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Department of Physiology, University of Bergen, 5009 Bergen, Norway
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Lack of thyroid hormones may affect the composition and
structure of the interstitium. Hypothyrosis was induced in rats by thyroidectomy 4-12 wk before the experiments. In hypothyroid rats (n = 16), interstitial fluid pressure measured with
micropipettes in hindlimb skin and muscle averaged +0.1 ± 0.2 and
+0.5 ± 0.2 mmHg, respectively, with corresponding pressures in
control rats (n = 16) of 
1.5 ± 0.1 (P < 0.001) and 0.8 ± 0.1 mmHg (P < 0.001). Interstitial fluid volume, measured as the difference between the
distribution volumes of 51Cr-EDTA and
125I-labeled BSA, was similar or lower in skin and higher
in hypothyroid muscle. Total protein and albumin concentration in
plasma and interstitial fluid (isolated from implanted wicks) was lower
in hypothyroid compared with control rats. Hyaluronan content
(n = 9) in rat hindlimb skin was 2.05 ± 0.15 and 1.92 ± 0.09 mg/g dry wt (P > 0.05) in hypothyroid and
control rats, respectively, with corresponding content in hindlimb
skeletal muscle of 0.35 ± 0.07 and 0.23 ± 0.01 mg/g dry wt
(P < 0.01). Interstitial exclusion of albumin in skin and
muscle was measured after 125I-labeled rat serum albumin
infusion for 120-168 h with an implanted osmotic pump. Relative
excluded volume for albumin (Ve/Vi) was calculated as 1 Va/Vi, and averaged 28 and 28% in hindlimb muscle (P > 0.05), 44 and 45%
in hindlimb skin (P > 0.05), and 19 and 32% in back skin
(P < 0.05) in hypothyroid and control rats, respectively. Albumin mass was higher in back skin in spite of a lower interstitial fluid albumin concentration, a finding explained by a reduced Ve/Vi in back skin in hypothyroid rats.
These experiments suggest that lack of thyroid hormones in rats changes
the interstitial matrix again leading to reduced interstitial
compliance and changes in the transcapillary fluid balance.
extracellular space; albumin space; extracellular matrix; interstitial fluid protein concentration; interstitial fluid albumin concentration; muscle; skin
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE INTERSTITIUM may be defined as the space located between the capillary walls and the cells. As recently reviewed by Aukland and Reed (3), the basic structure is similar in all tissues: collagen builds the fiber framework that contains a gel phase made up of glycosaminoglycans (GAGs), a salt solution, and proteins derived from plasma. The amount of interstitium varies from about 50% of wet weight in skin to 10% in skeletal muscle.
GAGs and their major component hyaluronan (3) are of importance for interstitial fluid volume control. Hyaluronan contributes to the gel-like structure of the interstitium and is in osmotic equilibrium with collagen and proteins in the interstitial fluid (34). Variation in the content and concentration of hyaluronan may therefore affect interstitial fluid characteristics like hydrostatic pressure and volume. In addition, changes in GAG content may also be of importance in disease conditions affecting the connective tissue.
In humans, lack of thyroid hormone may lead to a condition called myxedema, which is characterized by accumulation of GAGs in the interstitium [e.g., (30)]. The increased hyaluronan and chondroitin sulfate content in myxedematous lesions (32) has been suggested as explanation for the observed increased hydration and altered consistency of human skin (14) (see also Ref. 30). Lack of thyroid hormone has been shown to induce accumulation of hyaluronan also in rats (28). Another explanation for the fluid accumulation in myxedema was given by Parving and co-workers (22) who found an increased transcapillary escape rate of albumin and indications of extravascular albumin accumulation. Although hyaluronan has been measured in hypothyroid rats, few studies have addressed transcapillary fluid balance in this situation.
Hyaluronan may also influence other parameters relating to interstitial physiology. The presence of amorphous substances (like GAGs) in the interstitium limits the space accessible for plasma proteins and other macromolecules, a phenomenon called exclusion (8). As a result, the concentration of the plasma proteins in the accessible space is higher than calculated from tissue protein mass and interstitial fluid volume. The physiological importance of the exclusion phenomenon lies in its relationship determining the speed by which a new steady state is established after perturbations in interstitial fluid volume (Vi). Protein exclusion does, however, not determine steady-state protein concentration or colloid osmotic pressure of interstitial free fluid or lymph (3). Albumin is responsible for a major part of the colloid osmotic pressure in plasma and interstitial fluid, and previous studies have shown that this protein is excluded from a substantial part of many interstitia (8).
The distribution of a specific probe like albumin in the interstitial fluid is determined in part by both its size and charge (8, 12), but it will also be influenced by the composition of the tissue, i.e., by the amounts of structural components like collagen and hyaluronan. Because of the importance of GAGs in the regulation of tissue hydration, we studied the influence of changes in interstitial composition on interstitial fluid volume and some of its determinants in a hypothyroid animal. We found that lack of thyroid hormone resulted in increased interstitial fluid pressure in skin and muscle and a reduced protein concentration in interstitial fluid. Hyaluronan was increased in muscle and heart, but not in skin and interstitial fluid from skin and muscle. Interstitial exclusion of albumin was reduced in back skin. These experiments suggest that hypothyrosis leads to changes in the interstitial matrix and a reduced interstitial compliance. Preliminary reports of this work have been presented elsewhere (38, 41).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The experiments were performed in anesthetized female Wistar-Møller rats, 202-279 g, fed a standard laboratory diet. While the rats were anesthetized, body temperature was maintained at 37-38°C with a heat lamp. The rats were not fasted before or during the experiments. All experiments were performed in accordance with recommendations given by the Norwegian State Commission for Laboratory Animals and were approved by the local ethical committee.
