Methysergide reduces nonnutritive blood flow in normal and
scalded skin
Xiao-Jun
Zhang,
Øivind
Irtun,
Yaoqing
Zheng, and
Robert R.
Wolfe
Metabolism Unit, Shriners Burns Hospital for Children, and
Departments of Surgery and Anesthesiology, The University of Texas
Medical Branch, Galveston, Texas 77550
 |
ABSTRACT |
Methysergide is a serotonin antagonist and has been
demonstrated to reduce wound blood flow and edema formation. We have
determined the effect of methysergide on protein kinetics in normal and
scalded skin of anesthetized rabbits.
L-[ring-13C6]-
or
L-[ring-2H5]phenylalanine
was used to reflect skin protein kinetics by use of an ear model, and
L-[1-13C]leucine was used to
reflect whole body protein kinetics. The results were that infusion of
methysergide (2-3
mg · kg
1 · h
1)
reduced the blood flow rate in normal skin by 50% without changing skin or whole body protein kinetics. After scald injury on the ear,
administration of methysergide for 48 h reduced the weight of scalded
ears (43 ± 4 vs. 30 ± 5 g, P < 0.01) and ear blood flow
rate (42.6 ± 4.9 vs. 5.8 ± 1.0 ml · 100 g
1 · min
1,
P < 0.0001) and did not change wound protein kinetics.
Methysergide reduced arteriovenous shunting and maintained inward
phenylalanine transport from the blood to the skin pool. Using the
microsphere technique, we found that the infusion of methysergide
decreased blood perfusion by 33-36% in both normal and scalded
ear skin. We conclude that methysergide administration reduces
nonnutritive, as opposed to nutritive, blood flow in normal and scalded skin.
stable isotopes; mass spectrometry; arteriovenous balance; protein
metabolism; microspheres
 |
INTRODUCTION |
RECENT STUDIES HAVE SHOWN that administration of
methysergide (MS), a serotoninergic receptor blocking agent, blunts
blood flow and edema formation in scalded skin (12, 25). This finding has clinical implications because wound edema can cause secondary damage to the tissue and result in severe complications (11, 15, 16).
However, the reported short-term (2-4 h) observations on the
hemodynamic response are not sufficient to illustrate its clinical
usefulness because initial burn edema usually lasts 24-48 h before
declining. It is also not clear whether reduction of blood flow to the
skin wound would reduce amino acid supply and thereby inhibit protein synthesis.
Protein kinetics in wounded skin are closely related to the healing
process because wound healing requires degradation of dead tissue and
generation of new tissue; thus an important goal in burn treatment is
to facilitate protein anabolism in the wound. Whereas the hemodynamic
effects of serotonin (5-hydroxytryptamine, 5-HT) have been recognized
for several decades (13, 19, 26), only recently have studies attributed
changes in metabolism to vascular responses of serotonin (6,
8-10). For example, in the perfused rat hindlimb, serotonin was
found to decrease oxygen consumption by reducing nutritive flow to
muscle (20). However, this experiment was performed in a constant-flow
model, so an alternative explanation of the results is that serotonin
caused vasodilation in nonnutritive pathways, and flow in the nutritive pathways decreased because total blood flow was artificially prevented from increasing to accommodate the vasodilation. Moreover, oxygen consumption was the only index of muscle metabolism measured, and
changes in tissue protein kinetics would not necessarily be reflected
by oxygen consumption.
The present study was designed to investigate the role of serotonin in
controlling blood flow and protein kinetics in both normal skin and
scalded skin. We have used MS to block the action of serotonin, and
isotope tracer techniques and our recently developed ear model (30) to
measure the rate of skin blood flow and protein kinetics as well as
amino acid transmembrane transport between the blood and the tissue
intracellular pools. We also used colored dye-extraction microspheres
to measure blood perfusion in normal and scalded skin before and during
MS infusion. Because the microspheres have a diameter of 15 µm, the
results reflected the effect of MS on microcirculation. Thus the
results from this study not only provide insight into the physiological
action of serotonin, but also enable further evaluation of the
potential usefulness of MS in the management of early burn wounds.
 |
METHODS |
Animals.
Male New Zealand white rabbits (Myrtle's Rabbitry, Thompson Station,
TN), weighing ~4.5 kg, were used. The rabbits were housed in
individual cages and consumed Lab Rabbit Chow HF #5326 (Purina Mills,
St. Louis, MO) for weight maintenance. This study was approved by the
Animal Care and Use Committee of The University of Texas Medical Branch
at Galveston.
Isotopes.
L-[ring-2H5]phenylalanine
(L-[ring-2H5]Phe;
98% enriched) and
L-[ring-13C6]Phe
(99% enriched) were purchased from Cambridge Isotope Laboratories (Woburn, MA). L-[1-13C]leucine
(L-[1-13C]Leu; 99% enriched) was
purchased from Masstrace (Somerville, MA).
NaH13CO3 (98.8% enriched) was purchased from
Isotec (Miamisburg, OH). NaH14CO3 was purchased
from Sigma Chemical (St. Louis, MO).
