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im Erbil,
Yasemin Ozluk,
Murat Giri,
Artur Salmasl
oglu,
Halim Issever,
Umut Barbaros,
Yersu Kapran,
Selçuk Özarma
an and
Serdar Tezelman
Departments of General Surgery (Y.E., U.B., S.O., S.T.), Pathology (Y.O., Y.K.), Biochemistry (M.G.), Radiology (A.S.), and Public Health (H.I.), Istanbul Medical Faculty, Istanbul University, 34093 Capa/Istanbul, Turkey
Address all correspondence and requests for reprints to: Yeim Erbil, Department of General Surgery, Istanbul Medical Faculty, Istanbul University, 34093 Capa/Istanbul, Turkey. E-mail: yerbil2003{at}yahoo.com.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Objective: The aims of this clinical trial are to evaluate thyroid blood flow and microvessel density in patients with Graves’ disease who received Lugol solution treatment preoperatively.
Design: This was a prospective clinical trial.
Setting: This clinical trial took place at a tertiary referral center.
Method: Thirty-six patients were randomly assigned to receive either preoperative treatment with Lugol solution (group 1, n = 17) or no preoperative treatment with Lugol solution (group 2, n = 19).
Main Outcome Measures: Blood flow through the thyroid arteries of patients with Graves’ disease was measured by color flow Doppler ultrasonography. The microvessel density (MVD) was assessed by immunohistochemical and Western blot analysis of the level of expression of CD-34 in thyroid tissue. The weight and blood loss of the thyroid gland were measured in all patients.
Results: The mean blood flow, MVD, CD-34 expression, and blood loss in group 1 patients were significantly lower than those in group 2 patients. There was a negative correlation between Lugol solution treatment and blood flow (rs = –0.629; P = 0.0001), blood loss (rs = –0.621; P = 0.0001), MVD (rs = –0.865; P = 0.0001), and CD-34 expression (rs = –0.865; P = 0.0001). According to logistic regression analysis, Lugol solution treatment resulted in a 9.33-fold decreased rate of intraoperative blood loss.
Conclusion: Preoperative Lugol solution treatment decreased the rate of blood flow, thyroid vascularity, and intraoperative blood loss during thyroidectomy.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Preoperative treatment with Lugol solution for 10 d blocks the release of thyroid hormone and has the additional advantage of decreasing the vascularity of the thyroid gland (4, 5, 6, 7). Lugol solution causes a decrease in thyroidal iodide uptake, a decrease in iodide oxidation and organification, and a block in the release of thyroid hormones (3, 4). Although some endocrine surgeons administer Lugol solution to decrease thyroid gland vascularity, there is still not an agreement on its effectiveness (6, 7, 8, 9). Because studies are mostly based on empirical or indirect evaluations, such as the impression of the surgeon, objective results of the effects of Lugol solution on the gland are difficult to achieve (4, 5, 6, 7, 8, 9).
The availability of color flow Doppler ultrasonography (CFDS), a rapid, informative, and non-invasive technique, has recently allowed direct measurement of intrathyroidal blood flow and thyroid artery blood flow (10, 11). Several studies revealed that thyroid vascularity increased in patients with GD (10, 11, 12, 13, 14).
Angiogenesis is defined as the development of new blood vessels, and it is observed in various physiologic and pathologic conditions (15). Angiogenesis also occurs in the thyroid during disease processes, including goiter, GD, thyroiditis, and carcinoma (15, 16). Measurement of microvessel density (MVD) has been used to investigate angiogenesis in various tissues. MVD can be evaluated by immunohistochemistry and Western blot analysis. Vascular density has been most commonly assessed by counting vessels labeled using immunohistochemistry with antibodies to CD-34. The endothelial cells were highlighted by anti-CD-34, and the numbers of microvessels in the hotspots were counted (15, 16).