Surgical Procedures
Hypothyreosis was induced by thyroidectomy, 4-12 wk before the final experiments. After anesthesia with a 1:1 mixture of fentanyl/fluanisone (Hypnorm) and midazolam (Dormicum), 2.5 ml/kg, a midline skin incision was made in the neck, and the thyroid and parathyroid glands were identified under a dissection microscope. The thyroid gland was gently extirpated, and care was taken to leave the parathyroid glands in situ. A sham operation, i.e., skin incision only, was performed in control rats. Final experiments were performed 4 (n = 2), 8 (n = 20), and 12 (n = 4) wk after thyroidectomy.To verify complete extirpation of thyroid tissue, thyroxin (T4) was measured in a routine assay at a local hospital in plasma sampled at the end of the experiment. All rats where the thyroid gland had been removed had T4 levels below detection limit (<20 nmol/l), whereas the corresponding value in control rats was 48.4 nmol/l ± 1.8 (n = 19). Plasma Ca2+ averaged 2.65 ± 0.05 (n = 8) and 2.82 ± 0.09 (n = 8) mmol/l in hypothyroid and control rats, respectively (P > 0.05).
Series I: Interstitial Fluid Pressure and Composition
Measurement of interstitial fluid pressure. Interstitial fluid pressure (Pi) was measured with micropipettes as described in detail elsewhere (39). Punctures were performed with sharpened, siliconized glass pipettes with tip diameter 3-5 µm filled with 0.5 M NaCl colored with Evans blue, and the pressure measurements were obtained by connecting the micropipettes to a servo-controlled counter-pressure system.
The rat was placed in a supine position, and micropipettes were inserted on the medial aspect of the thigh through intact skin, or in the case of muscle in the fiber direction, through the fascia exposed by a 2- to 3-mm long skin incision. Pi was also measured in distal back skin after placing the rat in a prone position. Pressure measurements were performed during the first 2 h of isotope equilibration (see below).Measurement of distribution volumes. On the day of the experiment, the rat was anesthetized with pentobarbital, 50 mg/kg ip. After tracheotomy, a PE-50 catheter was inserted in the left carotid artery for measurement of blood pressure. Then both kidney pedicles were ligated via flank incisions, and a PE-50 catheter was inserted into the right jugular vein. A 150-µl blood sample was obtained by orbital puncture (exclusion series, see below), and 60-70 µCi 51Cr-EDTA injected i.v. for measurement of interstitial fluid volume (Vi).
To replace plasma loss during surgery, a bolus of 0.5 ml 5% BSA (35) was given i.v. and followed by a sustained infusion of 0.67 ml/h with a Harvard infusion pump. This procedure has been shown to maintain rat plasma total protein concentration and colloid osmotic pressure within 5% deviation of initial levels for several hours, and result in a stable protein concentration and colloid osmotic pressure in tail lymph (2). Four hours after the injection of 51Cr-EDTA, 3-4 µCi of 125I-labeled BSA (125I-BSA) [131I-labeled BSA (131I-BSA) in the exclusion series, see below] was injected after discontinuing the infusion of the BSA solution and allowed 5 min of equilibration. A final blood sample of 0.5-0.7 ml was obtained by cardiac puncture, and the rat was killed with saturated KCl administered intravenously. The skin was closely clipped on both hindlimbs and the back, and the rat was transferred to a chamber kept at 100% humidity at all times for implantation of wicks used to isolate interstitial fluid. Dry wicks were inserted postmortem in back subcutis and hindlimb skin and muscle as described in previous publications (36, 40). After a 20-min implantation period, the wick ends along with any bloodstained portions were cut off and the remaining sections transferred to preweighed glass vials filled with 1 ml 0.02% azide saline for elution. The wick-containing tubes were capped tightly with a screw cap and reweighed to the nearest 0.01 mg as soon as possible. After the wick implantation period, the rat was taken out of the humidity chamber, hair was carefully removed from the hind legs and lower back with fine clippers, and the skin was washed and dried. Paired (left and right side) 0.3- to 1.2-g samples were taken from hindlimb and back skin, and from both legs of lateral and medial gastrocnemius, tibialis anterior, and semimembranosus muscles and hindpaws. Tissue samples were placed in tared covered vials and weighed. Aliquots of plasma from the blood samples were made up to 1 ml in the same vials used for wick samples. Samples were counted in a LKB gamma counter (model 1282 Compugamma) using window settings of 530-690 keV for 51Cr, 700-860 keV for 131I and 120-320 keV for 125I. Standards were counted in every experiment to obtain spillover corrections, and counts were corrected for background and spillover and for 131I radioactivity decay during the period of measurement. After counting, tissue samples were dried at 85°C to constant weight (change < 1 mg in 24 h). Tissue water content (Vw) was taken to be the difference between wet and dry weights.Analysis of interstitial fluid and tissue samples. The wicks were eluted overnight. After vortexing was completed they were removed and dried allowing calculation of wick fluid weight (and volume) and wick water content. The fluid samples were stored in the refrigerator for later analyses.
Total protein in eluate and plasma was estimated by the Amidoschwartz method as described by Aukland and Fadnes (1), whereas the corresponding albumin concentration was measured with a fluorometric method using 1-anilinonaphthalene-8-sulfonic acid (ANS) as described by Rees et al. (27) and modified by Aukland and Fadnes (1). Analysis of hyaluronan in serum, wick fluid, and tissue was performed in nine rats 4 (n = 2) and 8 (n = 7) wk postthyroidectomy by a radioassay (HA-test 50, Pharmacia Diagnostics, Uppsala, Sweden) after papain digestion of freeze-dried specimens (24). Uronic acid was measured by using the method of Bitter and Muir (10) as described in a previous paper (23), whereas collagen was determined according to the method of Woessner (42), based on the determination of hydroxyproline content as described in a previous publication (23), using a hydroxyproline content of 0.91 µmol/1 mg collagen (9).Series II: Interstitial Exclusion of Albumin
Continuous isotope infusion. The present method for measurement of interstitial exclusion is based on reaching a steady-state tracer concentration in the interstitium by a slow, continuous infusion of tracer of 1 µl/h for 4-7 days. This method has been described in detail in a previous paper (35), and therefore only a brief description is given here. Rat serum albumin (RSA) (Cohn fraction V, Sigma) was labeled with 125I with Iodogen and purified on an ion exchange column and by dialysis in PBS.
