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Published online before print August 18, 2004, 10.1148/radiol.2331031340
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(Radiology 2004;233:165-171.)
© RSNA, 2004

Experimental Studies

Acute Rat Lung Injury: Feasibility of Assessment with Micro-CT1

Alexander Claus Langheinrich, MD, Boris Leithäuser, MD, Susanne Greschus, MD, Susanne von Gerlach, MD, Andreas Breithecker, MD, Fritz Reinhard Matthias, MD, Wigbert Stephan Rau, MD and Rainer Maria Bohle, MD

1 From the Departments of Diagnostic Radiology (A.C.L., S.G., A.B., W.S.R.), Pathology (S.v.G., R.M.B.), and Cardiology/Angiology (F.R.M.), Justus-Liebig-University, Langhansstrasse 10, D-35392 Giessen, Germany; and Department of Internal Medicine/Angiology, University of Bochum, Bochum, Germany (B.L.). From the 2003 RSNA scientific assembly. Received August 21, 2003; revision requested October 21; final revision received March 17, 2004; accepted April 1. Address correspondence to R.M.B. (e-mail: rainer.bohle@patho.med.uni-giessen.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To evaluate the feasibility of micro–computed tomography (CT) for analysis of the lung fine structure and its alterations during endotoxin-induced lung injury.

MATERIALS AND METHODS: Intravital perfusion-fixed rat lungs with (n = 5) and without (n = 5) endotoxin perfusion were scanned with micro-CT. Three imaging modalities (conventional histology, intravital microscopy, and electron microscopy) were used to document the effect of endotoxin and the in vivo application of contrast agent (a mixture of barium sulfate, gelatin, and thymol). The effect of endotoxin on structural changes of the lung was evaluated with analysis of variance.

RESULTS: Intravital microscopy, conventional histology, and electron microscopy demonstrated capillary perfusion of contrast agent, inflated alveoli, and no extravasation of barium sulfate in the extravascular space. Systemic application of endotoxin led to a significant increase in the soft-tissue volume of the lungs (ie, tissue edema) (58.09 µm3± 4.6 [standard error of the mean] vs 8.31 µm3± 1.63, P < .001) and significant thickening of the alveolar walls (34.01 µm ± 4.5 vs 14.83 µm ± 2.5, P < .001) at micro-CT. Simultaneously, endotoxin-treated rat lungs showed a significant reduction in total air space (49.74 µm3± 1.72 vs 100.99 µm3± 1.16, P < .001).

CONCLUSION: These findings indicate that micro-CT is feasible for structural evaluation of the lung fine structure and its alterations during endotoxin-induced lung injury.

© RSNA, 2004

Index terms: Animals • Computed tomography (CT), contrast enhancement • Computed tomography (CT), technology • Computed tomography (CT), tissue characterization, 60.1211 • Experimental study • Lung, CT, 60.1211


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many techniques and methods have been developed for examining and visualizing the lung microarchitecture (1–6). The validity of these methods is being discussed, because they are often complex and protracted and may themselves alter the vulnerable tissue. Until now, most examinations of lung microarchitecture involve instillation of a fixative through the airways or vascular system. These techniques possibly alter the normal pressure relationships of the capillaries and therefore their shape and size.

Mazzone et al (7) introduced a method of rapid freezing followed by freeze-substitution fixation and found good preservation of fine structure. This method was claimed to be superior to others because it allowed careful control of physiologic conditions.

Histologic analysis, as the standard method of studying lung architecture, has different limitations—for example, serial cutting of multiple thin slices from a tissue specimen is slow and expensive, and, once sliced, the intact volume is lost so further examinations with other methods are difficult or impossible. Moreover, histologic analysis does not provide three-dimensional views of lung microarchitecture and does not allow continuous, longitudinal measurements. Only analysis of serial sections in combination with the use of an electronic three-dimensional reconstruction technique might enable one to evaluate pulmonary microarchitecture.