L-[1-13C]Leu,
L-[ring-2H5],
and
L-[ring-13C6]Phe
were prepared in 0.45% saline as stock solutions and stored at
4°C, and then diluted with 0.9% saline before infusion. Both NaH13CO3 and NaH14CO3
were prepared as concentrated stock solutions in 0.01 N NaOH and
diluted with 0.9% saline immediately before infusion. When L-[ring-2H5]Phe
was used as a tracer,
L-[ring-13C6]Phe
was used as an internal standard to calculate Phe concentration, and
vice versa. The internal standard solution contained 29.7 µmol/l of
Phe tracer.
Partial thickness thermal injury.
After overnight fasting, the rabbits were anesthetized by the
intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg).
The left ear was shaved and disinfected with 70% isopropyl alcohol.
The ear was submerged in 72°C water for 3 s. This
procedure consistently produces partial thickness thermal injury on the ear skin, as was addressed in our previous publication (29). After the
thermal injury a single dose of antibiotic (Bicillin 50,000 U/kg, Wyeth
Laboratories, Philadelphia, PA) was intramuscularly injected. When
rabbits awakened, buprenorphine (0.03 mg/kg) was injected
intramuscularly twice daily as an analgesic.
Experimental design.
There were three protocols, with two groups in protocol 1,
three groups in protocol 2, and one group in protocol 3 (n = 5 each). Protocol 1 included the normal untreated
group and normal MS group. The rabbits in this protocol did not receive
scald injury, and the isotope infusion study was performed after an
overnight (16-h) fast. The purpose of this protocol was to investigate
the effects of MS on blood flow and protein kinetics in normal skin and
in the whole body. Protocol 2 was designed to investigate the
vascular and metabolic responses of scalded skin to continuous MS
administration and included the sham scald group, scald untreated group, and scald MS group. In the scald untreated group, the rabbits received the scald injury on the ear and did not receive MS treatment. After the scald injury, the rabbits had free access to food and water
during the first 32 h. During the following 16 h the animals were
denied access to food but water was freely available. The rabbits in
the sham scald group received the same doses of anesthetics, antibiotic, and analgesic as in the other two groups, except that no
scald injury was created on the ear. Because the rabbits in the scald
untreated and scald MS groups refused food during the first 32 h, the
rabbits in the sham scald group were given only water to make the
nutritional condition comparable in the three groups. In protocol
3, the thermal injury was created on the right ear skin using the
same procedure as in protocol 2. The rabbits had free access to
food and water after the scald injury. Forty-eight hours after the
scald, the rabbits received two injections of microspheres before and
during the MS infusion. Because the microsphere technique required
additional surgical procedures and large skin samples for analysis, it
was not possible to perform it at the same time as the isotope infusion
studies. Thus protocol 3 was designed only to investigate the
effect of MS on the microcirculation in the normal and scalded skin,
and no isotopes were used. The MS treatment and isotope infusion
protocols are outlined in Table 1.
The doses of MS were based on the effectiveness of the previously
reported single dose of 1 mg/kg (12) and our pilot studies. Two
different doses of MS were used. A higher dose was needed to acutely
reduce blood flow to the ear than was needed to reduce wound edema over
48 h. Thus, in the normal MS group a MS solution of 0.4 mg/ml was
infused intravenously at 3 mg · kg
1 · h
1
for the 1st h and about 2.0 mg · kg
1 · h
1
thereafter, until the end of the isotope infusion. In the microsphere group, MS was administered through intravenous infusion at 2-3 mg · kg
1 · h
1.
This higher dose was found necessary to acutely reduce skin blood flow
rate in our pilot studies. In the scald MS group, 1.5 mg/kg of MS was
infused via an ear vein in the contralateral ear over the first 1.5 h
after the scald injury. After the animals had awakened from anesthesia,
0.5-1.0 mg/kg of MS was injected intramuscularly every 3-5 h
until the 48th h. During the infusion of isotopes (49th-52nd h after
scald) MS was infused intravenously at 0.1-0.2
mg · kg
1 · h
1.
This dosing scheme was sufficient in our pilot studies to ensure a
constant inhibition of wound edema and blood flow rate throughout the
experimental period.
The anesthetic and surgical procedures were described in our previous
publications (30). In brief, the rabbits were anesthetized with
ketamine and xylazine. Polyethylene catheters (PE 90; Becton-Dickinson, Parsippany, NJ) were placed in the right femoral artery and vein. The
arterial line was used for blood collection and monitoring of arterial
blood mean pressure and heart rate; the venous line was for infusion. A
tracheal tube was placed via tracheotomy. A flow probe (1RB; Transonics
Systems, Ithaca, NY) was placed on the central ear artery of the
experimental ear and connected to a small animal blood flowmeter (model
T106; Transonics Systems). In protocol 3, additional procedures
were performed to quantify regional blood flow with microspheres (7).
In brief, the left carotid artery was dissected through an incision on
the neck. A 4.0 F polyurethane catheter (Cook Critical Care,
Bloomington, IN) was inserted into the artery and advanced retrograde
into the left ventricle. The location of tip was confirmed by
monitoring the blood pressure waveform on the screen of a pressure
monitor (model 78304A, Hewlett Packard, Palo Alto, CA). To prevent
coagulation in the catheter placed in the left ventricle, a dose of
heparin at 50 U/kg was injected intravenously.
The isotope infusion protocols are illustrated in Fig.