The aims of this clinical trial are to evaluate thyroid blood flow and MVD in patients with GD who received Lugol solution treatment preoperatively and to suggest that Lugol solution treatment might effect the reduction of thyroid vascularity and intraoperative blood loss. To our knowledge, this is the first study to evaluate the effect of Lugol solution treatment using immunohistochemistry and Western blot analysis.
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Patients and Methods |
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This prospective study included 36 consecutive patients undergoing surgical therapy for GD at the Department of General Surgery, Istanbul Medical Faculty, between September 2005 and September 2006. Patients were randomly assigned to preoperative treatment with Lugol solution. Patients in group 1 (n = 17) received Lugol solution for 10 d before surgical intervention, whereas patients in group 2 (n = 19) did not receive it. Lugol solution was administrated at 10 drops per day orally three times a day for 10 d. The patients in group 1 immediately underwent operations after these 10 d.
The diagnosis of GD was based on a history and signs of hyperthyroidism, elevated free and total thyroid hormone concentrations, undetectable or reduced levels of TSH, the presence of thyroid antibodies in the serum, and increased iodine uptake or diffuse uptake on a thyroid scan on admission.
Patients with GD were treated with an initial dose of propranolol (40–60 mg/d), propylthiouracil (PTU) (300–400 mg/d), or methimazole (MMI) (10–30 mg/d). These initial doses were reduced gradually to maintain euthyroidism as serum thyroid hormone concentrations declined. All patients were euthyroid before their operations.
The indications for surgical treatment were as follows: recurrent disease after antithyroid treatment (n = 11), recurrent disease in association with a large goiter (n = 15), severe Graves’ ophthalmopathy (n = 6), patient preference (n = 2), or findings suggestive of malignancy in a fine-needle aspiration biopsy (n = 2). Exclusion criteria were as follows: anticoagulant usage, a previous thyroid operation, and refusal to participate in this study.
Total and near total thyroidectomies were performed by the same surgeons in all patients. The volume of blood loss, which was measured as the amount of blood in the suction bottle and that absorbed by the gauze, was recorded for each patient. After thyroidectomy, a 1 x 1 cm of the tissue was excised and rinsed with ice-cold PBS. The tissue samples were immediately frozen and stored –80 C until they were used in studies. The remaining thyroidectomy specimens were subject to histopathological examination. Randomization and administration of Lugol solution were performed by residents. The surgical team did not know the randomization details. All patients were operated on by the same surgeons who were blinded to the randomization and to patient details.
In all patients, indirect laryngoscopic examination was used to evaluate vocal cord motility both before and after surgery. In cases of dysphonia with vocal cord injury, indirect laryngoscopy was also performed 1 and 6 months later. Persistent nerve palsy was defined as persistent dysfunction and clinical dysphonia that lasted for 6 months postoperatively. Hypocalcemia was defined as a serum calcium level less than 8 mg/dl after the operation. The presence of clinical symptoms or signs of hypocalcemia were noted. Persistent hypoparathyroidism was defined as serum PTH levels less than 10 pg/ml in patients requiring treatment for more than 3 months to maintain normocalcemia. The study plan was reviewed and approved by our institutional ethical committee, and informed consent was obtained for all patients.
Vascular studies
CFDS (Sonoline Antares; Siemens, Erlangen, Germany) using a high-frequency wideband linear transducer (frequency range, 7.3–11.4 MHz) was performed before treatment of group 1 patients with Lugol solution. Vascular studies were also performed on all patients 24 h before surgery. Thirty age- and sex-matched healthy medical staff volunteers were also analyzed using CFDS; they served as healthy control subjects. Each color Doppler examination was performed blindly by the same investigator who was unaware of the laboratory values at the time of the examination.