In the past decade, micro–computed tomography (CT) has become a powerful technique in laboratory investigation as technical advances in computer speed and memory have enabled micro-CT systems to generate high-spatial-resolution images of small specimens. Although the early investigations of micro-CT focused on the technical and methodologic aspects, more recent investigations have stressed the practical aspects of this technology. Feldkamp et al (8) established an x-ray–based micro-CT system to image a three-dimensional object with a spatial resolution of 50 µm. That technique to date has been successfully used to visualize the vasculature in intact isolated rodent organs (9) and the myocardial, renal, and hepatic vasculature (10–15) and trabecular bones (16) in surgical bone specimens. Recently, we were able to demonstrate the feasibility of x-ray micro-CT for analyzing the fetal placental villous tree (17). Results of several studies, including a relatively recent study in which excellent correlation between three-dimensional micro-CT stereologic parameters and standard two-dimensional histomorphometry was demonstrated (18), have been published.

Thus, the purpose of our study was to evaluate the feasibility of micro-CT for analysis of the lung fine structure and its alterations during pathophysiologic events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Protocol
Experiments were performed according to the German Animal Protection Law of 1993 (19). Approval of the institutional animal care and use committee of Justus-Liebig-University was obtained before the start of this study. After a 1-week period of acclimatization to the laboratory (including housing in stainless-steel cages in a temperature- and humidity-controlled room with free access to tap water and a standard laboratory animal chow), 300–400-g male Sprague-Dawley rats (Charles River, Sulzbach, Germany) were preanesthetized with inhaled 2.0-vol% enflurane (Abbott, Wiesbaden, Germany); general anesthesia was then induced with 6 mL per kilogram of body weight of urethane 20% (Ethylcarbamat 99%; Sigma, Deisenhofen, Germany) administered intramuscularly, and the rats underwent preparatory surgery. The anesthetized animals were all placed in the same vertical plane. The trachea was intubated to support spontaneous breathing. The right external jugular vein was cannulated for administration of endotoxin, volume replacement, and administration of contrast agent. Two groups of rats were allocated for micro-CT analysis.

Animals in the endotoxin group (n = 5) were infused with 0.5 mg/kg of lipopolysaccharide (Escherichia coli O55:B5, LPS; Sigma) in 1.0 mL of physiologic saline during 80 minutes (the beginning of the infusion was referred to as t = 0). The other group (the control group, n = 5) received an equal amount of physiologic saline. After the end of the lipopolysaccharide or saline infusion, all animals underwent volume replacement with 1.0 mL/kg/hr of physiologic saline until the end of the observation period. After 180 minutes and before the administration of a contrast agent, a left lateral abdominal incision was made, and a segment of the small intestine was exposed. Rats were placed on an adjustable heated inverted-camera microscope stage (Axiovert 135; Zeiss, Oberkochen, Germany), where body temperature, controlled with a rectal thermistor probe, was kept constant at 37°C. The ileocecal portion of the mesentery was fixed across an optically clear window on the stage. Animal preparation was performed by B.L. and A.C.L.

Intravital microscopy was used to clearly demonstrate that the intravenous infusion of contrast medium described below reached the microcirculation at the level of the capillaries in vivo. The technical aspects of intravital microscopy were recently described elsewhere (20). After the rats were placed on the microscope stage, a contrast agent—a barium sulfate–gelatin-thymol (BSGT) mixture consisting of 500 mL of barium sulfate (Micropaque; Guerbet, Sulzbach, Germany), 42 g of gelatin (Merck, Darmstadt, Germany), and 15 g of thymol (Merck)—was heated to 37°C and infused via the right jugular vein with constant pressure. With the beginning of infusion, the right or the left carotid artery was dissected, leading to exsanguination, replacement of nearly the entire blood volume by the contrast medium, and death. As soon as the BSGT mixture was detected in the mesenteric arteriolar branches, capillaries, and postcapillary venules (Fig 1), infusion was stopped. Intravital microscopy was performed and its results were analyzed by two experienced angiologists (B.L., with 12 years of experience with intravital microscopy, and F.R.M., with 10 years of experience).



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  		Figure 1. A-D, On successive images obtained at intravital microscopy performed after BSGT mixture infusion, contrast agent is clearly visible in the physiologic mesenteric artery (long arrow) and vein (short arrow). (Original magnification, x20.)

 
After the animals’ bodies were cooled in a refrigerator, the lungs were harvested from the chest and immediately scanned with micro-CT.