The radioactivity was adjusted to 125-140 µCi/ml, and the tracer was filled into an osmotic pump (model 2001, pumping rate 1 µl/h, Alzet). The filled pump was incubated overnight in a beaker containing azide saline (0.02%) kept at 37°C. The next day the rat was anesthetized with pentobarbital (40 mg/kg ip), and a PE-60 catheter was filled with isotope solution was inserted into the right jugular vein using a sterile technique. A bolus of 0.025 ml tracer was given, and the catheter was connected to the preincubated osmotic pump. The pump was tunneled to the interscapular region, the wound was closed with wound clips, and the rat was transferred to its cage to wake up. Blood samples, one hematocrit tube every 48 h, were obtained by scalpel incision of the distal part of one lateral tail vein with the rat in a perspex cylinder. On the day of the final experiment, the distribution volumes were measured, and wick fluid was processed for albumin analysis as described above.Elution of isotope from tissue. To see if the infused 125I-labeled RSA (125I-RSA ) was "free" in the interstitial fluid and not bound to the tissue, tissues from four rats were eluted (two hypothyroid and two control rats), and recovered isotope was calculated. Paired samples were obtained from hindlimb and back skin and hindlimb muscle. The tissues were minced with a scalpel and put in preweighed, capped tubes containing 6 ml of 0.02% azide in 0.15 M saline. After weighing was performed, the tubes were shaken vigorously and placed in an agitator for 24 h to stand overnight at room temperature and counted in the gamma counter. The tubes were then reweighed to correct for possible evaporation and centrifuged. As much supernatant as possible was removed and a 1-ml aliquot of it counted. An amount of azide saline corresponding to the volume removed was added to the tissue sample, and the extraction procedure was repeated. The counts recovered in the supernatant were compared with the initial total counts of sample and saline with correction for isotope decay occurring during the extraction procedure.
Calculations
The whole body plasma volume and extracellular fluid volume were calculated as the 5-min and 4-h, respectively, plasma equivalent distribution volumes of 131I-BSA and 51Cr-EDTA, each equal to the total counts injected divided by counts per ml terminal plasma.Intravascular plasma volume in a tissue sample (Vv) was
calculated as the 5-min 131I-BSA distribution volume
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(1) |
Tissue extracellular fluid volume (Vx) was calculated as
the 4-h distribution volume of 51Cr-EDTA
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(2) |
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(3) |
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(4) |
A more realistic estimate of extravascular albumin distribution volume
(Va,w) is obtained by assuming tracer albumin activity in
free interstitial fluid of the tissue is the same as in wick fluid from
that tissue
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(5) |
Albumin excluded volume Ve,a (ml/g) = Vi Va,w. Expressed as a fraction of Vi
(fractional excluded volume)
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(6) |
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(7) |
Specific activities of 125I-RSA in wick fluid and plasma (Sa,w, Sa,p counts/mg) were calculated from net counts of 125I and albumin concentrations measured by fluorimetric assay. Steady-state tissue albumin mass was calculated as the product of specific activity (equal to that in plasma or wick fluid) and counts of 125I per gram of tissue.
All distribution volumes are expressed in terms of wet tissue weight.
Statistics
Values for paired tissue and fluid samples (e.g., left and right leg skin) were combined and averaged. Each hypothyroid rat was assigned a unique control rat from the same batch of rats processed in parallel. Experiments in these pairs of rats were performed simultaneously and under similar conditions. Data are given as means ± SE and were compared with two-tailed t-tests, using paired comparisons when appropriate, i.e., each experimental rat was compared with its unique control. Differences were accepted as statistically significant at the P < 0.05% level.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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At the time of thyroidectomy, experimental and control rats weighed 223 ± 3 and 220 ± 4 g, respectively (P > 0.05). Lack of thyroid hormone affected body weight. Rats used for experiments 4 and 8 wk after thyroid extirpation weighed 207 ± 4 g compared with 260 ± 4 g in control rats (P < 0.001). Although the rat weight 4 and 8 wk after thyroid extirpation did not differ significantly, rats gained weight in the period from 8 to 12 wk. Thus, animals used 12 wk after the initial surgery weighed 233 ± 9 g, significantly different from the other thyroidectomized (P < 0.01) and control rats (P < 0.01).
The results for other parameters measured in the present paper have been related to time after thyroid extirpation and compared. Except for weight mentioned above, corresponding values did not differ, and therefore the results for various experiment durations have been pooled.
On the day of the experiment rectal temperature measured as soon as possible after induction of anesthesia averaged 36.8 ± 0.2 (n = 16) and 38.1 ± 0.2°C (n = 16) (P < 0.01) in hypothyroid and control rats, respectively, with corresponding mean blood pressures of 91.8 ± 5.0 (n = 16) and 123.8 ± 4.5 mmHg (n = 16) (P < 0.01).
Series I: Interstitial Fluid Pressure and Composition
Interstitial fluid pressure.
As evident from Fig. 1, induction of
hypothyrosis resulted in significantly higher interstitial fluid
pressures in the organs studied. Thus Pi averaged +0.1 ± 0.2 and +0.1 ± 0.2 mmHg in hindlimb and back skin of hypothyroid rats
(n = 16), respectively, with corresponding pressures of
1.5 ± 0.1 and 1.3 ± 0.2 mmHg in control rats
(n = 16) (P < 0.001 compared with control for both
tissues). In hindlimb muscle, Pi was +0.5 ± 0.2 and
0.8 ± 0.1 mmHg in hypothyroid and control rats, respectively
(P < 0.001).