Thyroid volume was measured by ultrasound and calculated using the ellipsoid model (width x length x thickness x 0.5233 for each lobe) (17). The superior and inferior thyroid arteries on each side of the neck were located first, and then peak systolic and diastolic velocities, vessel diameter, and the flow volumes were calculated using measurement tools installed within the Doppler unit. The study evaluated the resistance index (RI) of the four main thyroid arteries, sampled near their entrance into the gland. The RI was evaluated according to the following formula: (peak systolic velocity/end diastolic velocity)/peak systolic velocity (18, 19). A mean thyroid RI and blood flow rate were calculated from 12 measurements, with four for each artery.
Biochemical analysis
Thyroid hormone and thyroid antibody concentrations were determined by a chemiluminescent assay using the DPP Modular System (Hoffmann-La Roche, Basel, Switzerland). Normal ranges of the measured parameters were as follows: free T3 (FT3), 2.6–5.7 pmol/liter; free T4 (FT4), 9–18 pmol/liter; TSH, 0.3–4.9 mIU/liter; anti-thyroid peroxidase (TPO), 0–35 IU/ml; and TSH receptor antibody (TRAb), less than 10 IU/ml.
Western blot analysis
Thyroid issues were homogenized in 10 volumes of 50 mM Tris HCl (pH 7.4), 0.25% Na-deoxycholate, 10% SDS, 150 mM NaCl, 1 mM EDTA, 1% glycerol, 1% Triton X-100, 5 mM NaF, 2 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml soybean trypsin inhibitor. The homogenates were sonicated for 15 sec, passed through a 22-G needle, and then centrifuged at 15,000 x g at 4 C for 20 min. The supernatants were processed for the determination of total protein concentration using bicinchoninic acid (20). SDS-PAGE of the protein in the homogenates was performed using the Bio-Rad (Hercules, CA) Mini Protean III gel system according to Laemmli’s method (21). Equal amounts of protein from each sample (60 µg/well) were loaded onto a 7.5% SDS-PAGE gel. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked with PBS-Tween 20 (PBS-T) containing 5% nonfat dry milk overnight at 4 C. Membranes were incubated with mouse monoclonal anti-CD-34 at a 1:1000 dilution (GTX28536; Genetex, San Antonio, TX) and then with horseradish-peroxidase-conjugated secondary antibody (goat antimouse IgG-HRP; SC-2005; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:3000 dilution. After imaging, the membranes were stripped of primary and secondary antibodies by incubation in a solution of 0.2 M glycine and 0.05% Tween 20 (pH 2.5) for 30 min at 80 C. They were rinsed in PBS-T and reblocked in 5% nonfat dry milk in PBS-T. After blocking, membranes were reprobed using monoclonal anti-ß-tubulin antibody as the primary antibody (SC-5274; Santa Cruz Biotechnology). ß-Tubulin serves as an internal control for equal protein loading.
Immunohistochemistry
After an initial review of all available hematoxylin and eosin-stained slides of the surgical specimens, we selected one representative paraffin block from each case for additional study. Consecutive 5-mm sections were recut from each block. One section from each case was stained with hematoxylin and eosin, and additional sections were immunostained for CD-34. A standard avidin-biotin-peroxidase complex method was applied, and antigen retrieval was performed by pressure heating in citrate buffer. The primary monoclonal antibody was a monoclonal mouse anti-CD-34 (incubation for 60 min; RTU ready-to-use; clone QBEnd/10; catalog no. MS-363-R7; NeoMarkers, Fremont, CA). 3-Amino-9-ethyl carbozole was used as a chromogen, and counterstaining was performed with Mayer’s hematoxylin. Vessel counting was performed using x4, x10, x20, and x40 achrometric objectives and x10/20 left oculars on an Olympus Optical (Tokyo, Japan) BX51 microscope. Stained individual endothelial cells or endothelial cell clusters that were clearly separate from adjacent microvessels, follicular epithelial cells, and other connective tissue elements were regarded as capillary, even in the absence of lumen. Slides were scanned at low magnification (x40) to identify the most vascular areas (hotspots). In each case, capillaries in the hotspots were counted per field of view at x400 magnification (1.1 mm2) in five consecutive fields. The average MVD counts were recorded. Vessel counts were blindly assessed by two pathologists who were unaware of any clinical data before counting.