1. In protocol 1 (Fig. 1A),
after collection of a blood sample, a skin specimen from the incision
on the groin, and an expired-breath sample for background measurements,
priming doses of L-[1-13C]Leu (21 µmol/kg),
L-[ring-2H5]Phe
(6 µmol/kg), and NaH13CO3 (2.0 µmol/kg)
were injected intravenously. Immediately after the priming doses, a
continuous infusion of L-[1-13C]Leu
(0.35 µmol · kg
1 · min
1)
and
L-[ring-2H5]Phe
(0.15 µmol · kg
1 · min
1)
was started. The use of
L-[ring-2H5]Phe
instead of
L-[ring-13C6]Phe
as in protocol 2 (see below) and in our previous experiments (28, 29, 30) was because the 13C- labeled Phe would
contribute to CO2 enrichment and interfere with the
measurement of Leu oxidation. In a previous study from this laboratory,
two Phe tracers were verified to yield similar model-derived values
(3). A solution of MS (0.4 mg/ml) was also infused intravenously at an
initial dose of 3 mg · kg
1 · h
1
for the 1st h, and at ~2.0
mg · kg
1 · h
1
thereafter until the end of the isotope infusion. Ninety minutes after
the start of the isotope infusion, NaH14CO3 was
infused at a rate of 1,000 disintegrations · min
1
(dpm) · kg
1 · min
1,
with a prime of 85,000 dpm/kg, for measurement of CO2
production (
CO2). After
40-min infusion of NaH14CO3 solution to reach
an isotopic equilibrium, four expired-air samples were collected at
evenly spaced intervals over the next 50 min. The expired air was
collected from the endotracheal tube into a 3-liter air collection bag
(Quintron Instruments, Milwaukee, WI) via a 3-way valve connector.
During the 90 min of NaH14CO3 infusion we also
collected 6-min expired air into a 5-liter air collection bag and
measured the rate of CO2 expiration on a CO2
analyzer (model CD-3A, Ametek, Pittsburgh, PA). The rate of
CO2 production measured from the
NaH14CO3 infusion was used to calculate the
rate of whole body Leu oxidation. This approach accounts for the
so-called bicarbonate retention factor (27).

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Fig. 1.
Experimental protocols. x, Sampling of blood or skin. A: normal
untreated and normal methysergide groups in protocol 1;
B: sham injury, scald untreated, and scald methysergide groups
in protocol 2.
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Four simultaneous arterial and ear-venous blood samples were collected
every 15 min between 180 and 240 min for determination of Phe kinetics
in the skin. The blood flow rate was recorded at each arteriovenous
(a-v) sampling. After each a-v blood sampling, expired air was
collected into a 3-liter bag. The air was immediately transferred to
20-ml evacuated tubes (Becton-Dickinson, Franklin Lakes, NJ) for later
analysis of 13CO2 enrichment. At 240 min, a
skin sample was taken from the ventral side of the ear. After the final
samples had been obtained, the ear was cut off at the skin fold between
ear base and auricle to weigh the ear. In our previous study (28), we
dissected 10 normal ears and found that the ear contained 78% by
weight of skin. Thus the weight of the ear was multiplied by 0.78 to
get the weight of ear skin. The measured ear blood flow rate in
milliliters per minute per ear was then converted to the units of
millimeters per hundred grams per minute.
In protocol 2 (Fig. 1B), after collection of blood and
skin samples for background measurements,
L-[ring-13C6]Phe
was infused at a rate of 0.15 µmol · kg
1 · min
1
after a priming dose of 6 µmol/kg was injected. In the scalded MS
group, the MS solution was also infused at 0.1-0.2
mg · kg
1 · h
1
throughout the isotope infusion period to ensure a continuous inhibition of wound edema and blood flow. After 2 h of tracer infusion
to reach an isotopic equilibrium, four simultaneous a-v samples were
collected at an interval of 10 min over the 3rd h. Auricular blood flow
was recorded at each blood sampling. At 180 min, a skin sample was
taken from the ventral side of the ear. The ears were cut off as in
protocol 1. In the scald untreated and scald MS groups, both
ears were weighed. The weight difference reflected wound edema. Because
the weight of the scalded skin did not represent real tissue mass, the
weight of skin in the contralateral ear was used to estimate the weight
of skin in the scalded ear. The blood flow rate in the scalded ear was
divided by the weight of the ear skin to convert the units of
millimeters per minute per ear to millimeters per hundred grams per
minute. In both protocols 1 and 2, the blood samples
were collected into heparin tubes and placed in an ice-water bath until
the end of the study. The skin samples were immediately frozen in
liquid nitrogen and stored at
70°C for later analysis.
In protocol 3, the blood flow rate in the scalded ear was
recorded every 10 min for the first 40-60 min to obtain the
baseline value. When the blood flow rate reached a constant value, the colored dye-extraction microspheres (15-µm DIA Fluorescent
Microspheres, Interactive Medical Technology, Los Angeles, CA) were
injected into the left ventricle to measure tissue blood perfusion (7). The suspension of spheres was warmed to 37°C in a water bath. Immediately before injection, the spheres were vortexed vigorously for
1 min. Each injection contained 1.25 × 106 spheres in
randomized colors. After injection of the spheres, the infusion line
was flushed with 1 ml of warm saline. Reference withdrawal from a
femoral artery was at a rate of 2.0 ml/min by use of a syringe
infusion-withdrawal pump (sp210iw, World Precision Instruments,
Sarasota, FL), starting 5 s before the injection of the spheres and
continuing for 1 min after completion of the injection. The MS solution
was then infused at 2-3
mg · kg
1 · min
1.
