a b s t r a c t

Objectives: The objective of this study is to evaluate the diagnostic value of real-time 3-dimensional contrast- enhanced ultrasound in the hemorrhage of blunt renal trauma.

Methods: Eighteen healthy New Zealand white rabbits were randomly divided into 3 groups. Blunt renal trauma was performed on each group by using minitype striker. Ultrasonography, Color Doppler flow imaging, and contrast-enhanced 2-dimensional and real-time 3-dimensional ultrasound were applied before and after the strike. The time to shock and blood pressure were subjected to statistical analysis. Then, a comparative study of ultrasound and pathology was carried out.

Results: All the struck kidneys were traumatic. In the ultrasonography, free fluid was found under the renal capsule. In the color Doppler flow imaging, Active hemorrhage was not identified. In 2-dimensional contrast- enhanced ultrasound, active hemorrhage of the damaged kidney was characterized. Real-time 3-dimensional Contrast-enhanced ultrasound showed a real-time and stereoscopic ongoing bleeding of the injured kidney. The wider the hemorrhage area in 4-dimensional contrast-enhanced ultrasound was, the faster the blood pressure decreased.

Conclusions: Real-time 3-dimensional contrast-enhanced ultrasound is a promising noninvasive tool for stereoscopically and vividly detecting ongoing hemorrhage of blunt renal trauma in real time.

(C) 2013

Introduction

Blunt trauma is one of the chief killers among young adults in most countries in the world. As for blunt trauma, obscure symptom has increased the difficulty of diagnosis. Ongoing hemorrhage has been a significant signal in the diagnosis of blunt trauma and also usually been one of the main causes of death in blunt trauma patients. Thus, effective methods in diagnosis of hemorrhage, especially active hemorrhage, are important in the treatment of blunt trauma [1-4].

Clinically, contrast-enhanced computed tomographic scanning and angiography are often performed in the diagnosis of blunt trauma [5-7]. With the increasing data on the harmful effects of radiation [8-11], new imaging modalities are prompted to operate. Ultrasonography acting as a nonradioactive, rapid, reliable tool for the identification of free fluid with high specificity plays an increasing role in blunt trauma diagnosis [12,13]. However, it has difficulty in detecting active hemorrhage. With the development of technologies, many new

rabbit models of ongoing hemorrhage”>? Funding source: This study was supported by grants from Military Medical Scientific Research Plan Project (CWS12J076) and Guangdong Provincial Science and Technology Projects (2011B080701019, 2012B031800309).

* Corresponding author.

E-mail address: [email protected] (Y.-K. Li).

Sonographic methods such as contrast-enhanced ultrasound and 3-dimensional contrast-enhanced ultrasound have been put into use clinically, and such technologies have been reported to be successful in detecting the active bleeding in blunt abdominal trauma [14-16].

Recently, real-time 3-dimensional contrast-enhanced ultrasound (RT3D-CEUS) has been introduced as an advancement in sonographic technology [17]. It not only has RT3D-CEUS-rendered image informa- tion but also can study the injured lesion of blunt abdominal solid organs in 3 orthogonal planes and obtain information about the 3-dimensional vessel architecture in real time by glass body rendering, which provides new diagnostic information, although there are few reports on the diagnosis of active hemorrhage of blunt trauma by using this new imaging technique. Therefore, the objective of this study is to evaluate the RT3D-CEUS in detecting active hemorrhage of blunt renal trauma in a rabbit model, which will provide support for this application in diagnosis of blunt trauma in humans.

