 1.6 % (P = 0.05). Six mice are used on each run, and the original volume is measured at the start of the experiment; swelling is expressed as the percentage increase over this volume. Measurements are made immediately on removal from the apparatus (within 30 s) since recovery is rapid. For applying subatmospheric pressures to the ear,
134
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
AGE
ON
TRANSVASCULAR
FLUID
hfOVEMENT
WATER
FIG.
1. Apparatus
for
applying
decreased
atmospheric
pressure
to ear
or tail
of mouse.
In
apparatus
as used,
outlets
for
6 animals
were
em-
ployed
anesthesia is needed. Seventy-five milligrams per kilogram of a 0.75 % solution of sodium pentobarbital in 0.85 % NaCl is administered intraperitoneally. The anesthetized mouse is placed face down on the board, and the ear is inserted into the rubber tubing (G) with small curved forceps. Since edema of the ears is measured gravimetritally, petrolatum application was a source of error; it was found that satisfactory pressures could be maintained in the absence of grease by pressing the head against the rubber tubing and holding it in position by a nail behind the angle of the jaw, pushed into the board (Fig. 1). Gravimetric measurement of the ears was accomplished by excision immediately upon removal from the apparatus, while the mouse is still under anesthesia. Deep anesthesia is to be avoided. Since the edema subsides rapidly, the ear subjected to decreased pressure is excised first, within 30 s of removal. A technique of uniform excision presented a problem, which was largely solved in the following manner: a circular hole 0.7 cm in diameter was bored along the edge of a rigid plastic strip 1 mm thick. The
ear was put into this hole and pulled down by an artery clamp whose blades were enclosed in rubber tubing and to which was attached a 50-g weight. Excision was done with a single-edged razor blade, using a rapid sawing motion, with the blade pressed against the undersurface of the plastic. The plastic was fixed horizontally just above eye level, and the excision was performed with the mouse facing the investigator. The position of the plastic was reversed for the opposite ear so that excision could be performed at the same angle. The six ears subjected to Iowered pressures were pooled in one covered weighing vessel, and the six opposite ears were pooled in another. After obtaining wet weights, the opened vessels were placed in an oven at 90°C for 2-4 days, and then in a desiccator in vacuum over silica gel, before obtaining dry weights. Measurements of swelling and of increased water content were obtained by comparison of the ears subjected to decreased pressure with their opposite ears. An index of vascular permeability to macromolecules
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
136 was obtained by visual grading of the staining of the ear subjected to decreased pressure, following intravenous injection 1 h previously of Evans blue dye (Fisher Scientific Co.). A 2.5 % solution in 0.5 % NaCl was employed. Experiments were carried out at room temperatures of 22 to 25°C. RESULTS
Control Experiments Blood volume of tails ex@wd to decreased pressure. Exposure to pressures of -40 to 80 mmHg below atmospheric produced rapidly developing and transient swelling of the mouse tail and ear. In order to establish the role of vascular congestion in the swelling (by increased blood content of the tissues), experiments were with %hromiumlabeled mouse erythrocytes. Labeling was done with standard procedures (16), with counting by a Packard 578 scintillation counter. Washed labeled erythrocytes (0.1 ml) containing ZO-40,000 counts per minute were injected into the tail veins l-2 h prior to the experiment, and the tails were washed repeatedly in saline solution. Tail volume was measured in groups of six mice and then subjected to decreased pressures of -40 or -80 mmHg for 15 min. Upon removal from the apparatus volumes were again measured, and the animals were frozen by immersion in liquid nitrogen. The tails were then excised at the level of the anal rim and placed in individual counting vessels. Groups of six mice injected with labeled erythrocytes (but not exposed to decreased pressures) served as controls. Two groups ( 12 experimental and 12 control mice) were done at -40 mmHg decreased pressure and three groups were done at - 80 mmHg. At -40 mmHg the mean tail counts per minute were 368 (SE =tz 64.1) as compared with 281 (SE =t= 2 1.6) for controls (P = > 0.3) ; volumes averaged 6.7 YO above controls (SE & 0.80) (P = p > 0.05). The flow curves showed only slight decreases with time, indicating the large capacity of the subcutaneous tissues of the ear to adjust to fluid accumulation. For the tail, under positive pressure of a column of saline 80 cm in height, the flow rates were much slower, with less variation than in the ear; the flow curves showed decreases with time, indicating greater compliance than the ear. I Eight mice 3-4 wk old gave the following rates for the 15-min period: 0.27, 0.40, 0.89, 0.89, 1.02, 1.04, 1.12, and 1.17 pl/min (mean = 0.86). With eight mice 6-8 mo old the rates were: 0.46, 0.62, 1.02, 1.35, 1.46, 1.56, 1.71, and 1.87 pl/min (mean = 1.26) (P < 0.1). These results indicate a greater compliance in the subcutaneous tissues of young mice than in old (opposite to the results with swelling), although the results are not statistically significant.