The rate of MS infusion was adjusted to ensure a gradual decline of the
rate of blood flow in the scalded ear. When the rate of blood flow was
reduced to the range in normal skin, the second injection of spheres
was performed, by use of the same procedure as for the first injection
except that the color of the spheres was changed. The animals were
killed by intravenous injection of 5 ml saturated KCl solution under
general anesthesia. Skin samples were collected from the scalded and
contralateral ears, upper legs, and thorax. Both kidneys were taken via
laparotomy. The tissues were kept in individual tubes and the tissue
weight was recorded. The samples were shipped to Interactive Medical Technology (Los Angeles, CA) for analysis.
In all experiments, mean arterial blood pressure, heart rate, rectal
temperature, and ear blood flow rate were monitored continuously and
recorded every 30 min. The surface temperature of the experimental ears
was maintained at 37°C by means of a heating lamp. The room temperature and another heating lamp were adjusted to maintain a
relatively constant body core temperature.
Sample analysis.
After completion of the study, an internal standard, which contained
29.7 µmol/l of
L-[ring-13C6]Phe
(in protocol 1) or
L-[ring-2H5]Phe
(in protocol 2), was added to the blood (30). The samples were
deproteinized with sulfosalicylic acid and the supernatant was
processed to make the N-acetyl, n-propyl ester (NAP)
derivatives of amino acids (27). The blood samples for measurement of
-ketoisocaproate (KIC) enrichment were processed to make the
silylquinoxalinol derivative; 100 µl 1:1 N,O-bis(trimethylsilyl)
trifluoracetamide (BSTFA) with 1% trimethylchlorosilane (TMCS)
pyridine were added to each sample before analysis (27). Blood
hemoglobin (Hb) concentration was measured on an automated hematology
analyzer (Coulter JT3, Hialeah, FL). To determine the specific activity
(SA) of 14CO2 in the expired air, 1 mmol
CO2 was trapped in a solution of benzethonium hydroxide
(Sigma Chemical) and the dpm was determined on a scintillation counter
(model 1219 Rackbeta) (27). The amount of CO2 trapped was
verified by titration with HCl to calculate the SA in dpm per millimole
CO2.
Skin specimens of ~60 mg were homogenized in 5% perchloric acid
three times at 4°C. Phe in the supernatant was purified by HPLC
(30) and derivatized for the NAP derivatives (27). The skin precipitate
was washed and dried at 80°C as previously described (30). The dry
protein pellets were hydrolyzed in 6 N HCl. A cation exchange column
(Dowex AG 50W-X8, Bio-Rad Laboratories, Richmond, CA) was used to
purify the amino acids. The samples for determination of
L-[ring-13C6]Phe
enrichment were prepared for the
N-heptafluorobutyryl-n-propyl ester (HFBPr) derivatives
(21), and the samples for
L-[1-13C]Leu enrichment were
processed for the NAP derivative.
The isotopic enrichment in the blood and tissue supernatant was
measured on a Hewlett-Packard 5985 gas chromatograph mass spectrometer
(GC-MS) (Hewlett-Packard) with chemical (for Phe and Leu) or electron
impact (for KIC) ionization. Ions were selectively monitored at
mass-to-charge (m/z) ratios of 250, 251, 255, 256 for
Phe, at m/z ratios of 216 and 217 for Leu, and at
m/z ratios of 232 and 233 for KIC (27). Isotopic
enrichment of 13CO2 in the expired gas was
determined by isotope ratio mass spectrometry (VG Isogas, Middlewich,
Cheshire, UK) (3). In protocol 1,
L-[1-13C]Leu enrichment in the skin
protein hydrolysate was measured on a gas
chromatograph-combustion-isotope ratio mass spectrometer (GC-C-IRMS)
(Finnigan, MAT, Bremen, Germany). The measured
13CO2 enrichment was converted to Leu
enrichment by multiplying by 11 to account for the dilution of the
labeled carbon with the carbons in other positions of the derivatized
Leu. The protein hydrolysate in protocol 2 was analyzed for
L-[ring-13C6]Phe
enrichment by use of the method described by Patterson et al. (21).
Thus the ratio of m+6 to m+4 ions was measured on a
GC-MS (MD 800, Fison Instrument, Beverly, MA) and converted to the true
enrichment of
L-[ring-13C6]Phe
by means of a standard curve. Isotopic enrichments were expressed
as mole percent excess (MPE) after correction for the contribution of
the abundance of isotopomers of lower weight to the apparent enrichment
of isotopomers with larger weight (24). A skew correction factor was
also used to calculate
L-[ring-13C6]Phe
enrichment in the blood and skin supernatant (24).
The tissue and blood samples in protocol 3 were processed and
analyzed for spheres in a laboratory of Interactive Medical Technology
by use of the published method (7).
Calculations.