Methods

Rabbit models of ongoing hemorrhage of blunt renal trauma

Blunt renal trauma mode was obtained by the striking of self- made minitype striker (patent no. 201220162205.6; Fig. 1). It

0735-6757/$ - see front matter (C) 2013 http://dx.doi.org/10.1016/j.ajem.2013.06.013

Ultrasound imaging“>computed in accordance with a formula as follows,3.86 N/mm), strike top, and trigger.

k 1/4 G x d⁴ / 8

3

x MD x Nc

Fig. 1. Diagram of striker: arrow 1: pressure regulating screw; arrow 2: strike spring; arrow 3: strike top; arrow 4: trigger.

consists of pressure regulating screw, strike spring (rigidity modulus, 8000 kg/mm²; spring coil diameter, 1.5 mm; external diameter, 11 mm; internal diameter, 8 mm; intermediate diame- ter, 9.5 mm; effective number of circles, 15; spring constant was

where k is spring constant, G is rigidity modulus, d is diameter of the spring coil diameter, MD is intermediate diameter, and Nc is the effective number of circles.

To obtain different injury grades, the length of spring in the striker was adjusted to 20, 27.5, and 35 mm with a fixed strike depth of 10 mm. According to Hooke’s law (F = kx, F is the force of the strike, k is the spring constant, x is the length of spring), 77.2, 106.2, and

135.1 N were carried out among A, B, and C groups, respectively. This animal investigation was approved by the Institutional

Animal Care and Use Committee of Liuhuaqiao Hospital. Eighteen New Zealand white rabbits (weight, 3.0 +- 0.5 kg; age, 30.5 +- 1.0 weeks) were provided by the Animal experimental center of Liuhuaqiao Hospital. Male and female were not restricted randomly and evenly divided into 3 groups (A, B, and C).

The experimental rabbits were anesthetized with 3% pentobarbital sodium (Chinese medicine Shanghai chemical reagent company, Shanghai, China) through the auricular vein of the unilateral ear in which intravenous indwelling needle (22 G) acted as an injection channel. To achieve the model of active hemorrhage, 200 U/kg of heparin sodium was intravenously injected. Blood pressure was monitored through arteria femoralis. Dynamic observation of blood pressure was performed as soon as the experiment started. The fur of unilateral renal region was shaved. The animal was fastened on the plank and struck by minitype striker with different dynamics.

Ultrasound imaging

All studies were performed using a RIC 5-9 D sonographic scanner with high-resolution 5-9 MHz transducers (Voluson E8 Expert; GE

Fig. 2. A, Ultrasound displays local hyperechogenicity in the renal parenchyma (arrow1) and free fluid under renal capsule (arrow2). B, There is no signal of active hemorrhage in CDFI. C. Two-dimensional contrast-enhanced ultrasound shows irregular filling defect in the internal of kidney (arrow) after 11 seconds of injection of contrast agent. D, Filling defect was filled with enhance echo (long arrow), which were divided into 3 beams (short arrow).

Fig. 3. A, Real-time 3-dimensional contrast-enhanced ultrasound displays irregular Filling defects in the internal of kidney (arrow) after 11 seconds of injection of contrast agent, and the area was accordance with 2D-CEUS. B, Filling defect was filled with enhanced echo (long arrow), which were divided into 3 beams (short arrow) being accordance with 2D-CEUS. C, Pathologic examination reveals the lesion of kidney on the surface (arrow). D, Pathologic examination reveals the lesion of kidney in the interval (arrow).

Healthcare, Milwaukee, WI). The system uses code harmonic technology for US. Its mechanical indices ranged from 0.6 to 1.0.

Two-dimensional CEUS (2D-CEUS) and RT3D-CEUS were per- formed with a Bolus injection of 0.2-mL SonoVue (Bracco, Italy) and immediately followed by an infusion of 5.0-mL saline solution via a 20-G peripheral intravenous catheter under code pulse inversion mode with a low mechanical index (0.11-0.14) [18]. Each lasted 3 minutes. For RT3D-CEUS data acquisition, the probe was positioned on the surface of the injured side of the body to check the kidney. The data were stored digitally in the hardware of the machine and then reviewed and analyzed.

Ultrasound, then color Doppler flow imaging (CDFI), then 2D- CEUS, and RT3D-CEUS were applied before and after the strike, respectively. All of the above examinations were finished within 20 minutes after the strike.

The RT3D-CEUS data were reviewed and analyzed. The maximum lengths of the filling defects on the surface of the injured kidneys were measured. When hemorrhage was detected, the sonographer adjust- ed angles and rotated planes to a suitable section that showed the largest extent of bleeding and measured this.