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Interstitial fluid composition.
The protein concentration in fluid isolated from wicks implanted in
hypothyroid animals (n = 13) was generally lower than that
isolated from wicks from corresponding organs in control rats
(n = 13) (Fig. 2). Thus the average
protein concentration in back skin wick fluid in hypothyroid rats was
23.4 ± 1.0 with a corresponding value in control rats of 30.2 ± 2.1 mg/ml (P < 0.05). Similar concentrations were
found in fluid from wicks implanted in other sites. The average protein
concentration in plasma was 49.3 ± 1.7 and 55.8 ± 3.3 mg/ml in hypothyroid and control rats, respectively (P = 0.059).
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Fluid volumes. The total plasma and interstitial fluid volumes did not differ in hypothyroid and control rats. In hypothyroid rats, Vi and Vp were 20.1 ± 0.9 and 2.8 ± 0.5 ml/100 g rat, with corresponding volumes of 19.9 ± 0.9 and 2.7 ± 0.5 ml/100 g in control rats. The volumes in hypothyroid rats did not differ significantly from corresponding volumes in control rats.
Local interstitial fluid volume and total tissue water volume are documented in Table 2. Tibialis anterior is listed separately from the other skeletal muscles because it appeared to differ significantly (though slightly) in some respects (35). Whereas Vi in hindlimb skin was similar in hypothyroid and control rats, hypothyroid back skin Vi was 79% of control only. For muscle, volume change was in the opposite direction. Thus the pooled Vi for gastrocnemius and semimembranosus muscle was 21% higher in hypothyroid rats compared with control rats. Vi in tibialis anterior muscle in hypothyroid rats also tended to be higher than in control rats, but the difference was not statistically significant.
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Tissue hyaluronan, uronic acid and collagen content.
Hyaluronan content in skin was similar in hypothyroid and control rats,
~2 and 1.2 mg/g fat-free dry wt in hindlimb and back skin,
respectively (Fig. 5).
However, in pooled hindlimb muscle of hypothyroid rats, hyaluronan
content was 56% above control value. A significant increase in
hyaluronan content of 54% was also found in the heart. The hyaluronan
concentration (wick fluid) and content did not differ significantly in
rats killed 4 or 8 wk after thyroidectomy.
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Series II: Interstitial Exclusion of Albumin
The rats recovered within 1 h after the osmotic pump implantation, and they did not seem to be affected by the pump during the implantation period. Plasma levels of 125I-RSA were stable in plasma during the experiment in hypothyroid as well as in control rats. Figure 8 shows the time course of average plasma tracer concentrations normalized to individual terminal values (nine at 120 h and two at 164 h). Except for the values at 24 h, the relative plasma concentration did not differ significantly from 1.0 at the other intervals for hypothyroid or control rats, showing a stable tracer concentration during the experiment.
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Comparison of distribution volumes. As stated in methods, plasma equivalent spaces may serve to indicate that a steady state has been achieved. Whereas most experiments were terminated at 120 h (n = 9, both types of rats), two control and two hypothyroid rats were infused for 168 h. Plasma equivalent space for albumin did not differ significantly depending on infusion time for any tissue. Thus for hypothyroid rats, Va,p averaged 229.1 ± 17.7, 369.7 ± 25.5 and 39.1 ± 3.3 µl/g wet wt after 120 h of isotope infusion in hindlimb skin, back skin and pooled gastrocnemius and semimembranosus muscle, respectively, with corresponding averages after 168 h of infusion (n = 2) of 232.3, 379.3, and 37.5 µl/g wet wt. None of the corresponding volumes differed significantly. In control rats, Va,p averaged (n = 9) 199.3 ± 17.8, 183.6 ± 14.4 and 33.1 ± 3.3 µl/g wet wt in hindlimb skin, back skin and pooled gastrocnemius and semimembranosus muscle, respectively, with corresponding values after 168 h of infusion (n = 2) of 204.0, 206.8, and 35.4 µl/g wet wt (P > 0.05 for all respective comparisons). Because the volumes for 120 and 168 h did not differ significantly, the corresponding data from these rats with different duration of 125I-RSA infusion have been pooled
The local plasma equivalent distribution volume of 51Cr-EDTA (Vi) and wick fluid equivalent volume of RSA (Va,w) in skin and muscle are shown in Fig. 9. The local interstitial fluid volumes compared well with those obtained in series I (see Table 1) but are shown here for comparison to Va,w. Mean wick fluid equivalent volumes ranged from 260.7 to 277.6 µl/g wet wt in hindlimb and back skin in hypothyroid as well as control rats. In muscle, the corresponding range was 43.8-53.5 µl/g wet wt (Fig. 9). None of the volumes in hypothyroid rats differed significantly from that obtained in the respective tissues in control rats.
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Albumin concentration and mass. Data for albumin concentration in plasma and wick fluid isolated from skin and muscle are included in Fig. 3.
The ratio of specific albumin activity in plasma and wick fluid did not differ significantly from 1.0 for any of the tissues where wick fluid was sampled. Albumin masses in the various tissue samples are shown in Fig. 10. Whereas the average masses in hindlimb skin in hypothyroid and control rats were similar (3.66 and 3.74 mg/g wet wt, respectively), the mass of 6.03 mg/g in back skin of hypothyroid rats was significantly higher than the corresponding value of 5.15 mg/g in control rats (P < 0.05). In muscle the average albumin masses ranged from 0.75 mg/g in tibialis anterior in hypothyroid rats to 1.26 mg/g in gastrocnemius and semimembranosus muscles, also in hypothyroid rats. None of the masses in muscle differed significantly from the corresponding values in control rats.
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Interstitial albumin distribution ratios.
The values of fractional albumin exclusion
(Ve,a/Vi) calculated by Eqs. 6 and 7 are displayed in Fig. 11.