S. M.
ROSENTHAL
AN’D
L. A,
JOHN
LA
120
0
l/Z
I
Iv-2
2
HOURS
-
0
0
l/2
I
2
HOURS FIG. 2. Influence of age on swelling of mouse tail subjected to decreased pressure. Initial readings made immediately upon removal from apparatus; later readings made during 2 h after removal. Numbers in parentheses represent numbers of animals. A: pressure of - 80 mmHg for 15 min. 23; pressure of - 80 rnmHg for 60 min.
Relutian to Age Tail volume after decreased pressure. Swelling of the tail was measured in mice of various ages after exposure of the tail to a decreased pressure of - 80 mmHg for 15 or 60 min. The ages were 3-4 wk (13-15 g wt), 7-8 wk (19-22 g), 12-16 wk (25-28 g), and 24-36 wk (30-38 g)* An inverse relationship to age was obtained at both exposures (Fig. 2). The results indicate that the greater swelling in young animals is not a difference in early rate, since the differences persist after 1 h of vacuum, when the swelling has apRecovery curves were variable, proached its maximum. but reduction in swelling was more rapid in young animals during the early recovery period, suggesting more rapid absorption of fluid. To follow the relation to age in greater detail, measurements on a group of 22 mice were made every 2 wk for 5 mo, beginning at 3 wk of age. Tails were exposed to decreased pressures of - 80 mmHg for 15 min. The results are shown in Fig. 3, along with weight curves. A rapid decline in the extent of swelling occurred between the 3rd and 8th wk, followed by much smaller declines until the end of the experiment. Weight curves showed a close inverse correlation. Thus, the large increases of swelling
occurred during the first 2 mo of age and are correlated with rapid growth. This suggests that transvascular Auid movement is related to the nutritional needs of rapidly growing tissues, rather than the age of the animal. Swelling of the ear under decreased pressure. The resistance to swelling afforded by tissues subjected to increased transmural pressure gradients is well known since the experiments of McMaster (11) and of Landis and Pappenheimer (9). Because the circular structure and thickening skin of the tail might play a role in the relationship to age on swelling, studies were carried out on the ears of mice, where the flat surface and thin skin would exert less reIt was indeed found that swelling sistance to swelling, from a given negative pressure was approximately twice as great in the ear as the tail. Decreased pressures of -80 mmHg produced considerable vascular congestion in the ear, so that most experiments were done at -40 mmHg for 15 min. The results with eight groups of six mice each for each age group are shown in Table 1. In all experiments the left ear was subjected to decreased pressure, and swelling was calculated by comparison with the right ear (left/
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
AGE
ON
TRANSVASCULAR
FLUID
139
MOVEMENT
right wet weight). A relationship to age similar to that obtained in the tail experiments was found This was confirmed by dry weight measurements, which showed corresponding changes in water content (left/right Hz@. The water content of the normal right ears varied from 6 1.5 % in weanlings to 52.7 % in 5-mo-old animals; this is in accord with the relationship of body water to age.