Statistical analysis
Data are reported as the mean ± SD. Analysis was performed with the statistical package SPSS 10.1 (SPSS, Chicago, IL). Differences between parameters were compared with the t test, Mann-Whitney U test, Wilcoxon’s test, and 
2 test. Relationships among parameters were tested by the Spearman’s correlation coefficient. Receiver operating characteristic (ROC) curves were designed to identify the cutoff values for age, thyroid hormone, thyroid antibodies, thyroid gland volume, MVD, blood flow, and CD-34 expression to determine the efficiency of Lugol solution. The predictive factors for intraoperative blood loss were evaluated by logistic regression analysis. Stepwise logistic regression analysis was performed with intraoperative blood loss as the dependent variable. The following continuous variables were selected for the stepwise regression model: age, gender, FT3, FT4, THS, anti-TPO, TRAb, Lugol solution usage, thyroid gland volume, MVD, blood flow, and CD-34 expression. Study parameters that had non-Gaussian distributions were transformed to log values. Results were considered statistically significant at P < 0.05.
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Results |
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The mean age of the whole group was 41.9 ± 11 yr (range 14–61 yr). The female/male ratio was 5:1 (n = 30 and 6). The mean serum FT3, FT4, TSH, anti-TPO, and TRAb levels were 4.3 ± 0.6 pmol/liter, 13.9 ± 1.9 pmol/liter, 2.1 ± 1.1 mIU/liter, 234 ± 67 IU, and 374 ± 48 IU, respectively. Of 36 patients, 20 patients were treated with PTU and 16 patients were treated with MMI. Mean doses of PTU and MMI were 350 ± 50 and 23 ± 7 mg/d, respectively. The mean thyroid gland volume, blood flow, RI, MVD, CD-34 expression, and intraoperative blood loss were 50 ± 28 ml, 103.17 ± 44 ml/min, 0.49 ± 0.09%, 91.34 ± 45 vessels, 83.31 ± 30 U, and 83 ± 50 ml, respectively. The normal controls consisted of 30 healthy subjects (7 males, 23 females) aged 19–61 yr (mean of 40.3 ± 10 yr). There was no operative mortality. The incidence of transient vocal cord paralysis and hypoparathyroidism were 5.5 and 11.1%, respectively. Persistent vocal cord paralysis and hypoparathyroidism were not encountered in our series. Histopathological examination revealed GD in all patients.
Effect of Lugol solution treatment on Doppler evaluations
The mean ages were 40.7 ± 10 yr in group 1 and 42.9 ± 11 yr in group 2. The female/male ratios were 14:3 and 16:3 in groups 1 and 2, respectively. There was not a significant difference between age, gender, the ratio of ophthalmopathy, the choice of antithyroid drugs, preoperative serum thyroid hormone levels, thyroid antibodies, or thyroid gland volumes between groups 1 and 2 (Table 1
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). There was not a significant difference between thyroid gland volume and thyroid hormone levels in patients before and after treatment with Lugol solution (Table 1
).
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The mean blood flow and blood loss in the group 1 patients (74.7 ± 26 ml/min and 54.4 ± 22 ml) were significantly lower than those in the group 2 patients (128.62 ± 42 ml/min and 108.68 ± 54 ml; P = 0.0001). The mean RI in the group 1 patients of (0.60 ± 0.02%) was significantly higher than that of the group 2 patients (0.40 ± 0.01%; P = 0.0001) (Fig. 2).
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). The mean MVD by immunohistochemistry in the group 1 patients (56.80 ± 19 vessels) was significantly lower than that of the group 2 patients (122.25 ± 39 vessels; P = 0.0001) (Fig. 4).
