| ObjectiveAt present,alcohol has been widely used in people's daily life,and its accompanying health problems have gradually become prominent.Besides,the reproductive system problems caused by alcohol have also become increasingly serious which lack of real-time,non-invasive and objective evaluation methods in current clinical practice.This study aims to explore the clinical value of contrast-enhanced ultrasonography(CEUS)and its quantitative analysis techniques in the evaluation of testicular microvascular injury caused by chronic alcoholism and its relationship with testicular spermatogenesis.MethodsThe general lipid Microbubbles(MB),a kind of ultrasonic contrast agent,was prepared and its related physical properties and performance characteristics were detected.72 healthy New Zealand rabbits were randomly divided into 12 groups of 6animals each,which were the control group S(S1,S2,S3 group),the low-dose alcohol group L(L1,L2,L3 group),the medium-dose alcohol group M(M1,M2,M3group),and the high-dose alcohol group H(H1,H2,H3 group).All groups were given the same fodder with the equal quantity for 7 days.Then,apart from the normal diet daily,the L,M and H group were given 60%alcohol 5ml/kg,7.5ml/kg and 10ml/kg every other day by gavage,respectively,which aimed to establish models of chronic alcoholism injury in rabbit testis with different alcohol dose.The S group was given7.5ml saline per kilogram every other day by gavage.The S1,L1,M1 and H1 groups were fed for 30 days,the S2,L2,M2,and H2 groups were fed for 60 days,and the S3,L3,M3,and H3 groups were fed for 90 days.Each group of New Zealand rabbits went through two-dimensional gray-scale and color doppler examination on the following day after the feeding period was completed,and the sonographic features were observed.All rabbits underwent MB CEUS to observe the performance and quantitatively analyze the changes of various parameters of each group in order to obtain accurate and dynamic perfusion information of testicular tissue.At the end of the experiment,about 10ml heart blood was obtained by ultrasound-guided puncture and serum endothelin-1(ET-1)and nitric oxide(NO)were measured.One side of testicular tissue was extracted for hematoxylin-eosin(HE)staining section,Johnsen's score,malondialdehyde(MDA)content detection and transmission electron microscopy(TEM)specimen.Histopathology,MDA content and ultrastructural changes of TEM were observed,as well as their differences of each group.ResultsMB physical characteristics and performance characteristics detection:MB appeared as milky white liquid by naked-eye observing,and regular circular microbubbles with relatively uniform size and distribution under the light microscope.The microbubbles concentration were about 4.9×10~8 cells/ml.After laying aside for0.5 hours,the number of microbubbles decreased.After laying aside for 24 hours,the number of microbubbles decreased significantly and the particle size became smaller.Testicular acoustic image findings:The pre-CEUS findings were almost similar among the control group,the alcohol groups not only with different alcohol dose but also with different time.The size of bilateral testes was similar,the echo of parenchyma was uniform,and the color doppler flow signal was between the grade I-II according to the semi-quantitative grading standard of Adler flow.CEUS findings:1.Control group:there were no statistically significant difference in CEUS parameters among S1,S2 and S3 group(P>0.05).2.The alcohol groups with different dose and time:With the increase of alcohol dose and time,CEUS parameters,PI and AUC,gradually decreased(P<0.05)and the flow of filling blood also gradually decreased.Slope decreased gradually(P<0.05)due to the decrease of PI,but the difference in TP,MTT and DT/2 was not statistically significant(P>0.05).Differences in ET-1,NO and MDA:There were no statistically significant difference in ET-1,NO and MDA levels in control group(P>0.05).The levels of ET-1,NO and MDA were growing in the alcohol-treated group with the increase of alcohol dose and time(P<0.05).Pathological findings of light microscopy:1.Control group:the basement-membrane of the seminiferous tubule was intact,the epithelium was thick and the spermatogenic cells were orderly arranged.Besides,a large amount of sperm could be seen in the proximal lumen surface.The interstitial structure was clear,no swelling and widening.The microvascular morphology of the interstitial is normal,and red blood cells are visible inside.2.The alcohol group with different dose and time:With the increase of alcohol dose and time,the lumen of spermatogenic tubules gradually expanded,the spermatogenic epithelial layer gradually became thinner,the sperm gradually decreased,and even no sperm is formed.The early manifestation of interstitial microvasculature was microvascular dilatation.With the increase of alcohol dose and time,the wall of microvasculature gradually became thicker,the inflammatory cells in the lumen gradually increased,and the mesenchyme gradually became swollen and wide.3.Johnsen's score:The Johnsen's score was statistically insignificant among the groups in the S group(P>0.05).Except for S1 vs L1 group,L1 vs M1 group,and M1 vs H1 group(P>0.05),there were significant differences among the S group,the alcohol group with different dose(L,M,H group)and the alcohol group with different time(30d,60d and 90d)(P<0.05).Johnsen's score decreased with the increase of alcohol dose and time.Ultrastructural performance of TEM:1.Control group:The basement-membrane of the seminiferous tubule was intact,the spermatogenic cells were arranged clearly and orderly,the spermatogenic cells and their cytoplasmic mitochondria and endoplasmic reticulum were normal in morphology,the nucleus was intact.A large amount of sperm was observed in the lumen.The interstitial structure was intact,the morphology of microvascular endothelial cells,pericytes and interstitial cells was normal,the membrane structure and the nucleus were intact,the staining was uniform,the cytoplasmic mitochondria and endoplasmic reticulum were normal,and the microvascular basement-membrane was intact.2.The alcohol group with different dose time:With the increase of alcohol dose and time,the damage degree of seminiferous tubules gradually increased,the number of necrotic spermatogenic cell gradually increased,the sperm in the lumen gradually decreased,and even spermatogenic cells had differentiated and matured obstacle,with no sperm finally.The degree of structural damage of interstitial microvasculature was gradually aggravated.At the early stage,the mitochondria in the cytoplasm of vascular endothelial cells were swollen and vacuolated.With the increase of dose and time,the cytoplasm and nucleus of microvascular endothelial cells and pericytes were dissolved and necrotic,or even a large number of them fell off.In severe cases,the basal membrane was dissolved and disrupted.The interstitial structure was gradually disordered,with extensive mitochondrial swelling,endoplasmic reticulum expanding and vacuolating,and the destruction of interstitial cells was increasingly serious.Conclusions1.Chronic alcohol intake can cause testicular microvascular injury,which is manifested as the change of testicular hemodynamics with reduced blood perfusion,and then cause tissue ischemia and hypoxia,resulting in spermatogenic dysfunction.2.Alcohol has a dose-effect and time-effect relationship on the damage of testicular microvasculature and spermatogenic function.The changes of testicular microvascular function are synchronous with the ultrastructural changes,and the testicular microvascular injury and spermatogenic function are consistent.Alcohol had a dose-effect and time-effect relationship with the damage of testicular microvasculature and spermatogenic function.The changes of testicular microvasculature and its ultrastructure were synchronous,while the changes of testicular microvasculature and spermatogenic function were consistent.3.CEUS can be used to evaluate testicular microvascular injury caused by chronic alcoholism and reflect changes in spermatogenic function.Overall,the quantitative parameters PI and AUC of CEUS decreased with the increase of alcohol dose and time.The more severe the microvascular injury is,the lower the level of the PI and AUC is,and the more severe the spermatogenic function injury was. |
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