Average Ve,a/Vi in hindlimb skin of hypothyroid
and control rats was 0.44 and 0.45 (P > 0.05), with
corresponding figures for back skin of 0.19 and 0.32, respectively
(P < 0.05). All values for hindlimb skin differed significantly from back skin (P < 0.01).
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Characteristics of tracer albumin in plasma. Samples of stock 125I-RSA and of plasma after 120 h of 125I-RSA infusion run on a Sephacryl 300 (Pharmacia) column produced nearly identical elution patterns.
Tissue elution. The tissue elution experiments showed that practically all 125I-RSA and 51Cr-EDTA could be eluted in tissues from hypothyroid (n = 2) as well as control rats (n = 2). In hindlimb and back skin and hindlimb muscle of hypothyroid rats, the eluted fractions of 125I-RSA were 98.5 ± 0.9, 98.8 ± 2.1 and 94.8 ± 1.0% respectively, with corresponding numbers for 51Cr-EDTA of 99.5 ± 1.5, 95.5 ± 1.3 and 97.8 ± 1.4%. In control rats, the extraction of 125I-RSA from hindlimb and back skin and hindlimb muscle was 98.8 ± 1.1, 97.8 ± 1.3 and 94.3 ± 1.0% respectively, with corresponding numbers for 51Cr-EDTA of 99.3 ± 0.9, 96.5 ± 1.2 and 99.3 ± 1.3%.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study has shown that induction of hypothyrosis by thyroidectomy in the rat results in several changes in the composition of the interstitium and interstitial fluid balance. In all the hypothyroid tissues studied, the interstitial fluid pressure was higher (more positive) than in control tissues, and the interstitial fluid albumin and total protein concentrations were lower. A uniform change is what may be expected in a situation of hormonal changes. It was therefore somewhat surprising to find different directions of changes in interstitial fluid volume and total tissue water of back skin and hindlimb skeletal muscle. Also, while hypothyrosis is associated with accumulation of hyaluronan and other GAGs in human skin (32), this finding was not confirmed in rat skin. However, in rat hindlimb muscle and heart the hypothyroid state resulted in a more than 50% increase in hyaluronan content. A significantly increased hyaluronan content in muscle was not sufficient to affect excluded volume in that tissue. A reduction in back skin collagen and a possible reorganization of the collagen fibers in hypothyroid rats, however, resulted in a reduced excluded volume for albumin and may partly explain previous findings of an increased extravascular albumin mass in hypothyroid patients (22).
Evaluation of Methods
We chose thyroidectomy as a method to create a hypothyroid state. In humans hypothyroidism develops over a long period of time, often years. One may argue that 8 wk of exposure to a low level of thyroid hormones is short and not relevant for comparison to human studies. Studying influence of maturation and aging of connective tissue in rats, Vogel (31) found that rats are senescent at the age of 2 years. If we say humans reach the same stage of development at 60 years of age, 8 weeks in rats would correspond to more than 4 years in humans. This is a longer duration than for all the patients in the study by Parving and co-workers (22). Even though the comparison between age in humans and rats is not exact, it indicates that our study period was sufficient.The presently used method for measurement of interstitial exclusion and potential sources of error has been evaluated for infusion of tracer albumin into control rats, and the same considerations apply for hypothyroid rats (35). The most fundamental requirement of this method is that steady-state distribution of tracer between plasma and tissue is established. In control rats, stable levels were reached in skin and muscle at 72 h (35). It could be that longer time was needed to reach steady state in the generally affected hypothyroid rats, but the similar 125I-RSA distribution volume after 120 and 168 h of isotope infusion suggests that steady state was reached in these rats, too. Our data on relative specific activities of 125I-RSA in wick fluid and plasma also support the assumption that steady-state conditions existed when the tissues were sampled. Specific activity ratios for muscles, leg skin and back skin were not significantly different from unity. Another fundamental requirement is that we obtain a sample representative for interstitial fluid. The wick method has previously been evaluated thoroughly for use in the tissues studied here (1, 17, 36, 40). Fluid isolated from wicks in the present study should thus be representative for interstitial fluid.
Comparison to Previous Studies
In humans, lack of thyroid hormones lead to accumulation of GAGs in skin and to myxedema (see Ref. 30). Previous studies have shown that this occurs in rat skin too. Schiller and co-workers (28) made rats hypothyroid using propylthiouracil and then measured hyaluronan and chondroitin sulfate in pooled skin samples. Hyaluronan was significantly higher in hypothyroid animals compared with control. The increase was 28 to 68% depending on the duration of the treatment, while chondroitin sulfate was 10-28% lower in hypothyroid rats, strongly suggesting a differentiated effect of thyroid hormone on synthesis and/or turnover of hyaluronan and GAGs. In our experimental model we did not find increased content of hyaluronan in hindlimb and back skin, while there was a significant increase in hindlimb muscle and heart. We have not been able to find data on muscle and heart hyaluronan content in hypothyroid rats, but the difference between Schiller et al. (28) and our data is puzzling. In addition, studies on human fibroblast cultures deprived of thyroid hormone have shown an increased accumulation of hyaluronan, suggesting that this may occur in vivo, too (29).We measured uronic acid to be able to quantify total content of GAGs. In skin, the content of hyaluronan as well as uronic acid did not differ in hypothyroid and control rats, which indicates that the ratio between hyaluronan and total GAGs was the same in the two experimental conditions and not lower as suggested in Schiller and co-workers' study (28). However, the similar uronic acid and different hyaluronan content in hindlimb muscle in the two experimental situations indicates that the total proteoglycans are lower in hypothyroid than in control rats, in agreement with their data from skin.
One might argue that we did not use sufficient time for the increase in hyaluronan to develop, but our studies were done in the same time range of hypothyrosis as theirs. In addition, there was no indication in our experiments that the tissue content increased with duration after thyroidectomy. There are two other factors that may partly explain the discrepancy between the studies. First, whereas they used a chromatographic method, we used a radioassay (11, 20) to measure hyaluronan. This method is sensitive and specific for hyaluronan, and there is no interference from fibronectin, chondroitin sulfate or keratan sulfate (11).