Injury assessment

Animals were sacrificed by an intravenous injection of potassium chloride after their blood pressure declined below 40 mm Hg. Histopathologic analyses were performed. The maximum lengths of the lesions on the surface of the kidneys corresponding to the filling defects showed in RT3D-CEUS were measured. The injured kidney was graded by the reviewing surgeon independently in accordance with the American Association for the Surgery of trauma guidelines [19].

Data analysis

A senior sonographer performed the above imaging examinations. Another one reviewed the images. They were blinded to the category

of strike force. The ultrasound results were analyzed and compared with the results of pathology. Blood pressure was recorded every 10 minutes, and duration of shock was calculated by mean +- SD.

Results

Before strike, in US, internal structure of kidney was identified vividly. In CDFI, the renal vessels were showed clearly. In 2D-CEUS and RT3D-CEUS, no abnormal enhanced signal was found.

After the strike, US showed that the kidney was swelling immediately, and part of parenchyma was hyperechogenicity. Free fluid was identified under renal capsule and had an increasing trend, which had an expression of Uniform distribution of focus (Fig. 2A). However, the information of position and the extent of active

Fig. 4. Blood pressure decreased with the lost of blood. The larger the hemorrhage area in RT3D-CEUS was, the faster the blood pressure decreased.

hemorrhage could not be identified. There was no signal of active bleeding in CDFI, and the renal blood signal disappeared in the suspicious lesion area (Fig. 2B).

In our study, we found that, in all cases within 10 to 12 seconds after injection of contrast agent, 2D-CEUS showed wedge-shaped and irregular enhanced area in the kidney, which presented an obvious difference with normal renal cortex (Fig. 2C). After 13 +- 2 seconds of injection of contrast agent, consistent enhanced signal gushed from the injured site (Fig. 2D).

We also found that, in all cases, after 10 to 12 seconds of injection of contrast agent, RT3D-CEUS showed that the injured site of the kidney was enhanced with irregular filling defect (Fig. 3A). The pictures of the renal lesions were displayed intuitively. The maximum lengths of filling defects in the 3 groups were 15.1 +- 1.8 mm of group A, 18.2 +- 2.1 mm of group B, and 22.9 +- 2.8 mm of group C. Several small groups of bleeding signals gushed from the damaged tissue in real time, and this came to a confluence under the capsule (Fig. 3B). The bleeding was strip shaped or cloud shaped. Real-time 3-dimensional contrast-enhanced ultrasound depicts a more stereo- scopic and panoramic image than 2D-CEUS. The maximum lengths of hemorrhage area in the 3 groups were 14.6 +- 1.6 mm of group A,

16.5 +- 1.9 mm of group B, and 20.5 +- 3.2 mm of group C. In our experiment, the larger the hemorrhage area in RT3D-CEUS was, the faster the rabbits went into shock state (Fig. 4). The times of shock were 247 +- 14 minutes in group A, 103 +- 9 minutes in group B, and 50 +- 10 minutes in group C.

According to the American Association for the Surgery of Trauma, pathologic examination revealed that the injury severity scale of groups A, B, and C was as follows: 2 cases in group A of grade I, 4 cases of grade II; all cases in group B of grade III; 1 case in group C of grade III, 5 cases of grade IV (Fig. 3C and D). The maximum lengths of the lesions on the surface of the kidneys were 14.8 +- 2.4 mm of group A,

19.5 +- 2.6 mm of group B, and 23.7 +- 3.3 mm of group C.