Micro-CT
Image acquisition.—Sections of lung tissue were scanned in a micro-CT unit (SkyScan1072  80 kV; SkyScan, Aartselaar, Belgium). The x-ray system is based on a microfocus tube (20–80 kV, 0–100 µA) that reaches a minimum spot size of 8 µm at 8 W for generation of projection images and that produced x-rays in a cone-beam geometric magnification. Technical aspects of this micro-CT scanner were recently described elsewhere (17). Similar systems have been described in detail before (21–23). For this system, the manufacturer gives a maximum spatial resolution of 8 µm at 10% modulation transfer function, which is an accepted method for defining the experimentally determined spatial resolution and performance of an optical system (24).

In our experimental setting, samples were positioned on a computer-controlled rotation stage and scanned 180° around the vertical axis in rotation steps of 0.45°. The tube is operated at a 60-keV peak and 100 µA. The exposure time for each view is typically 2.4 seconds. Three-dimensional volume images are reconstructed from the angular views by using a modified Feldkamp filtered back-projection algorithm (25). However, with this system, an entire rat lung (field of view, approximately 2.5 cm) may be studied, with images having typical cubic voxel dimensions as small as 5–10 µm. The opacity of each voxel is represented by a 16-bit gray-scale value.

Image reconstruction was performed with a dual IntelXeon processor (2 x 1800 MHz) (Intel, Santa Clara, Calif) equipped with 1024 megabytes of random access memory. Three radiologists (A.C.L., S.G., and W.S.R.) with 4, 3, and 20 years of experience, respectively, performed image reconstruction and analyzed the micro-CT images.

Image processing.—Image analysis was performed by using the Analyze software package version 4.0 (Biomedical Imaging Resource, Mayo Foundation, Rochester, Minn) and 3D-Calculator (Skyscan), a commercial-potential software that includes various three-dimensional visualization tools. In our study, we basically used spatial filtering, multiplanar reformations, and volume rendering. Binarized images were used for object extraction and region-of-interest measurements. Volume measurement of objects was enabled by adding the voxel size to the image header.

Quantification of Lung Tissue at Micro-CT
After the perfused lungs were cooled to 4°C, full-depth tissue samples were randomly taken from 10 different regions of the lung (two from each lobe) and cut into approximately 2-mm3 blocks.

Measurements were carefully performed by using the 3D-Calculator software. The rectangular region of interest was established manually (by A.C.L.) in the area of lung tissue within the volume of interest with a side length of 0.5 mm (0.125 mm3).

Morphometric indexes were determined directly as follows:

1. The total air volume represented the sum of all pixels marked as air after binary thresholding.

2. The total contrast agent volume (TCAV) represented the sum of all pixels marked as contrast agent after binary thresholding.

3. The total soft-tissue volume represented the sum of all pixels inside the region of interest after the TCAV was excluded with binary thresholding.

4. The soft-tissue surface (STS) represented the sum of all bordering pixels marked as soft tissue after binary thresholding and was used to calculate the median alveolar wall thickness.

5. The total volume fraction was calculated (as a percentage) as the sum of the TCAV and the total soft-tissue volume within the volume of interest.

Based on the soft-tissue surface, the median alveolar wall thickness (AVW) was calculated as

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Histologic Examination
After CT scanning was completed, the lungs were fixed in 4% neutral-buffered formalin, embedded in paraffin, sliced, and stained with hematoxylin-eosin. Histopathologic analysis was performed independently by two experienced pathologists (R.M.B., with 20 years of experience with lung histology, and S.v.G., with 8 years of experience) who were blinded to the results of micro-CT analysis.

Electron Microscopy
Small pieces of lung tissue (two per animal), each of which was 1 mm3 in size, were fixed in 2.3% glutaraldehyde (Merck) in a sodium cacodylate buffer (Merck) (pH 7.2) at 4°C for 2 hours and then transferred into a sodium cacodylate buffer. The specimens were postfixed in 1.25% osmium tetroxide (Merck) at room temperature for 3 hours. After being rinsed with cacodylate buffer and dehydrated for 20 minutes each in 20%, 50%, 70%, 80%, and 90% ethanol, the specimens were then immersed in 100% ethanol for 30 minutes, withdrawn, immersed again in 100% ethanol for another 30 minutes, and transferred into a 1:1 mixture of Spurr medium (Serva, Heidelberg, Germany) and 100% ethanol for 30 minutes (26).

