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Am J Physiol Endocrinol Metab 278: E452-E461, 2000;
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Vol. 278, Issue 3, E452-E461, March 2000

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
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  		Table 1.   Methysergide treatment and isotope infusion protocols

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 (VCO2). 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.

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 alpha -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 (VCO2) was calculated from NaH14CO3 infusion (27) as follows: VCO2 = 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 × VCO2)/(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).

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
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  		Table 2.   Physiological responses of rabbits

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.

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 VCO2 (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.

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.

                              
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  		Table 3.   Concentration and enrichment of phenylalanine


                              
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  		Table 4.   Protein kinetics in the ear skin


                              
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  		Table 5.   Phenylalanine transport data in the ear skin

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.

                              
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  		Table 6.   Fractional synthesis rates of skin protein

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
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RESULTS
DISCUSSION
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4.   Clark, M. G., E. Q. Colquhoun, S. Rattigan, K. A. Dora, T. P. D. Eldershaw, J. L. Hall, and J. Ye. Vascular and endocrine control of muscle metabolism. Am. J. Physiol. Endocrinol. Metab. 268: E797-E812, 1995[Abstract/Free Full Text].

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Am J Physiol Endocrinol Metab 278(3):E452-E461
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