| Objectives:To evaluate the value of intraoperatively judging testicular viability by Color Doppler Ultrasonography (CDU) and Contrast-Enhanced Ultrsonography (CEUS) in experimently induced acute testicular ischemia.Methods:1. Animal Model of Testicular Torsion56 healthy male New Zealand rabbits, 2.0-2.5kg in weight, were randomly assigned into 7 groups, there were eight rabbits in each of the following experimental groups: (1) Normal control, (2) Sham-operated, (3) 0.5h group,(4)1.5h group, (5) 3h group,(6) 6h group,(7)12h group. In all experiments the right testis served as the internal control while the left testis served as the experimental side. The rabbits were anaesthetized jointly by chloral hydrate enema and pentobarbital i.v. (30mg/kg body weight). In normal control group, not any operation was done; in Sham-operated group, the left spermatic cord was delivered to the operating field via a transverse skin incision, 1 cm above the left scrotum in the distance. After 10 minutes of exposure, cuts were sutured. In groups 3-7, ischemia of the left testis was produced by clamping the spermatic cord structures by arterial clamps for 0.5h, 1.5h, 3h, 6h and 12h respectively, with no color flow signal detected by CDU in the left testicle, and the clamped spermatic cord and the clamp were buried together inside the sutured wounds. When the experiments began with CDU and CEUS, the rabbits were anaesthetized by pentobarbital i.v, the successful ischemia models were confirmed by the CDU with no color signal detected in the testis. After the cut was reopened and the clamp was removed, the left scrotum was coated with wet warm gauze for ten minutes before CDU, CEUS and testicular bleeding test (TBT) were performed in order.2. CDU, CEUSGray-scale and Doppler parameters were kept constant throughout the study and were set so as to maximize slow flow detection. Gray-scale examinations consisted of testis shape, echogenicity and the taking of 3 sets of orthogonal diameter measurements of the maximal longitude plane of each testis. And the testicular volume was calculated with the formula: V=Height×Length×Width×π/4×0.9; Bilateral intratesticular flow and peripheral flow were detected and recorded. The image, richest in color flow, was selected to calculate the area of color flow display (a) and maximal longitudinal plane (S) by the software of Image-Pro 5.0, and the ratios of r (r=a/S×100%) of both testes were calculated respectively, the values of the ratios of left r versus right r were analyzed statistically. The probe was vertically fixed on the biggest longitudinal plane of left testis with instrument to small angiography mode, imaging parameter frequency 3.2/6.4 MHz, MI 0.10, TIs 0.0. Contrast medium SonoVue was injected in bolus into rabbit ear marginal veins at a dose of 0.2ml/kg, then 1ml saline was followed quickly. At the same time, the built-in timer was started, and testicular perfusion images were stored. Perfusion results were classified into four types: (1) fully homogeneous perfusion: the contrast was distributed diffusely and homogeneously in the whole testicular section; (2) fully inhomogeneous perfusion: the contrast was distributed diffusely and inhomogeneously in the whole testicular section; (3) perfusion defect: the contrast was distributed in the local section of testis; (4) no perfusion: no contrast was filled into the testis. Select the whole longitudinal section of testicle as a region of interest (ROI), a computer-based time-intensity curve was produced with three parameters of arriving time (AT) , time to peak intensity (TTP) and increased peak intensity (⊿PI) analyzed by the statistical software SAS V8.1.3. TBT and Pathological MorphologyFor each group, scrotal skin was incised and the testis was exposed by a sharp dissection down to tunica albuginea, and a 5mm-long wedge cut into testicular medulla was made to observe the testicular bleeding. According to Arda's classification criteria, the results were divided into three levels: level I, fully bleeding: the active bleeding can be observed in incising the testis; level II , not fully bleeding: no immediate bleeding was observed in incising, but was in ten minutes; level III, no bleeding: no bleeding or errhysis was observed in ten minutes.We observe the shapes and colors, sizes of ischemia testes and select the left harvested testes as specimens, a representative slice was then stained with hematoxylin and eosin (HE stain), and a specimen 1-2 mm3 in size from the deep testicular medulla was picked to perform Transmission Electron Microscopy (TEM).Gross pathology and Ultrastructure of ischemia testes were observed.4. Data Evaluation and Statistical AnalysisGray-scale, CDU and CEUS images were obtained after experimental models examined. All gray-scale and CEUS images were reviewed by 2 senior radiologist experienced in testicular ultrasonography and who were unaware of the surgical procedure used and the pathologic results. They determined the echogenicities and heterogeneities of echoed by consent, and classified contrast medium perfusion results into four types: fully homogeneous perfusion, fully inhomogeneous perfusion, perfusion defect and no perfusion. The