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There was a negative correlation between Lugol solution treatment and blood flow (rs = –0.629; P = 0.0001), blood loss (rs = –0.621; P = 0.0001), MVD (rs = –0.865; P = 0.0001), and CD-34 expression (rs = –0.865; P = 0.0001). Moreover, there was a negative correlation between the RI and CD-34 expression (rs = –0.765; P = 0.0001) and MVD (rs = –0.788; P = 0.0001). A significant positive correlation was found between Lugol solution treatment and RI (rs = 0.869; P = 0.0001) (Fig. 5). There was a positive correlation between blood loss and thyroid volume (rs = 0.779; P = 0.0001), blood loss and blood flow (rs = 0.928; P = 0.0001), blood loss and CD-34 expression (rs = 0.385; P = 0.02), and blood loss and MVD (rs = 0.505; P = 0.002), as well as between blood flow and thyroid volume (rs = 0.745; P = 0.0001), blood flow and CD-34 expression (rs = 0.410; P = 0.01), and blood flow and MVD (rs = 0.442; P = 0.007) (Fig. 6).
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ROC curve analysis demonstrated a significant relationship between Lugol solution treatment and blood flow (area under the curve, 0.136; P = 0.0001), MVD (area under the curve, 0.995; P = 0.0001), CD-34 expression (area under the curve, 0.996; P = 0.0001), and blood loss (area under the curve, 0.858; P = 0.0001). The cutoff values derived from the ROC curves for the best sensitivity and specificity were 80.6 ml/min, 89 vessels, 93 U, and 67.5 ml for blood flow, MVD, CD-34 expression, and blood loss, respectively. Lugol solution treatment resulted in a 15.5-fold [odds ratio (OR), 15.5; 95% confidence interval (CI), 2.6–91.5] decrease in the rate of blood flow, a 21-fold (OR, 21; 95% CI, 3.4–126.32) decrease in the rate of blood loss, a 44.8-fold (OR, 44.8; 95% CI, 4.65–430.90) decrease in the rate of MVD, and a 60-fold (OR, 60; 95% CI, 6.0–599.49) decrease in the level of CD-34 expression.
Stepwise regression analysis
When age, gender, thyroid hormone, thyroid antibodies, blood flow, MVD, CD-34 expression, and Lugol solution treatment were included as independent variables, Lugol solution treatment (OR, 9.33; 95% CI, 1.13–76.67; P = 0.03) was found to be the only significant independent determinant of intraoperative blood loss. Lugol solution treatment resulted in a 9.33-fold decrease in the rate of intraoperative blood loss.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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GD is one of the most common causes of hyperthyroidism (1, 2). Hyperthyroidism is associated with hemodynamic changes, including high output state, increased heart rate and cardiac contractility, and decreased peripheral resistance that are related to both direct cardiostimulatory effects of thyroid hormone and increased peripheral oxygen consumption (2, 3). Preoperative preparation of the patient is crucial to avoid intraoperative or postoperative thyroid storms and to decrease the vascularity of the gland (1, 2, 3, 4).
Inorganic iodide (Lugol solution) decreases the synthesis of thyroid hormone and release of hormone from the thyroid in the short term. It is used to treat patients with thyroid storm or, more commonly, to reduce thyroid vascularity before thyroidectomy (3, 4). Iodine also reduces thyroid cellularity and vascularity and therefore is used in the preparation of patients for thyroidectomy (5, 6, 7). This effect transiently blocks thyroid hormone generation, with thyroid hormone synthesis recovering in a few days or weeks. There is no consensus that reductions in vascularity, glandular friability, and blood loss are obtained preoperatively (6, 7, 8). Most studies related to the effects of Lugol solution are based on indirect evaluations, such as the impression of the surgeon (5, 6, 7, 8, 9).