Another important point in this connection is the site of sampling. In our experiments we found almost twice the content of hyaluronan per dry weight in hindlimb compared with back skin in control as well as hypothyroid rats. The hyaluronan concentration in wick fluid was also significantly different between these two sampling sites. These findings show that there are differences in hyaluronan content and concentration in interstitial fluid depending on the location. Schiller et al. (28) did not specify from where the skin was sampled. They pooled skin from 10-20 rats before analysis. Whether we sampled skin from the same area cannot be determined from their paper, but differences in sampling site does influence hyaluronan content as seen by the present study. Even though the whole tissue is exposed to the same hormone concentration, the sensitivity between areas may vary as shown by the increase in collagen in hindlimb skin and decrease in back skin of hypothyroid rats. We used hindlimb skin and distal back skin for analysis because pressure measurements were performed in this area, but hyaluronan may have been different in other areas.
Concentration of hyaluronan estimated from tissue content and interstitial fluid volume is 1-2 mg/ml in skin as well as in skeletal muscle. Hyaluronan concentration in prenodal lymph from dog skin is 5-10 µg/ml (25), while no corresponding data are available from skeletal muscle. However, the hyaluronan concentration in the wick fluid is between these values, i.e., 50-200 µg/ml. That wick fluid concentration is higher than found in lymph is not explained by a high-plasma concentration, which is two to three orders of magnitude less than in prenodal lymph from skin. The most likely reason for the high wick fluid concentration of hyaluronan would be physical disruption of hyaluronan normally bound to cells during the wick implantation procedure.
The figures for interstitial exclusion of albumin in control rats compare well with previous data obtained with the same method in skin and muscle (35), but these numbers for excluded volume fraction are generally lower than those obtained in other studies. In rabbits, Bell and Mullins (5, 6) found excluded volumes close to 50% in both tissues. We have previously discussed the variation in numbers and concluded that the discrepancy between their and our values may be due to interspecies variations of the interstitial matrix composition and short equilibration time for exogenous tracer albumin (35). However, the much higher exclusion of 45% in back skin found by Reed and co-workers (23) may seem more problematic to explain. As in the present study they used wicks to isolate interstitial fluid, 51Cr-EDTA as extracellular tracer, and rats were of the same size and sex as the ones used in the present study. To measure tissue albumin they used an immunological method after elution. An incomplete albumin extraction will result in a higher exclusion, but unless the native albumin is more difficult to extract than 125I-RSA, this phenomenon cannot explain the higher excluded volume for albumin found in their study. A closer look at their albumin data may give an explanation. Their albumin concentration of 46.6 and 33.3 ml/g in plasma and interstitium, respectively, is 60 and 52% higher than corresponding figures in our study. Although not reflected in the interstitial fluid volume of back skin, which actually was lower in the present study, the high albumin concentration may suggest that their rats were more dehydrated than ours at the time of study. As shown by Reed et al. (23) dehydration will increase the excluded volume fraction, suggesting that state of hydration is the explanation for the difference in excluded volume for albumin. That plasma and interstitial fluid protein concentration varies substantially between batches of euhydrated rats has been addressed previously (36).
Fluid Volumes
From the appearance of skin in hypothyroid patients, we had expected that the fluid volumes were increased in skin, but this was not the case. Actually, while Vi and total tissue water were unchanged, these volumes were smaller in back skin of hypothyroid animals. A possible explanation for the reduced volumes in back skin could be changes in composition or changes in transcapillary fluid balance (see below).In muscle, hypothyroidism resulted in an increase in interstitial fluid volume as well as in total tissue water. Even if this change may seem small, it represents a reduction in dry weight of 6.4% in the pooled sample of semimembranosus and gastrocnemius muscle. Again, an increase in muscle volume may be a result of changes in transcapillary fluid balance, but in the cell- rich muscle the reduced efficiency of the Na+-K+-ATPase may also be of importance. As reviewed by Everts (13), skeletal muscle is one of the major target organs for the action of thyroid hormones. Hypothyroidism results in a substantial reduction in Na+-K+-ATPase in skeletal muscle cells. However, this does not seem to appreciably affect the Na+-K+ content in muscle because the concentration of these ions was similar in hypothyroid and control rats (15).
Our data suggest that swelling occurs in muscle interstitium as well as cells. If we consider the pooled data for semimembranosus and gastrocnemius muscle, the Vi averaged 63.0 and 70.0 µl/g wet tissue in control and hypothyroid rats, respectively, with corresponding total tissue water of 750 and 767 µl/g wet tissue. Thus of a total increase in volume of 17 µl/g in the actual tissue, the largest absolute volume increase (10 µl/g) occurred intracellularly while the largest relative increase (11.1%) took place in muscle interstitial fluid. Even if the concentration of ions is unchanged intracellularly (15), these data show that the cell volume regulation is affected by lack of thyroid hormone.
While skeletal muscle interstitial fluid and total tissue volumes increased significantly, no changes were found in hindlimb skin while corresponding back skin volumes were actually reduced in hypothyroid rats. This is surprising considering that we are looking at a hormonal effect. In that case we might expect similar directions of volume change in the tissues studied. There might be two explanations for the differential effect on volume in the two tissues studied: their number of cells and their hyaluronan content. In skin, interstitial fluid volume is two-thirds of the total tissue water, while in muscle the corresponding fraction is less than 10%. Thus if cell volume regulation is affected, as suggested above, the influence will be greatest in cell-rich tissues like muscle. It might well be that our method is not sensitive enough to detect a relatively small change in cell volume of less than 1.5% as observed here. The other factor is the hyaluronan content, which was increased in muscle but unchanged in hypothyroid skin. The interstitial fluid volume during normal hydration is largely determined by the hyaluronan content of the tissue (33), which can explain part of the observations with respect to Vi. The normal volume in skin thus supports the finding of similar skin hyaluronan content in control and hypothyroid rats.