Permeability
in Ems to Evans Blue Dye
Staining of tissues with dyes which attach themselves to plasma proteins has been used extensively as a measure of permeability of the capillaries to macromolecules ( 14). Evans blue dye in an intravenous dose of 125 mg/kg did not appreciably color normal ears for 4 h or more, although the snout is rapidly colored and normal ears showed a slight coloration the next day. The dye was injected 0.5-l - 30 h prior to exposure to decreased pressure. With subatmospheric pressures applied to the ear, the staining was dependent upon the pressure; little staining occurred at -40 mmHg while an intensity 5-8 times as great was observed at -80 mmHg. This relationship of macromolecular permeability to capillary hydrostatic ,dO pressure has been described by Haddy et al. (6). - 20 -0 /P’ The intensity of staining was scored by visual observa/ tion, using a grading score of O-4. The scores of each group E $ CT c of six mice were averaged, and 12 mice were used for each 4 $2 age group and each pressure. Application of decreased pressure for 15 min resulted s in the following staining scores: 3- to 4-wk age group: - f0 0.4 (SE of mean & 0.15) at -40 mmHg and 2.75 & 0.25 at -80 mmHg; 7- to 8-wk group: 0.33 & 0.15 at -40 mmHg and 2.83 & 0.18 at -80 mmHg; 24- to 36-wk group: 0.66 & 0.18 at -40 mmHg and 2.66 =tz:0.25 at -80 mmHg. It is thus observed that only slight staining resulted from 1 I I I exposure to -40 mmHq < for 15 min, with no significant 0 0 I 2 3 4 5 6 diierences in the three -age groups (scores of 0.33-O-66). AGE MONTHS At a decreased pressure of -80 mmHg, marked staining FIG. 3. Influence of age on swelling of tai1. Tails subjected to a was present in all age groups, with no significant differences pressure of -80 mmHg for 15 min. Consecutive readings upon same between them (scores of 2.66-2.83). Considerable conmice for 5 mo, beginning at 3 wk of age. Solid line represents average increases in volume in 22 mice compared with volume obtained begestion was often encountered, particularly in the 3- to fore each exposure to decreased pressure. Only initial reading made 4-wk age group, but scorings can be delayed for 2 or 3 immediately upon removal from apparatus was used for comparison. h, when the interference from congestion has largely disInterrupted line represents weight curve.
Increase Ear
Wet
Dry
mg H20/mg Dry- L/R Wet
Hz0
%
L/R
Hz0
Mice
P Value
2-3 wk (13-15 g) L
126.5
zt 2.65*
42.6
=t= 1.13”
83.9
zt
1.96*
1.97
R
104.9
zt 2.57
40*9
&
64.0
zk 1.63
1.56
1.12
20.0
26,
47
in U.S.A.
Effect of age on transvascular
fluid
movement
SANFORD M. ROSENTHAL AND LAWRENCE A. LAJOHN Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014
SANFORD transuascular@d
M., AND movement.
ROSENTHAL,
of age on
LAWRENCE A. Am. J Physiol.
LAJOHN. Effect 228(X > : l34-
140. 1975.-Swellings of the mouse tail and ear were produced by subjecting them to subatmospheric pressures of -40 to - 80 mmHg for 15-60 min. Increase in volume was measured volumetrically in the tail and gravimetrically in the ear. Blood volume increases in the tail, as measured with 51Cr erythrocytes, contributed a minor part of the fluid increase. Comparison of mice
from
3 to 36 wk
in
age
showed
a large
decrease
of fluid
movement with age, with major changes during the growth period. Study of permeability of the ear under decreased pressure, to intravenously administered Evans blue, showed no influence of age on permeability to the protein-bound dye. Measurement of transmission of the applied negative pressure through the skin, and of compliance of the tissues of the ear and tail in mice of different age groups, indicated that these factors were not responsible for the observed changes with age. hydrostatic pressure and edema; influence interstitial fluids; capillary permeability
of age on swelling;
THE LARGE LITERATURE on capillary permeability and transvascular fluid movement contains little reference to the influence of age (9, 12). A decreasing local edema with age, in response to trauma, was demonstrated by Little (10) in the hindlimbs of rabbits, under conditions where damage to the capillary wall had destroyed its selective permeability. In the present study local edema has been produced by exposure of the mouse tail or ear to relatively mild degrees of subatmospheric pressure. The influence of age on the resulting edema has been investigated. While most previous studies have employed increased arteriolar or venous pressure to effect transcapillary fluid movement, the vascular reactions and fluid movement in human extremities subjected to subatmospheric pressure have been reported by Greenfield and Patterson (4) and in subsequent reports from this laboratory (1, 2, 3), by Yamada and Burton (17), and by Guyton et al. (5). The local vascular reactions to hydrodynamic changes have been recently reviewed (12). METHODS