CO2 production
(
CO2) was calculated from
NaH14CO3 infusion (27) as follows:
CO2 = F/SA, where F is the
infusion rate of NaH14CO3, and SA is the
specific activity of 14CO2 at isotopic
equilibrium. In all of the experiments in protocol 1, a plateau
was achieved for plasma KIC enrichment, so the standard steady-state
equations were used for the calculation of whole body Leu kinetics
(27): rate of appearance (Ra) of Leu = F/Ep, where F is the infusion rate of
L-[1-13C]Leu, Ep is the
plasma KIC enrichment; rate of Leu oxidation = (IECO2 ×
CO2)/(IEKIC),
where IECO2 is the enrichment of
13CO2 in the expired air and IEKIC
is the enrichment of plasma KIC; nonoxidative Leu disposal = Leu
Ra
Leu oxidation.
Protein kinetics and Phe transport in the normal and scalded skin were
calculated from a three-compartment model (2). The three pools are
arterial pool (pool A), venous pool (pool V), and
tissue intracellular free pool (pool T) (Fig.
2). Other definitions necessary for the
description of Phe kinetics are Fin, the rate of Phe
entering the a-v unit via arterial flow (i.e., inflow); FT,A, the rate of delivery from pool A to pool
T (i.e., inward transport); FV,A, the rate of delivery
directly from pool A to pool V (a-v shunting);
FV,T, the rate of delivery from pool T to pool
V (i.e., outward transport); Fout, the rate of Phe
leaving the a-v unit; FO,T, the rate of disappearance
(Rd) from pool T; and FT,O,
Ra of Phe from endogenous source. Because Phe is neither synthesized nor degraded in the peripheral tissues, endogenous Ra represents protein breakdown and Rd
represents protein synthesis. Total Ra into pool T
is the sum of inward transport and proteolysis. By measuring Phe
enrichment and concentration in the three pools and the blood flow
rate, we can calculate the rates of protein kinetics and Phe
transmembrane transport (2, 30).

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Fig. 2.
Three-compartment model of phenylalanine kinetics. FV,A
indicates direct phenylalanine flow from artery to vein without
entering intracellular pool; FT,A and FV,T
refer to inward and outward phenylalanine transport from artery to
tissue and from tissue to vein, respectively. FT,O
indicates intracellular phenylalanine appearance from proteolysis;
FO,T is rate of disappearance (Rd) of
intracellular phenylalanine via protein synthesis. Free phenylalanine
pools in artery (A), vein (V), and tissue (T) are connected by arrows
indicating unidirectional phenylalanine flow between compartments.
Phenylalanine enters arterial-venous (a-v) system via artery
(Fin) and leaves system via vein (Fout).
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Phe concentration in the arterial and venous blood was calculated using
the internal standard method (27). We corrected venous Phe
concentration for the change of Hb concentration between the arterial
and ear-venous blood that occurred because of loss of water vapor from
the surface of the skin. We assumed that the rate of water evaporation
from the ear skin was constant under the same physiological conditions.
Thus the faster the blood flow, the smaller the a-v difference in Hb
concentration, and vice versa. In the normal untreated group and the
sham scald group, we used a correction factor of 98.2% of measured
venous concentration. This correction factor was derived from our
previous study (28) under comparable physiological conditions. In the
normal MS group, the blood flow rate was reduced by 50%, so the
correction factor was accordingly estimated to be 96.4% of the
measured venous blood concentration. In the scald MS group, Hb
concentration measured in the arterial blood and ear-venous blood was
11.3 ± 0.3 and 11.8 ± 0.3 g/dl (n = 5), respectively, which
resulted in a correction factor of 95.6%. The corresponding blood flow
rate was 6.5 ± 0.9 ml · 100 g
1 · min
1.
Because the blood flow rate in the scalded ear varied over a large
range, we corrected the venous Phe concentration against the blood flow
in each individual rabbit: correction factor = [1
(6.5 × 4.4%/BF)], where 6.5 (ml · 100 g
1 · min
1)
is the average blood flow rate at the time of collecting a-v blood for
Hb measurement; 4.4% is the average increase in Hb concentration from
arterial to venous blood; BF (ml · 100 g
1 · min
1)
is the individual blood flow rate in the scalded ear skin. This correction equation was also based on the assumption that the rate of
water evaporation from the 48-h-scalded ear skin surface was constant
under the same physiological conditions. The Phe concentration in the
venous blood was then multiplied by the correction factor to eliminate
the influence of water evaporation.
The fractional synthesis rate (FSR) of skin protein was calculated by
means of the direct incorporation method (27). The blood flow rate by
use of the colored microsphere method was calculated by the following
equation: blood flow rate
(ml · min
1 · g
1) = [(total tissue spheres)/(tissue weight in g) × (reference spheres · ml
1 · min
1)]
(7).
Statistical analysis.
Data are expressed as means ± SD. Differences between the two groups
of protocol 1 were evaluated using the Student's
t-test. Differences among the three groups in protocol
2 were evaluated using a one-way ANOVA. Post hoc testing was
accomplished using the nonpaired Student's t-test combined
with Bonferroni's inequalities. A P value <0.05 was
considered statistically significant.
 |
RESULTS |
The physiological parameters are presented in Table
2. The data of the sham scald group were
presented in our previous publication (28). There were no significant
differences in body weight, heart rate, or rectal temperature between
groups. Mean arterial blood pressure was significantly higher
(P < 0.01) in the MS groups of both protocols 1 and
2 compared with their corresponding untreated groups.