Excluding Agents in the Interstitium
We had expected that the increased hyaluronan content in the muscle interstitium would have influenced interstitial exclusion in hypothyrosis, but this was not the case. It might be that interstitial exclusion is not sensitive enough to be influenced by changes in hyaluronan content even as big as 56%. According to studies by Laurent (19), exclusion of albumin by hyaluronan solutions or gels is simply proportional to the amount of hyaluronan per unit volume as 0.05 ml is excluded per mg hyaluronan. An increase in average hyaluronan content in muscle from 0.23 to 0.35 mg per g fat free dry wt corresponds to 0.06 to 0.08 mg/g wet wt in control and hypothyroid rats, respectively. If we assume that all hyaluronan is distributed in the interstitial fluid, this will exclude 0.003 and 0.004 ml/g wet wt or 4.7 and 5.7% of the interstitial fluid volume. Such an increase is small and barely recognizable if a change in hyaluronan concentration was the only change in interstitial composition in hypothyrosis. Because the increase in hyaluronan was accompanied by a reduction in proteoglycans, changes in interstitial exclusion were not due to a change in the amount of GAGs.However, a suggested effect of hypothyrosis on interstitial compliance (see below) indicated an alteration in interstitial composition compared with the control situation, and we therefore measured the collagen content to look for changes in other excluding agents. The only tissue where excluded volume fraction differed significantly between control and hypothyroid rats was back skin. In skin, the major excluding agent is collagen (8), and the reduced amount of collagen in back skin of hypothyroid rats may explain the lower exclusion observed in this tissue compared with control rats. The exclusion due to collagen will depend on the organization of the fibers, and randomly oriented fibers will exclude a greater fraction than more organized ones (7). The measured collagen in back skin corresponds to 127 and 143 mg/g wet wt in hypothyroid and control rats respectively. If we assume an exclusion by collagen as in human dermis of 1.6 ml/g collagen (7), collagen excluded a volume of 0.20 and 0.23 ml per g wet wt in hypothyroid and control rats, respectively. Using the numbers for hyaluronan in back skin [1.2 mg/g dry wt in both types of rats corresponding to 0.5 mg/g wet wt (also both types of rats) and adding the volume excluded by hyaluronan (0.05 ml/mg, see Ref. 19)], we arrive at a contribution from hyaluronan of 0.03 ml/g wet wt and a total excluded volume fraction in back skin of 0.23 and 0.26 ml/g wet wt in control and hypothyroid rats, respectively. In addition, use of the data for uronic acid and the data from Aukland et al. (4) to estimate the excluding effect of proteoglycans suggests that GAGs contribute to albumin exclusion to an equal extent as collagen. According to these estimates, albumin is excluded from more than 100% of the total back skin interstitial fluid volume in hypothyroid as well as control rats, i.e., obviously not in accordance with the measured respective values of 19 and 32%.
A lower measured excluded volume than expected from calculations using concentrations of excluding agents was also found in a previous study (35). We then concluded that part of the collagen in rat skin probably is more densely organized than in human dermis thereby excluding less albumin than anticipated from in vitro experiments. Aukland and co-workers (4) have suggested that lack of correspondence between experimentally observed volumes and volumes calculated from tissue constituents shows that there is a considerable overlap of excluded volumes in the interstitium.
Even though such phenomena as discussed above may also explain the discrepancy between observed and calculated exclusion in the present study, the difference between hypothyroid and control back skin exclusion is puzzling. This suggests an effect from thyroid hormone on collagen fiber size and/or orientation, which is not unlikely considering the observed differences in collagen content in control and experiment group rats. In the myocardium of hypothyroid rats, Klein and co-workers (16) found a significant remodeling of collagen compared with age- and sex-matched control rats. They observed an increased thickness of individual type I collagen fibers. A more condensed organization of collagen may result in a reduced excluded volume (7), and such phenomena may be the explanation for the reduced excluded volume for albumin observed in back skin in hypothyroid rats.
Physiological Implications
The present study clearly shows that hypothyrosis leads to several changes in the transcapillary fluid balance in rats. Pi was 1.3-1.6 mmHg more positive than in control rats in the tissues studied, and the reduced plasma and interstitial fluid protein concentration show that the colloid osmotic pressure in plasma and interstium is reduced compared with control. If we use the presently measured albumin concentration and assume that the difference between total protein concentration and albumin is globulin, we can use the Landis and Pappenheimer equations to calculate the colloid osmotic pressures in plasma and interstitial fluid (18). By such calculations we obtain a colloid osmotic pressure in plasma, hindlimb skin and medial muscle of hypothyroid rats of 13.6, 6.5, and 6.5 mmHg, respectively. The corresponding pressures in control rats were 17.0, 9.1, and 9.0 mmHg. Using their equation for plasma yields plasma colloid osmotic pressures that are 1.7 and 1.3 mmHg higher in hypothyroid and control rats, respectively, while the corresponding pressures in skin and muscle interstitial fluid will be 0.3 to 1.1 mmHg lower. Because of a significantly higher albumin-to-globulin ratio in the present experiments than their value of 1.1, the calculations based on summing albumin and globulin probably are most representative of the actual colloid osmotic pressure. As reviewed by Levick (21), there may be gradients in the interstitial fluid protein concentration, and accordingly the pericapillary colloid osmotic pressure is the most relevant parameter for transcapillary fluid balance. Tissue may also be heterogeneous with respect to filtration or absorption of fluid (21). Considered in this context, wicks sample fluid from a large area of the interstitium, and wick fluid represents a time and site average of interstitial fluid.The observed changes in plasma and interstitial fluid colloid osmotic pressure in hypothyroid rats suggest that some long-term compensatory changes have occurred. A reduction in plasma colloid osmotic pressure will reduce resorption of fluid into the capillaries, thereby tending to dilute the interstitial fluid. This will stimulate lymph flow and washout of proteins (3), resulting in a reduced interstitial fluid colloid osmotic pressure and a constant interstitial fluid volume, as observed in hindlimb skin. Thus the transcapillary colloid osmotic gradient may be unchanged, as observed here. Such changes in tissue protein concentration were also observed in skeletal muscle, but in this tissue hyaluronan was increased, which increased Vi as discussed above.