areas of intratesticular blood flow and longitudinal plane of the testes were quantified by the computerized software. Statistical analysis was performed by using SAS V8.1 software packet with Paired-Samples T test, Nonparametric Test (Kruskal -Wallis H), Variance analysis and Chi-Square test, the numerical data was shown with Mean±SD.Results:1. Animal Model of Testicular TorsionFive group models of testicular ischemia were made successfully and confirmed by the detection without blood supply after the spermatic cord clamped before the artery clamps removed. Testicular ischemia model success rate was 100% (40/40).2. CDU, CEUSThe left testicular volume (unit: mm3) was 0.62±0.102 in normal control group, and 0.63±0.069 in sham-operated group. The ratios of the volumes of the left and contralateral testis were not significant difference for the 2 groups (P > 0.05). In five ischemia groups, the volumes of the left testes were larger than the contralaterals. Of 0.5 h, 1.5h ,3h, 6h and 12h groups, the left testicular volumes were 1.09±0.184 mm3, 1.23±0.099, 1.28±0.280, 1.12±0.092 and 1.14±0.141 respectively, and the ratios of the volume of the left and contralateral testis were significant difference for the 5 groups (P < 0.0001). In 0.5h and 1.5h groups, CDU showed a homogeneous echo and iso-echogenicity similar to normal control and sham–operated groups. In 3h, 6h and 12h groups, CDU showed a heterogeneous hypo-echogenicity, which was gradually extended in range with the ischemia time prolonged. Between normal control and sham-operated groups, the R values were 1.00±0.185, 1.04±0.196 respectively and not statistically significant (P>0.05). In 0.5h, 1.5h,3h, 6h and 12h groups, the R values were 1.57±0.193, 1.67±0.175, 0.74±0.267, 0.54±0.100 , 0 respectively and were statistically significant (F = 110.22, P < 0.0001 ).In 0.5 h and 1.5h groups, 16 cases were fully homogeneous perfusion similar to normal control and sham- operated groups. In 3h group, 8 cases were fully inhomogeneous perfusion. In 6h group, 7 cases were perfusion defect, and 1 case no perfusion. And in 12h group, all of 8 cases were no perfusion.In the results of TICs, the AT values (unit: s) from normal control group to 6h group were 4.1±0.83, 4.5±0.76, 6.3±1.04, 8.3±2.12, 12.1±3.91 and 20.7±6.32 respectively, ( x 2 = 50.0846, P < 0.0001) the TTP values (unit: s) were 9.5±0.76, 10.4±1.41, 14.3±2.76, 22.4±3.96, 36.8±4.4, 50.0±3.11, respectively( x 2 = 52.7090, P < 0.0001), and the values of⊿P I (unit: dB) were 4.1±0.83, 7.5±1.27, 7.5±1.26, 4.9±0.90, 3.9±0.65 and 1.81±0.88 respectively(F = 83.73, P < 0.0001). Of 12h group, 8 cases were all no perfusion, the value⊿ PI of which was decreased to 0.3. TBT and Pathological MorphologyAccording to Arda's criteria of TBT, 32 cases from normal control, sham-operated, 0.5h and 1.5h groups were graded Level I, 8 cases of 3h group , 4 cases of 6h group and 2 cases of 12h group were graded LevelⅡ, 4 cases of 6h group and 6 cases of 12h were graded LevelⅢ.In normal control and sham operated groups, all testes were oval in shape and pink in color, on the surface of which a lot of small vessels were visualized. In all ischemia groups, all the testes were dark red or black in color with diffuse swelling and showed globular or hooklike in shape. After coated warmly with wet gauze, the color became pink from dark red or black in 0.5h and 1.5h groups, dark red from black in 3h and 6h groups, and all cases showed no changes in black in 12h group.In normal control and sham-operated groups, microscopic examinations showed normal interstitial structure and fine seminiferous tubules lined with a stratified epithelium. In 0.5h and 1.5h groups, microscopic images showed a slightly change, even without change similar to normal control and sham-operated, and mild congestion in small interstitial vessels, and TEM images showed slightly swollen smooth endoplasmic reticulum and mitochondria. Under the microscope of 3h group, marked interstitial congestion and detached tubular epithelium can be found in tubular cavity, and ultrastructures detected by TEM showed chromatin condensation, aggregation at the periphery of the nucleons, and platelet adhesiveness and platelet aggregation in small interstitial vessels. In 6h group, microscopic images showed obvious detachment of tubular epithelium from the base memerane, occlusion of tubular cabvity, spermatoza and epithelial cell lay out of cracked tubular cavity with interstitial hemorrhage. Generalized edema of endoplasmic reticulum and mitochondria was accompanied by a part of pyknotic nuclei and disrupted nuclei membrane. In 12h group, only the outlines of seminiferous tubules were at an optic field with a large mount of hemorrhage. The ultrastructures showed fragmentation and dissolution of nuclei with disappeared organelles.Conclusion:We found that we were able to judge testicular viability in torsed testes by intraoperatively using CDU and CEUS technology. Viable testes had significantly higher vascularity display than nonviable testes, even than normal testes in CDU. And viable testes had fully homogeneous perfusion, and better values of parameters in TICs.
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