In protocol 1, the ear blood flow rate in the normal untreated
group was basically stable over the 4 h of tracer infusion (Fig.
3A). In the normal MS group the ear
blood flow rate decreased during the 1st h of MS infusion and was
relatively stable over the remainder of the tracer infusion period at a
value that was ~50% of the value in the normal untreated group (Fig.
3A). The blood flow rate in normal skin was responsive to
physical and chemical stimuli, because massaging or alcohol wiping
elicited instant and temporary increase in the flow rate. However, 30 min after the start of MS infusion, the ear blood flow rate no longer responded to such stimuli.

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Fig. 3.
Effect of methysergide administration on blood flow rates in normal and
scalded skin. Bars represent standard deviation. A: blood flow
rates in normal skin with or without methysergide infusion. B:
blood flow rates in scalded skin with or without methysergide
treatment. * P < 0.05 vs. untreated group.
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In protocol 2, the scalding time of 72°C water in the two
groups was almost identical (3.25 ± 0.09 and 3.24 ± 0.07 s, P > 0.05). The partial thickness thermal injury was
confirmed by both clinical and microscopic observations. In the scald
untreated group, the scald injury caused increasing redness, edema, and blisters on the ear, which reached maximal intensity by the 24th h and
lasted until 48 h. Light-microscopic observation of the 48-h-scalded
skin revealed the same responses in both untreated and MS groups: areas
of necrotic epidermis as shown by karyolysis of cells and
hypereosinophilia, and patches of acute inflammatory infiltrate within
the necrotic epidermis.
The rabbits in the scald MS group received MS treatment as described in
METHODS. The total dose of MS used in the 48-h postinjury period was 10.1 ± 1.0 mg/kg. Over the 3 h of the isotope infusion, an
additional 2.1 ± 0.8 mg/kg of MS were infused intravenously. The MS
treatment significantly reduced the vascular response. The weight of
the scalded ears was significantly lower in the scald MS group than in
the scald untreated group (30 ± 5 vs. 43 ± 4 g, P < 0.01). By use of the weight of the contralateral ear as a reference,
the weight increase in the scalded ear was 26 ± 9 and 74 ± 6%
(P < 0.01) in the scald MS and scald untreated groups,
respectively. The blood flow rate in the scald untreated ears was 6- to
7-fold greater than that in the ears of the sham injury group (Table
2). In the scald MS group, the ear blood flow rate was reduced to a
rate comparable to that of the sham injury group (Fig. 3B).
Isotopic plateaus were achieved during blood sampling (Fig.
4). In protocol 1, there were no
differences between the normal untreated and normal MS groups in the
whole body rates of appearance (Ra) of Leu (5.3 ± 0.4 vs.
5.0 ± 0.4 µmol · kg
1 · min
1)
and Phe (1.2 ± 0.3 µmol · kg
1 · min
1
in both groups), Leu oxidation rate (1.7 ± 0.2 µmol · kg
1 · min
1
in both groups), nonoxidative Leu disposal rate (3.5 ± 0.3 vs. 3.3 ± 0.6 µmol · kg
1 · min
1),
and
CO2 (232 ± 14 vs. 230 ± 7 µmol · kg
1 · min
1).
The rates of total CO2 expiration measured were 219 ± 32 and 202 ± 39 µmol · kg
1 · min
1
(P > 0.05) in the normal untreated and normal MS groups,
respectively. The corresponding bicarbonate retention rates were 7 ± 13 and 12 ± 18% (P > 0.05) of total CO2
production. These results indicated that the MS infusion did not change
the whole body protein kinetics.

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Fig. 4.
Phenylalanine enrichment in arterial blood reached plateaus during four
pairs of a-v samples. A-1, A-2, A-3, and A-4 are 4 enrichment values in
arterial blood. In protocols 1 and 2, a-v sampling
started after 3- and 2-h primed constant infusion, respectively.
Interval between two samplings was 10 min.
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|
Phe enrichment and concentration in the arterial and ear-venous blood
and enrichment in the skin free amino acid pool are presented in Table
3. Calculated skin protein kinetics are
presented in Table 4. There were no
significant differences in protein synthesis, breakdown, or net balance
between groups in protocols 1 or 2. The rates of skin
protein synthesis and breakdown in protocol 2 were lower than
those in protocol 1, but the rates of net balance were not
different. These data are consistent with our previous results (28),
indicating parallel decreases in both skin protein synthesis and
breakdown in response to fasting. In the scald MS group, the rate of
net protein balance in the scalded skin was not significantly different
from that in the sham scald and scald untreated groups. Phe transport
data are presented in Table 5. In both
protocols 1 and 2, whereas the rates of Phe inflow into the ear were significantly greater (P < 0.01 or P < 0.0001) in the untreated groups than in the MS groups, the rates of
inward transport were not significantly different, due to the
significant (P < 0.01 or P < 0.0001) reduction in
a-v shunting in the MS-treated groups.