Doppler techniques seem to be the best method to evaluate blood flow in the thyroid gland, because thyroid vascularity has been quantitatively assessed by counting the number of vessels per square centimeter in the longitudinal plane and by measuring volumetric blood flow in the thyroid artery (10, 11, 12, 22). Higher intrathyroidal blood flow and increased peak systolic velocity have been documented in GD (11, 12, 23). In this study, the blood flow of thyroid arteries was higher than the values obtained in control subjects. Ansaldo et al. (9) aimed to evaluate the effects of Lugol solution therapy in a series of patients with GD through analysis of the RI of thyroid arteries. After Lugol solution treatment, RI reached a mean value similar to that of the normal subjects reported our results. RI is a semiquantitative, easy-to-obtain, and reproducible measurement reflecting the vascular impedance within the vascular bed distal to the site of sampling. Changes in vascular impedance after drug administration have been widely studied using this method. Chang et al. (7) demonstrated a reduction of blood volume flow within the superior thyroid arteries after Lugol solution treatment in patients with Graves’ hyperthyroidism. In our study, Lugol solution treatment resulted in a significant decrease of the mean basal blood flow in patients with GD relative to values obtained before treatment.
Thyroid vascularity in GD patients can be evaluated by monitoring CD-34 expression using immunohistochemistry and Western blot analysis as well as by CFDS (15, 16). The vascular density of different tissues has been assessed by counting vessels labeled using immunohistochemistry with antibodies against different endothelial markers on both frozen and paraffin-embedded sections. Antibodies that are most commonly used are directed against the endothelial antigens factor eight-related antigen, CD-31 (platelet endothelial cell adhesion molecule), CD-34, and the lectin ulex europaeus agglutinin 1. CD-34 is the most sensitive and specific marker currently available for the detection of angiogenesis. The use of these markers reflects total vascular density (15, 16).
In practice, immunohistochemistry is the most frequently used method for the assessment of CD-34 status. However, results can be affected by a number of variables, including the antibody that is used, the detection technique, and the use of frozen sections or paraffin-embedded tissue. The use of fresh tissue homogenates in Western blot analysis results in a higher confidence in the results compared with other methods. Yamada et al. (24) show that iodide inhibits vascular endothelial growth factor-A expression in cultured human thyroid follicles, and, consequently, they suggest that proangiogenic and antiangiogenic factors may at least partly account for the iodide-induced decrease in thyroid blood flow. Currently, there are not any published reports on the effect of Lugol solution on patients with GD using MVD and Western blot analysis. In our study, after Lugol treatment, MVD as determined by immunohistochemistry and CD-34 expression were significantly lower than values obtained before treatment. Moreover, there was a negative correlation between Lugol solution treatment and blood flow, blood loss, MVD, and CD-34 expression.
The main aim of our study was to investigate the effect of Lugol solution on vascular density in GD. We hypothesized that Lugol solution might act through two mechanisms. It probably decreases both angiogenic stimuli and blood flow in GD. Decreased angiogenesis and blood flow resulted in a significantly decreased number of vessels. Therefore, it is reasonable to presume that increased vascular density could reflect increased angiogenic stimuli in this disease.
In conclusion, preoperative Lugol solution treatment, blood flow, MVD, and CD-34 expression were found to be significant independent determinants of intraoperative blood loss. Moreover, preoperative Lugol solution treatment decreased the rate of blood flow, thyroid vascularity, and intraoperative blood loss during thyroidectomy. The reduction of intraoperative bleeding allows better visualization and preservation of the surrounding nerves, vasculature, and parathyroid glands.
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Footnotes |
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Disclosure Statement: All authors have nothing to declare.
First Published Online March 27, 2007
Abbreviations: CFDS, Color flow Doppler ultrasonography; CI, confidence interval; FT3, free T3; FT4, free T4; GD, Graves’ disease; MMI, methimazole; MVD, microvessel density; OR, odds ratio; PBS-T, PBS-Tween 20; PTU, propylthiouracil; RI, resistance index; ROC, receiver operating characteristic; TPO, thyroid peroxidase; TRAb, TSH receptor antibody.
Received January 31, 2007.
Accepted March 16, 2007.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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