Embedding was performed by using gelatin capsules (Fa. Plano, Wetzlar, Germany) in Spurr medium (100%) overnight within an exsiccator at –10 to –15 mm Hg. Polymerization was performed for 24 hours at 60°–70°C in an oven (Memmert, Schwabach, Germany). When polymerization was completed, thick slices were prepared in a routine fashion (ie, with an Autocut 1140 device [Reichert & Jung, Bensheim, Germany]) and were stained with Azur-II-Methylenblue (Merck). Tissue blocks were selected for thin slicing with an ultramicrotome (Om U2; Reichert, Vienna, Austria). Slices were then mounted on a 300 mesh hexagonal copper/palladium grid (Fa. Plano), stained with 5% uranyl acetate in methanol (Merck) for 5 minutes and Reynolds lead citrate (Merck) for 1 minute, and studied with a Zeiss Leo 906 transmission electron microscope (Zeiss, Oberkochen, Germany). Electron microscopic analysis was performed by a pathologist with 12 years of experience with electron microscopy (R.M.B.).

Statistical Analysis
All data are presented as means ± standard errors of the mean. Data were analyzed by using unpaired t testing and one-way analysis of variance to establish differences among groups. P < .05 was considered to indicate a significant difference in all analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
General Considerations
After administration of the BSGT mixture via the right jugular vein in spontaneously breathing animals, the contrast agent was detected in the mesenteric vasculature by using intravital microscopy (Fig 1), which demonstrated capillary perfusion after in vivo application of BSGT. The pulmonary arteries were opacified by BSGT, indicating that the entire lung circulation had been filled (Fig 2). Electron microscopy revealed no extravasation of BSGT into the extravascular space (Fig 3).



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  		Figure 2. A, Micro-CT maximum intensity projection shows homogeneous filling of the pulmonary arteries after in vivo perfusion of BSGT. B, Unfiltered inverted two-dimensional micro-CT image shows well-extended alveolar lung tissue with slim alveolar walls and pulmonary capillaries (arrows) containing BSGT. (For both images, section thickness = 6 µm, x-ray source acceleration voltage = 60 kV, current = 100 µA, spatial resolution = 6 µm, and magnification = x80.)

 


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  		Figure 3. A, Electron micrograph of pulmonary alveolar capillary containing vacuoles of contrast medium (CM), electron-dense barium granules (solid arrows), erythrocytes (Er), an endothelial cell (Ec), and some plasma. The capillary wall (dotted arrow) is intact. No contrast medium deposits are seen outside the capillary. (Original magnification, x4646.) B, Electron micrograph of small pulmonary artery containing vacuoles of contrast medium (CM) within the lumen. No extraluminal contrast medium can be detected. (Uranyl acetate and lead citrate stain; original magnification, x2784.)

 
The alveoli of the lungs were in an inflated state (Fig 4) owing to in vivo fixation through hardening of gelatin. Therefore, because in vivo perfusion of BSGT prevents atelectasis, the lung microarchitecture and its vascular interconnectivity could be visualized and quantified with micro-CT (Figs 2, 5).



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  		Figure 4. A, B, Photomicrographs show pulmonary parenchyma with well-extended alveolar walls and pulmonary arteries that contain large amounts of BSGT (arrow in B). B represents an enlargement of the portion of A that is circumscribed by the box. (Hematoxylin-eosin stain; original magnification of A, x25; original magnification of B, x100.)

 


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  		Figure 5. Typical inverted micro-CT images of a rat lung obtained in approximately the same position in different angles (A and C are sagittal; B and D are transverse) after intravenous infusion of BSGT and normal saline. A and B were obtained after additional infusion of endotoxin; C and D were obtained from the control group (without endotoxin infusion). Capillaries are filled with BSGT (arrow in C and D). In contrast, note the interstitial edema designated by the swollen connective tissue space in A and B and the "unsharped" capillaries (arrow in A and B) that occurred after additional intravenous challenge with 5 mg/kg of lipopolysaccharide and BSGT. (For all images, section thickness = 6 µm, x-ray source acceleration voltage = 60 kV, current = 0.12 mA, spatial resolution = 6 µm, and magnification = x80.)

 
Quantitative Micro-CT Data Analysis
In addition to enabling visual assessment of the structural changes depicted in Figure 5, micro-CT enabled the determination of morphometric indexes.

Using tomographic reconstruction algorithms, we obtained three-dimensional images (Fig 2) with micro-CT that enabled total stereoscopic visualization and continuous quantitative analysis over the entire length of the investigated sample.