To confirm the protein synthesis data derived from the ear model, we
measured the FSR of protein in the normal and scalded skin (Table
6). Because the measurement of FSR did not
include blood flow rate, and the ear model did not involve measurement of the rate of incorporation of tracer into protein, the FSR data provided an independent means to confirm the results derived from the
ear model. As was the case with the ear model, there was no significant
difference in FSR between groups in protocols 1 and 2.
Thus the FSR data confirmed that skin protein synthesis rates were
essentially maintained when blood flow was greatly reduced by the MS
administration.
In protocol 3, the scalding time in 72°C water was
3.4 ± 0.1 s, which was not different (P > 0.05)
from that in protocol 2. The first injection of microspheres
was performed 40-60 min after completion of surgical procedures
when the blood flow rate (from flowmeter) in the scalded ear was 41.6 ± 12.8 ml · 100 g
1 · min
1.
The blood flow rate began to decrease within 5 min of the MS infusion
and reached the normal skin rate in 15-30 min. At the time of the
second injection of the spheres, the blood flow rate was 7.3 ± 1.1 ml · 100 g
1 · min
1.
During the 15-30 min of MS infusion, there were slight but
significant (P < 0.01 by paired t-test) increases in
rectal temperature (from 38.8 ± 0.5 to 38.9 ± 0.5°C)
and in mean arterial blood pressure (from 73 ± 7 to 79 ± 7 mmHg). The microsphere data showed that blood perfusion rates in the
right and left kidneys were 3.72 ± 0.51 and 3.75 ± 0.48 ml · g
1 · min
1
for the first injection and 4.06 ± 0.50 and 4.10 ± 0.51 ml · g
1 · min
1
for the second injection. These values indicated that the injected spheres were uniformly distributed in the bloodstream. The baseline blood perfusion was 17.7 ± 15.7, 3.4 ± 0.8, 4.3 ± 1.9, and
3.6 ± 1.4 ml · 100 g
1 · min
1
in the scalded ear skin, normal ear skin, upper leg skin, and thorax
skin, respectively. The infusion of MS caused decreases in blood
perfusion by 33 ± 29% in the scalded ear skin, 38 ± 21% in the normal ear skin, 32 ± 12% in the upper leg skin, and 8 ± 18% in the thorax skin (Fig. 5).

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Fig. 5.
Skin blood perfusion was determined by microsphere method. Values
before methysergide (MS) infusion served as baseline. Values
during MS infusion were obtained after 15- to 30-min infusion of
MS at 2-3
mg · kg 1 · h 1,
when blood flow rate in scalded skin was reduced to range of
normal skin blood flow rate. * P < 0.05 vs. baseline
values.
|
|
 |
DISCUSSION |
The results from the present study showed that MS administration
reduced the rate of blood flow in both normal and scalded skin without
affecting the rate of protein synthesis. This was so because there was
an increased proportion of amino acids extracted as the blood flow was
reduced. In our model, this was reflected by a reduction in a-v
shunting and a maintenance of the rate of inward transport.
Arteriovenous shunting is defined as a route for amino acids to pass
from the arterial pool directly to the venous pool (see Fig. 2), thus
being functionally equivalent to nonnutritive blood flow as defined by
Clark et al. (4). In contrast, inward transport measures the rate of
delivery of amino acids from the arterial pool to the tissue
intracellular pool. Thus the rate of inward transport reflects
nutritive flow. Because MS administration markedly reduced a-v shunting
without affecting the rate of inward transport, we can conclude that
serotonin selectively caused vasodilation in nonnutritive blood vessels.
The lack of changes in the rate of wound protein synthesis in the face
of reduced wound blood flow was also found during the hyperinsulinemic
clamp in the same animal model (29). In that study, insulin infusion
reduced wound blood flow by 41-44%, resulting in a significant
decrease in the rates of both inward transport and total amino acid
availability in the wound fluid. The maintenance of wound protein
synthesis was attributed to the increased efficiency of protein
synthesis, because a greater percentage of Phe was incorporated from
the wound fluid into protein than in the untreated group. In contrast,
in the present study the inward transport was not reduced by the MS
infusion, and neither was the total amino acid availability in the skin
free amino acid pool (see Table 5). Thus the unchanged nutritive flow
explains the maintenance of protein synthesis in both normal and
scalded skin during the MS-mediated low blood flow.
The concept that serotonin affects functional vascular shunting was
proposed by Rippe and Folkow (23), who found that serotonin infusion
decreased both capillary diffusion and filtration capabilities in the
rat hindlimb preparation. Recently, Newman et al. (20) investigated the
vascular and metabolic effects of serotonin and norepinephrine using
the constant-flow perfused rat hindlimb. They observed a transient
washout of red blood cells when low-dose norepinephrine (LDNE) was
added to the perfusion solution. When serotonin was added to the
perfusate, no red cell washout was observed. Furthermore, vascular
entrapment of fluorescent-labeled dextran (Fx) perfusions indicated
that LDNE recruited a new vascular space and serotonin closed off a
previously perfused vascular space. Corrosion casting of the arterial
tree with methyl methacrylate showed a decrease in cast
weight by serotonin administration, as opposed to an increased cast
weight by LDNE. Thus they concluded that serotonin increased functional
shunting and decreased capillary surface area. Because the flow rate
was artificially fixed in that model, an increase in nonnutritive flow
was necessarily accompanied by a decrease in nutritive flow, and vice
versa; therefore, it was not possible to ascertain whether the primary
response to serotonin infusion was increased nonnutritive flow or
decreased nutritive flow. Furthermore, there was controversy regarding
the existence of muscular a-v shunts, because a search for anatomical a-v shunts in the skeletal muscle was not as successful as in other
tissues (14).