The apparatus for obtaining graded subatmospheric pressures to the mouse tail or ear is shown in Fig. 1. It consists of a column of mercury in a 100-ml stoppered cylinder, through which air is drawn. It is connected to a vacuum source through a T tube inserted into the stopper (B). The intake of air is through a second glass tube (A)
and
in the stopper inserted to any desired depth in the mercury column; the depth of the insertion determines the pressure at which air will be drawn in through the mercury. The second tube (A) is connected to another loo-ml cylinder with a similar arrangement, but filled with water; this serves as a fine adjustment for pressure regulation (Fig. 1). The T tube from the mercury cylinder (B) is connected to a series of six T tubes (C) joined together with rubber tubing, which are fastened to a board of cork or insulating material of such consistency that small nails can be pushed into it by hand. The outlets of the six T tubes are connected to glass tubes (D) (0.5 cm OD) 10 cm in length by strips of thick-walled rubber tubing, each fitted with a metal clamp. The open ends of the six tubes (D) are fitted with a segment of thin-walled flexible rubber tubing (G) (ss ID X 552 inch wall thickness), about 3 cm in length, protruding about 1.5 cm. The open end of the last T tube (E) is connected to a mercury manometer. For applying subatmospheric pressure to the tail, the mouse is placed head first in a containing cylinder (F) closed at one end with aluminum wire screening; the animal is held within by three no. 30 rubber bands enclosing the length of the cylinder, The cylinder should be of such size that the mouse cannot turn around, but not too tight to restrict respiration. An assortment of sizes should be available to fit individual mice. The protruding tail is inserted to the base into the rubbertipped tube (G), and sealing is effected by applying petrolatum jelly to the hair around the base of the tail. The animal is positioned with slight pressure against the rubbertipped tube, and the cylinder is held in place by three small nails around the containing cylinder, pushed into the board. With the apparatus adjusted to a given pressure, a continuous bubbling of air through the mercury occurs, and any leakage is indicated by a decrease or cessation of bubbling, as well as by a fall in the level of the manometer. With a vigorous flow of air, minute leakages do not affect the pressure values No anesthesia is required. Swelling of the tail is measured by a previouslv published procedure for measuring tail volume (13); it iS a plethysmographic method based on fluid displacement* The accuracy with groups of 10 mice is & 1.6 % (P = 0.05). Six mice are used on each run, and the original volume is measured at the start of the experiment; swelling is expressed as the percentage increase over this volume. Measurements are made immediately on removal from the apparatus (within 30 s) since recovery is rapid. For applying subatmospheric pressures to the ear,
134
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
AGE
ON
TRANSVASCULAR
FLUID
hfOVEMENT
WATER
FIG.
1. Apparatus
for
applying
decreased
atmospheric
pressure
to ear
or tail
of mouse.
In
apparatus
as used,
outlets
for
6 animals
were
em-
ployed
anesthesia is needed. Seventy-five milligrams per kilogram of a 0.75 % solution of sodium pentobarbital in 0.85 % NaCl is administered intraperitoneally. The anesthetized mouse is placed face down on the board, and the ear is inserted into the rubber tubing (G) with small curved forceps. Since edema of the ears is measured gravimetritally, petrolatum application was a source of error; it was found that satisfactory pressures could be maintained in the absence of grease by pressing the head against the rubber tubing and holding it in position by a nail behind the angle of the jaw, pushed into the board (Fig. 1). Gravimetric measurement of the ears was accomplished by excision immediately upon removal from the apparatus, while the mouse is still under anesthesia. Deep anesthesia is to be avoided. Since the edema subsides rapidly, the ear subjected to decreased pressure is excised first, within 30 s of removal. A technique of uniform excision presented a problem, which was largely solved in the following manner: a circular hole 0.7 cm in diameter was bored along the edge of a rigid plastic strip 1 mm thick. The