Systemic application of endotoxin led to a significant increase in the soft-tissue volume of the lungs (ie, tissue edema) (P < .001) and thickening of the alveolar walls (P < .001).

Simultaneously, endotoxin-treated rat lungs showed a significant reduction in total air space (P < .001) and showed swollen, "unsharp" connective tissue spaces (Fig 5, Table). Morphologic quantitation revealed equal spatial capillary density and volume in endotoxin-challenged rats compared with density and volume in control rats (17.18 µm3± 0.76 vs 15.69 µm3± 0.5). The total volume fraction, which represents the entire soft-tissue volume, showed a 41% increase in animals treated with the endotoxin (P < .001). The Table shows the relative changes in each parameter (total air volume, total soft-tissue volume, total contrast agent volume, alveolar wall thickness, and total volume fraction) in each group at the end of observation.


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  		Results of Quantitative Computerized Digital Analysis of Micro-CT Images Obtained in Saline-infused and Endotoxin-infused Rats

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study illustrates a methodologic approach to preserving and examining the fine structure of rat lung tissue in physiologic and pathophysiologic conditions. We evaluated the ability of a BSGT mixture to pass the microcirculation after intravenous injection in vivo, together with the technical feasibility of micro-CT for assessing lung microarchitecture. In vivo application of BSGT is sufficient to prevent atelectasis of the lungs and to preserve their microarchitecture and three-dimensional interconnectivity. Furthermore, because the lungs were fixed in situ and examined immediately, the results of this study show that micro-CT can enable detection and quantification of the changes in interstitial lung water after injection of endotoxin. To our knowledge, this is the first study in which the micro-CT technique was evaluated for imaging and enabling the nondestructive quantification of lung tissue and its alterations in a standard model of endotoxin priming.

Rats were chosen for this study with reference to an established endotoxin shock model (20) and with respect to their importance in studies of pulmonary vascular remodeling (27–29) and genetics (30,31). Moreover, use of small lungs offers the advantage of the geometric magnification possible with microfocal imaging.

The implementation of novel vascular contrast agents has resulted in several new applications for micro-CT in the evaluation of microvascular anatomy (32). In vitro imaging of vascular specimens has been developed by casting the vasculature in situ with a silicon-based compound containing lead chromate (9). This approach has been used to investigate the vasculature of rat kidney (11,12), heart (10,13,15), and liver (14). Possibly owing to the convincing results for visualization and quantification of the vasculature with the above method, BSGT was never used for in vivo application. Ex vivo administration of BSGT via the pulmonary artery can be used for parameterizing the rat pulmonary arterial tree structure and vessel distensibility with micro-CT images, as reported previously (32,33). Our findings clearly demonstrate the fact that BSGT passes the microcirculation of the lung after intravenous injection and reaches the systemic arterial circulation. Thereby, the gelatin portion of BSGT fixes the vascular tree.

Microscopic examination of lung sections has been very successful for correlating pulmonary structure and function and has been established as the standard of reference for analysis of lung tissue (1–6). However, it also has its inadequacies. One problem with this approach is the difficulty in registration of adjacent slices and distortions that occur during the slicing process. Both tissue fixation through extraction of water with formaldehyde solution and mechanical treatment (grinding) involve the risk of artifacts occuring during soft-tissue histologic analysis. In addition, only a few micrometers of lung tissue can be analyzed histologically. Histomorphometric results are reported by slices, and for volumetric determinations, the results are often estimated by using interpolation. Most importantly, once sliced, the intact volume of the specimen is lost and further examinations with other methods are difficult or impossible.

Micro-CT not only enables a more complete analysis without disturbing the tissue or creating sectioning artifacts but, because it minimizes the need for data interpolation, also yields results that are probably more quantitatively accurate. Histologic analysis, including immunohistochemical staining, can be performed on the same sample after micro-CT scanning is performed. Unlike magnetic resonance imaging (34), thin-section CT (35), or near-infrared spectroscopy (36), micro-CT provides the advantage of allowing one to perform a rapid and continuous analysis over the entire length of the investigated specimen.

Systemic application of endotoxin leads to an increase in soft-tissue volume (ie, tissue edema) with thickening of the alveolar wall. This effect is due to a disturbance of the microcirculation with loss of endothelial barrier function, increased vascular permeability, and subsequent extravasation of fluid and proteins. It is based on an uncompensated interplay between endothelial and circulating blood cells and humoral mediator systems, in particular the coagulation and complement cascade.