In the present study, we have documented for the first time that
serotonin regulates functional shunts (e.g., nonnutritive flow) in the
skin. The skin has abundant a-v anastomoses, which serve as
preferential channels to move blood past the capillaries, principally
for the purpose of thermoregulation. The microsphere technique was
performed in an attempt to distinguish between physiological and
anatomical shunts. Because the microspheres have a diameter of 15 µm,
entrapment in the tissue reflects capillary flow. Before the MS
infusion, the wound perfusion rate was 17.7 ± 15.7 ml · 100 g
1 · min
1,
which was 43% of the flow rate measured by the flowmeter. The additional 57% of wound blood flow can be reasonably regarded as
anatomical shunts. The infusion of MS caused a decrease of wound blood
perfusion by 33%, which accounted for 14% of the total blood flow.
Thus, in the scalded ear skin, 57% of the blood flow was directed to
the anatomical shunts, 14% was directed to the physiological shunts,
and 29% was directed to the nutritive route.
After injury, the vascular response is an important component of
inflammation reaction in the wound (22). Increased blood flow and
capillary permeability allow easier access for inflammatory cells to
enter the injured area; however, the role of increased blood flow in
wound protein metabolism is not clear. The rapid blood flow rate might
be useful in supplying more nutrients for wound repair; however, the
results from this study do not support this hypothesis, because protein
kinetics in the 48-h-scalded skin were not increased compared with the
sham injury group (see Table 4). Moreover, protein kinetics were
unaffected when blood flow was reduced. Rather than delivering an
increased amount of amino acids to the metabolically active tissues in
the wound, as much as 92 ± 1% of Phe was directed to a-v shunting in
the scalded untreated skin (see Table 5). It is possible that the results would have been different had we studied the animals at a
different time after injury. In our previous study (29) of the
7-day-scalded skin, the rate of protein synthesis increased to twice
the value in the normal skin. However, the wound blood flow increased
to 15-fold the normal skin rate, suggesting an excessive vascular
response. Although we have little evidence that the rapid blood flow
served a beneficial purpose, excessive vasodilation does increase heat
loss and does decrease blood supply to the vital organs; consequently,
reducing nonnutritive blood flow in the skin wound may have clinical benefits.
In conjunction with decreased blood flow, MS administration reduced
wound edema formation over the first 48 h after the scald injury.
Serotonin has been known to increase vascular permeability, especially
in postcapillary venules (17). Serotonin characteristically causes the
endothelial cells of postcapillary venules to contract, thus opening
the intercellular junctions between endothelial cells, permitting the
leakage of plasma out of the venules (1). In a patient with a large
body surface area burn, the capillary leak can lead to rapid
hypovolemic shock. In the burn area, edema may inhibit cell-mediated
immune responses (11) and contribute to respiratory insufficiency (15)
and vascular compression in close-space areas (16). Thus edema is
believed to increase the risk of tissue ischemia and infection
(18, 22). Our observation substantiates the important role of serotonin
in the development of initial burn edema. The continuous reduction of
wound edema did not change protein kinetics in the scalded skin (see
Tables 4 and 6). The protein kinetics in the scald MS group, although
measured from the 3-h isotope infusion, can be reasonably considered to
be representative of the first 48 h after injury. Although minimizing
wound edema may have a beneficial effect on wound healing, caution must
be exercised in using MS clinically. Serotonin is known to play an important role in the regulation of a wide variety of neurobiological functions and behaviors, including appetite, pain, mood, circadian rhythm, and the like. (5). Therefore, possible disturbances of the
physiological and psychiatric responses would need to be minimized if
MS is to be used in burn patients.
In summary, MS administration reduced blood flow in normal and scalded
skin, indicating the primary role of serotonin in increasing wound
blood flow. MS selectively reduced nonnutritive blood flow and
maintained the nutritive blood flow, so its administration did not
inhibit protein synthesis in either normal or early scalded skin, or at
the whole body level. After a partial thickness thermal injury,
immediate and continual MS treatment for 48 h largely reduced edema
formation in the skin wound. The MS-mediated reduction of wound edema
and wound blood flow can have clinical benefits if cautions are taken
to avoid its possible side effects.
 |
ACKNOWLEDGEMENTS |
The authors are grateful to Hal K. Hawkins for the histologic
diagnosis of scalded skin, and to John J. Ferrara and
associates for advice in preparing methysergide solution. We thank
Zhanpin Wu, David Doyle, Jr., Guy Jones, Yunxia Lin, and Zhiping Dong for technical assistance.
 |
FOOTNOTES |
This work was supported by Grants 8630 and 8490 from the Shriners Hospital.
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: R. Wolfe,
Shriners Burns Institute, 815 Market Street, Galveston, TX
77555-1220 (E-mail: rwolfe{at}utmb.edu).
Received 2 April 1999; accepted in final form 28 October 1999.
 |
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