Discussion

Ultrasound is widely accepted as an integral part of the primary examination for patients with trauma. It has been shown to be effective in identifying hemoperitoneum. However, it cannot show the ongoing hemorrhage. Contrast-enhanced ultrasound is sensitive to diagnose the hemorrhage and its origin in the injured abdominal solid organs, and our previous study has testified its value in the diagnosis of active bleeding in Blunt hepatic trauma vs unenhanced ultrasound [20]. However, a limitation of information coming from sectional plane makes it unable to provide a comprehensive visualization of the hemorrhage. Three-dimensional contrast-en- hanced ultrasound acquires a volume of the enhanced capabilities of sonographic imaging, which the operator can use for retrospective image analysis as well as to render 3-dimensional sonographic image from a variety of rendering algorithms. Based on 2D-CEUS, this technology effectively eliminates many limitations and presents the information more comprehensively. It can provide spatial relation- ships of anatomical and pathologic features. It has been shown to be useful in evaluating the intra-Abdominal hemorrhage. However, its Image quality is easily affected by the movement of organs and breath. Real-time 3-dimensional contrast-enhanced ultrasound proven in our study could present the active bleeding of renal trauma vividly as well as provide real-time and 3-dimensional information. If the renal tissues are damaged and swollen, blood overflows from the wound around the kidney. If the capsule is ruptured, blood will flow into retroperitoneal space even into peritoneal cavity after the strike. The wounded lesions appear as slightly sporadic hyperechoic or anechoic areas, and the swelling kidney can also be presented in the conventional US; a girdle-shaped fluidity density at peri-kidney area indicates active bleeding while the baseline US may not discover the signal. The decrease of the renal perfusion after the strike may be the

result of the activation of fight-or-fight response. Two-dimensional contrast-enhanced ultrasound can only provide 2-dimensional infor- mation; some important slices may be theoretically missed, and it depends on the expertise of operator. Real-time 3-dimensional contrast-enhanced ultrasound contains all the information in 2D-CEUS and lessens the manipulator’s burden in missing the information, and it provides a more comprehensive visualization than ultrasonic images. In our study, it presented ongoing hemor- rhage of renal trauma vividly in the cases as well as provided real-time and 3-dimensional information.

In our experiment, we found that the wider the hemorrhage scale was in RT3D-CEUS, the faster the blood pressure decreased. To achieve ideal animal models, the experimental rabbits were injected with heparin sodium before strike. So after the strike, once there was bleeding, it would last until the animal died. We established several renal injury grades; 2D-CEUS showed irregular anechoic area in the renal parenchyma with clear margin, but 2D-CEUS is limited in offering integrated bleeding signals. Fortunately, the RT3D-CEUS achieves that. The extent of bleeding had a close relationship with the pressure as mentioned above.

We also discovered in the experiments that RT3D-CEUS displayed the origin of bleeding real timely and stereoscopically. Blood flowed out of the damaged renal vessels especially arteries after the kidney was hurt. The traumatic area in baseline US showed ill-shaped and irregular margin in renal parenchyma, while doctors usually cannot find the bleeding origin. Two-dimensional contrast-enhanced ultra- sound could show the lesions demonstrated as unenhanced defects in artery phase, after that, instantaneous extravasation of the microbubbles indicated the active bleeding. Two-dimensional contrast-enhanced ultrasound is unable to provide sufficient spatial information. Real-time 3-dimensional contrast-enhanced ultra- sound stereoscopically presents the anechoic enhancement in the traumatic parenchyma at initial phase, and RT3D-CEUS could display bleeding information dynamically, 3-dimensionally, and in real-time, thus making it easier to diagnose the bleeding origin. Real- time 3-dimensional contrast-enhanced ultrasound had indicated its value in rabbit models in renal trauma about the active hemorrhage, which was reliable and obviously specific. Real-time 3-dimensional contrast-enhanced ultrasound proven in this study can provide more important information to estimate the injury grades for clinicians.

Yet, imaging in our experiment was done with a high-resolution 5-9 MHz probe. This would not be possible in an adult patient. The results with the improvements in resolution seen with the higher frequency probes in a smaller animal (rabbit) may give outcomes that are not applicable to the adult human where imaging would have to be done with a lower frequency probe due to the greater depth of the kidney in the human.

To sum up, RT3D-CEUS is a promising noninvasive tool for stereoscopically and vividly detecting ongoing hemorrhage of blunt renal trauma in real time, and its clinic value is ready to be further studied.

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