The activation of endothelial cells is a crucial matter in a circle of cellular and humoral reactions (37). Furthermore, endothelial cells are involved in the activation of leukocytes followed by the expression of adhesion molecules, enhanced endothelial-leukocyte interaction, the release of cytokines, and the subsequent activation of the coagulation and fibrinolytic system and the complement cascade (38). The pulmonary microvascular endothelium in particular is a principal target for activated leukocytes and other components of systemic inflammatory reactions. In clinical aspects, these phenomena may also appear as a generalized vascular leak syndrome (39,40). Endotoxin-treated rats in our study showed a significant reduction in total air space and an increase in soft-tissue volume with swollen, "unsharped" connective tissue. These findings of lung edema are confirmed by published reports (6).

Using electron microscopy, we demonstrated that there is no extravasation of BSGT or its barium sulfate (Ba2SO4) component into the extravascular space. This finding is of particular interest, as this excludes a methodical error in the measurement of capillary density.

Animal models of endotoxin-induced lung injury resulting in microvascular leakage are well established (41). Assessment of lung edema with gravimetric analysis is a standard method for evaluating the severity of experimentally induced ischemia/reperfusion injury. Fehrenbach et al (42) showed that gravimetry is of minor functional importance compared with assessment with stereologic methods for the evaluation of lung injury in different experimental settings. Several techniques have been developed to evaluate these pathologic changes in endotoxin-induced lung injury, but none has proved entirely satisfactory for visualization and quantification of pulmonary edema and microarchitectural changes (41–43). The observations drawn from these various studies are not entirely consistent. This may result from substantial limitations of the different approaches.

Our study had several limitations. Our method is based on in vivo perfusion with a radio-opaque contrast medium; this can cause methodologic limitations. Thus, the state of pulmonary vessel segments is not well controlled, the pressure of BSGT infusion may be locally altered because the mixture of blood and contrast medium may cause unequal hardening, and, moreover, complete homogeneous filling with BSGT of all pulmonary capillaries cannot be ensured. Moreover, perfusion of contrast agent may cause quantitative artifacts like reactive constriction of the vascular wall after injection. In our study, micro-CT was used for quantification of vascular structures, but it has to be taken in consideration that only structures delineated with sufficient contrast material can be visualized and quantified exactly. Here, histologic analysis shows clear advantages over micro-CT. Micro-CT could be considered as an additional tool with histologic analysis that has advantages but also disadvantages. Neither of these methods can fully replace the other.

There are also several known artifacts in CT imaging (ie, cone-beam, ring, and shading artifacts), each of which can interfere with quantitative analysis (44).

Practical application: This report presents a method for in situ fixation of rat lungs with intravenous in vivo application of a BSGT mixture in combination with micro-CT scanning and three-dimensional image reconstruction for visualizing lung microarchitecture. Three-dimensional micro-CT measurements allow evaluation of diseased lung tissue. With its ability to enable measurement of diseased pulmonary microstructures, this technique may be performed in a large number of conceivable experimental or therapeutic examinations that specifically target the microarchitecture of the lung.


   FOOTNOTES
 
Abbreviation: BSGT = barium sulfate–gelatin thymol

Authors stated no financial relationship to disclose.

Author contributions: Guarantors of integrity of entire study, R.M.B., A.C.L., W.S.R.; study concepts, B.L., A.C.L., R.M.B.; study design, B.L., A.C.L., R.M.B., W.S.R.; literature research, A.C.L., B.L., S.v.G., R.M.B.; experimental studies, A.C.L., B.L., R.M.B.; data acquisition, A.C.L., S.G., R.M.B., S.v.G., F.R.M.; data analysis/interpretation, W.S.R., A.C.L., S.v.G., R.M.B., B.L., A.B.; statistical analysis, A.C.L., R.M.B., W.S.R.; manuscript preparation, B.L., A.C.L., R.M.B., S.v.G., S.G.; manuscript definition of intellectual content, A.B., R.M.B., W.S.R., B.L.; manuscript editing and final version approval, A.C.L., R.M.B., B.L.; manuscript revision/review, S.G., R.M.B., B.L., A.C.L.

A.C.L. and B.L. contributed equally to this work.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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