More puzzling than the changes in interstitial protein concentration was the rise in interstitial fluid pressure in skin of hypothyroid rats. With a similar, or even reduced Vi, one might expect a similar or lower Pi in the groups studied. In previous studies we have measured the relationship between interstitial fluid volume and pressure (compliance) in skin of rats and found that Vi changed by 14% per mmHg change in Pi (37). If compliance in skin of hypothyroid and control rats was similar and compliance in back and hindlimb skin is similar, the observed interstitial fluid pressures would have corresponded to about 20% higher Vi in hypothyroid than in control rats. When such a change was not observed, the present data suggest that the tissue pressure-volume relationship is altered by hypothyrosis. This assumption is supported by recent measurements of interstitial compliance in hypothyroid rats (Lund and Wiig, unpublished observations). Our measured increase in volume (11%) is slightly smaller than the 17% rise in Vi predicted from compliance data for this tissue (26), suggesting that interstitial compliance may be changed in this organ, too, again influencing the response of the hypothyroid tissue to changes in tissue hydration.
Our finding of a reduced excluded volume for albumin in back skin in hypothyrosis may explain previous observations in hypothyroid patients. Parving and co-workers (22) assessed extravascular albumin accumulation in patients with myxedema by measuring metabolic turnover and transcapillary escape rate for 131I human serum albumin and compared the data to what was observed in the same patients after treatment with thyroxin, i.e., in an euthyroid state. They found that hypothyrosis significantly affected albumin turnover and concluded that a reduced catabolism and turnover led to extravascular accumulation of albumin again leading to the general edema found in myxedema. Although local tissue distribution was not measured in their study, it is reasonable to assume that a significant portion of the calculated 73% increase in extravascular albumin mass was found in skin.
By measurement of the specific activity of albumin, we were able to estimate the tissue albumin mass. Assuming that the intravascular mass is negligible (35), we found that the interstitial albumin content was 17% higher in back skin of hypothyroid than in control rats. An alternative calculation using wick fluid albumin concentration, interstitial fluid volume and excluded volume fraction also showed that the albumin mass was higher in back skin of hypothyroid rats, although the numbers were not identical (5.8 and 5.4 mg/g in hypothyroid and control rats respectively). Thus even if the interstitial fluid albumin concentration was lower, the reduced excluded volume fraction and thereby increased available volume fraction resulted in an increased tissue albumin mass.
Although there are quantitative differences between the present study and that of Parving and co-workers (22), they both show that albumin may accumulate in the interstitium in hypothyrosis. We found that this was not due to an increase in interstitial fluid albumin concentration, which is actually reduced. The data from Parving et al. (22) do not allow a quantitative analysis of the distribution of albumin, but our findings suggest that at least part of the observed increase in interstitial albumin mass in patients with hypothyrosis can be a result of a reduced excluded volume fraction for this protein.
The observations discussed above show the important physiological implications of a change in interstitial exclusion. Albumin (and most likely other proteins) can accumulate as a result of an altered interstitial structure. However, even if the change in structure stems from a hormonal, and thereby a universal, stimulus to all cells, the response is different, e.g., in collagen content in the same type of tissue (skin) in different parts of the body (hindlimb and back). As discussed above, such differentiated responses between tissues in hypothyroid rats were also found for interstitial fluid volume and total tissue water and show that whole body responses cannot be deduced from changes in single organs.
In conclusion, we have shown that induction of hypothyrosis leads to changes in interstitial fluid balance and composition in rats. A higher hyaluronan concentration in muscle can explain the observed increase in interstitial fluid volume. The increased cell volume probably resulted from hormonal influence on cell volume regulation. In skin, hyaluronan was unchanged, and interstitial fluid volume and total tissue water were unchanged (hindlimb) or reduced (back). In both tissues the albumin and total protein concentration was reduced and the interstitial fluid pressure was higher than in control rats. These findings suggest that hypothyrosis results in a washout of interstitial proteins and leads to a change in interstitial compliance. Interstitial exclusion of albumin in hypothyroid was similar to control rats for most of the tissues studied, meaning that the more than 50% increase in hyaluronan observed in skeletal muscle did not influence exclusion. The only tissue where exclusion differed between the two groups was back skin, where the fractional excluded volume in hypothyroid rats was almost half of that in control rats. A likely explanation for at least part of this discrepancy is a reduced collagen content in hypothyroid back skin, but difference in collagen content alone cannot account for this finding, a structural change may be of importance. One implication of the observed altered exclusion is that more protein can accumulate in the tissue without a concomitant increase in interstitial fluid protein concentration, and this may be part of the explanation for the interstitial accumulation of albumin found in hypothyroid patients.
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ACKNOWLEDGEMENTS |
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This work was supported by The Norwegian Council on Cardiovascular Diseases and The Research Council of Norway. Expert technical assistance from Sigrid Lepsøe, Else Elsayed and Kristin Mesteig is gratefully acknowledged
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Wiig, Dept. of Physiology, Univ. of Bergen, Årstadvn. 19, N-5009 Bergen, Norway (E-mail: helge.wiig{at}pki.uib.no).
Received 19 July 1999; accepted in final form 10 November 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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) and control (
)
rats. Values are means ± SE; n = 11 rats for experiments
lasting for up to 120 h, n = 2 for 168 h.