ear was put into this hole and pulled down by an artery clamp whose blades were enclosed in rubber tubing and to which was attached a 50-g weight. Excision was done with a single-edged razor blade, using a rapid sawing motion, with the blade pressed against the undersurface of the plastic. The plastic was fixed horizontally just above eye level, and the excision was performed with the mouse facing the investigator. The position of the plastic was reversed for the opposite ear so that excision could be performed at the same angle. The six ears subjected to Iowered pressures were pooled in one covered weighing vessel, and the six opposite ears were pooled in another. After obtaining wet weights, the opened vessels were placed in an oven at 90°C for 2-4 days, and then in a desiccator in vacuum over silica gel, before obtaining dry weights. Measurements of swelling and of increased water content were obtained by comparison of the ears subjected to decreased pressure with their opposite ears. An index of vascular permeability to macromolecules
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
136 was obtained by visual grading of the staining of the ear subjected to decreased pressure, following intravenous injection 1 h previously of Evans blue dye (Fisher Scientific Co.). A 2.5 % solution in 0.5 % NaCl was employed. Experiments were carried out at room temperatures of 22 to 25°C. RESULTS
Control Experiments Blood volume of tails ex@wd to decreased pressure. Exposure to pressures of -40 to 80 mmHg below atmospheric produced rapidly developing and transient swelling of the mouse tail and ear. In order to establish the role of vascular congestion in the swelling (by increased blood content of the tissues), experiments were with %hromiumlabeled mouse erythrocytes. Labeling was done with standard procedures (16), with counting by a Packard 578 scintillation counter. Washed labeled erythrocytes (0.1 ml) containing ZO-40,000 counts per minute were injected into the tail veins l-2 h prior to the experiment, and the tails were washed repeatedly in saline solution. Tail volume was measured in groups of six mice and then subjected to decreased pressures of -40 or -80 mmHg for 15 min. Upon removal from the apparatus volumes were again measured, and the animals were frozen by immersion in liquid nitrogen. The tails were then excised at the level of the anal rim and placed in individual counting vessels. Groups of six mice injected with labeled erythrocytes (but not exposed to decreased pressures) served as controls. Two groups ( 12 experimental and 12 control mice) were done at -40 mmHg decreased pressure and three groups were done at - 80 mmHg. At -40 mmHg the mean tail counts per minute were 368 (SE =tz 64.1) as compared with 281 (SE =t= 2 1.6) for controls (P = > 0.3) ; volumes averaged 6.7 YO above controls (SE & 0.80) (P = p > 0.05). The flow curves showed only slight decreases with time, indicating the large capacity of the subcutaneous tissues of the ear to adjust to fluid accumulation. For the tail, under positive pressure of a column of saline 80 cm in height, the flow rates were much slower, with less variation than in the ear; the flow curves showed decreases with time, indicating greater compliance than the ear. I Eight mice 3-4 wk old gave the following rates for the 15-min period: 0.27, 0.40, 0.89, 0.89, 1.02, 1.04, 1.12, and 1.17 pl/min (mean = 0.86). With eight mice 6-8 mo old the rates were: 0.46, 0.62, 1.02, 1.35, 1.46, 1.56, 1.71, and 1.87 pl/min (mean = 1.26) (P < 0.1). These results indicate a greater compliance in the subcutaneous tissues of young mice than in old (opposite to the results with swelling), although the results are not statistically significant.
S. M.
ROSENTHAL
AN’D
L. A,
JOHN
LA
120
0
l/Z
I
Iv-2
2
HOURS
-
0
0
l/2
I
2
HOURS FIG. 2. Influence of age on swelling of mouse tail subjected to decreased pressure. Initial readings made immediately upon removal from apparatus; later readings made during 2 h after removal. Numbers in parentheses represent numbers of animals. A: pressure of - 80 mmHg for 15 min. 23; pressure of - 80 rnmHg for 60 min.
Relutian to Age Tail volume after decreased pressure. Swelling of the tail was measured in mice of various ages after exposure of the tail to a decreased pressure of - 80 mmHg for 15 or 60 min. The ages were 3-4 wk (13-15 g wt), 7-8 wk (19-22 g), 12-16 wk (25-28 g), and 24-36 wk (30-38 g)* An inverse relationship to age was obtained at both exposures (Fig. 2). The results indicate that the greater swelling in young animals is not a difference in early rate, since the differences persist after 1 h of vacuum, when the swelling has apRecovery curves were variable, proached its maximum. but reduction in swelling was more rapid in young animals during the early recovery period, suggesting more rapid absorption of fluid. To follow the relation to age in greater detail, measurements on a group of 22 mice were made every 2 wk for 5 mo, beginning at 3 wk of age. Tails were exposed to decreased pressures of - 80 mmHg for 15 min. The results are shown in Fig. 3, along with weight curves. A rapid decline in the extent of swelling occurred between the 3rd and 8th wk, followed by much smaller declines until the end of the experiment. Weight curves showed a close inverse correlation. Thus, the large increases of swelling
occurred during the first 2 mo of age and are correlated with rapid growth. This suggests that transvascular Auid movement is related to the nutritional needs of rapidly growing tissues, rather than the age of the animal. Swelling of the ear under decreased pressure. The resistance to swelling afforded by tissues subjected to increased transmural pressure gradients is well known since the experiments of McMaster (11) and of Landis and Pappenheimer (9). Because the circular structure and thickening skin of the tail might play a role in the relationship to age on swelling, studies were carried out on the ears of mice, where the flat surface and thin skin would exert less reIt was indeed found that swelling sistance to swelling, from a given negative pressure was approximately twice as great in the ear as the tail. Decreased pressures of -80 mmHg produced considerable vascular congestion in the ear, so that most experiments were done at -40 mmHg for 15 min. The results with eight groups of six mice each for each age group are shown in Table 1. In all experiments the left ear was subjected to decreased pressure, and swelling was calculated by comparison with the right ear (left/
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
AGE
ON
TRANSVASCULAR
FLUID
139
MOVEMENT
right wet weight). A relationship to age similar to that obtained in the tail experiments was found This was confirmed by dry weight measurements, which showed corresponding changes in water content (left/right Hz@. The water content of the normal right ears varied from 6 1.5 % in weanlings to 52.7 % in 5-mo-old animals; this is in accord with the relationship of body water to age.
Permeability
in Ems to Evans Blue Dye
Staining of tissues with dyes which attach themselves to plasma proteins has been used extensively as a measure of permeability of the capillaries to macromolecules ( 14). Evans blue dye in an intravenous dose of 125 mg/kg did not appreciably color normal ears for 4 h or more, although the snout is rapidly colored and normal ears showed a slight coloration the next day. The dye was injected 0.5-l - 30 h prior to exposure to decreased pressure. With subatmospheric pressures applied to the ear, the staining was dependent upon the pressure; little staining occurred at -40 mmHg while an intensity 5-8 times as great was observed at -80 mmHg. This relationship of macromolecular permeability to capillary hydrostatic ,dO pressure has been described by Haddy et al. (6). - 20 -0 /P’ The intensity of staining was scored by visual observa/ tion, using a grading score of O-4. The scores of each group E $ CT c of six mice were averaged, and 12 mice were used for each 4 $2 age group and each pressure. Application of decreased pressure for 15 min resulted s in the following staining scores: 3- to 4-wk age group: - f0 0.4 (SE of mean & 0.15) at -40 mmHg and 2.75 & 0.25 at -80 mmHg; 7- to 8-wk group: 0.33 & 0.15 at -40 mmHg and 2.83 & 0.18 at -80 mmHg; 24- to 36-wk group: 0.66 & 0.18 at -40 mmHg and 2.66 =tz:0.25 at -80 mmHg. It is thus observed that only slight staining resulted from 1 I I I exposure to -40 mmHq < for 15 min, with no significant 0 0 I 2 3 4 5 6 diierences in the three -age groups (scores of 0.33-O-66). AGE MONTHS At a decreased pressure of -80 mmHg, marked staining FIG. 3. Influence of age on swelling of tai1. Tails subjected to a was present in all age groups, with no significant differences pressure of -80 mmHg for 15 min. Consecutive readings upon same between them (scores of 2.66-2.83). Considerable conmice for 5 mo, beginning at 3 wk of age. Solid line represents average increases in volume in 22 mice compared with volume obtained begestion was often encountered, particularly in the 3- to fore each exposure to decreased pressure. Only initial reading made 4-wk age group, but scorings can be delayed for 2 or 3 immediately upon removal from apparatus was used for comparison. h, when the interference from congestion has largely disInterrupted line represents weight curve.
Increase Ear
Wet
Dry
mg H20/mg Dry- L/R Wet
Hz0
%
L/R
Hz0
Mice
P Value
2-3 wk (13-15 g) L
126.5
zt 2.65*
42.6
=t= 1.13”
83.9
zt
1.96*
1.97
R
104.9
zt 2.57
40*9
&
64.0
zk 1.63
1.56
1.12
20.0
26,
47
