Evaluation Of The Focal Hepatic Lesion In Rats Using Ultrasonic Contrast Agent And Its Effects On Gene Delivery

PART I Ultrasonography of Wistar rats' Liver and corresponding applicationsObjectiveTo confirm the ultrasound findings of the liver in normal Wistar rats and to explore its' application in ultrasonic experiments.MethodsForty adult Wistar rats weighted about 200g were studied. Two-dimensional ultrasound with high frequency scan head and CDFI were used to detect the liver of rats after anesthesia. The rats were randomly divided into 2 groups. Methylene blue about 0.05-0.1 ml were injected into left exterior hepatic lobe of rats in group 1 and right hepatic lobes in group 2 individually; Pulse inversion harmonic imaging was used to evaluate the process of enhancement of liver after bolus injection of ultrasonic contrast agent(0.05ml/200g) through caudal vein; The rats were then killed and examined if there were leaking out of methylene blue between hepatic lobes; The size of each lobes was also studied with slide gauge.ResultsThe liver of Wistar rats contains 6 lobes: left exterior lobe, left interior lobe, median lobe, right lobe, caudate lobe and papillary lobe; There was notleaking out of methylene blue in cases of group 1, while 5/20 cases were found leaking out in group 2; Hepatic artery stage, portal vein stage and balance stage last for 2-4s, 3-8s and 3-5min individually could be seen clearly during contrast enhancement of liver in all the cases.ConclusionHigh-frequency ultrasonography can be used to discern parts of hepatic lobes in Wistar rats. Left exterior lobe was easier to identified in ultrasound imaging. Penetration seldom happened during puncture operations on left exterior lobe; Liver of rats could be used in experiment of contrast enhancement imaging. While, the relatively short duration of artery stage and portal vein stage may limite the use of Wistar rats in the experiment of hepatic tumor contrast enhancement to some extent.PART II Evaluation of the focal hepatic lesion in rats using new type of ultrasonic contrast agentObjectiveTo evaluate the focal hepatic lesion in rats caused by local injection of absolute alcohol using ultrasonic contrast agent.MethodsTwenty adult Wistar rats weighted about 200g were injected with absolute alcohol for 0.05-0.1ml each one on the exterior left lobe of the liver under monitoring of ultrasound. The focal lesions were scanned with conventional B-mode ultrasound, color Doppler flow imaging and color Doppler energy. Seven days later, the focal lesions were studied again as before. Then, pulse invasion harmonic imaging was used to evaluate the focal lesions after bolus injection of ultrasonic contrast agent(0.05ml/200g) through caudal vein. The exterior left lobe of liver with focal lesion was incised and underwent pathologic examination.ResultsThe focal lesions couldn't be identified clearly by traditional B-mode ultrasound. 6/20 of the subjects were detected blood signals before intrahepatic injection of absolute alcohol. In all of the subjects, the focal lesions could be defined clearly after bolus injection of the ultrasonic contrast agent under mode of pulse invasion harmonic imaging. There were good correlation between the size of the focal lesions measured byultrasound under mode of pulse invasion harmonic imaging and that gotten by pathologic examination with significant difference(P=0.39).ConclusionThe role of traditional B-mode ultrasound, color Doppler flow imaging and color Doppler energy is limited in identification of the focal lesions caused by local injection of absolute alcohol in liver. Ultrasonic contrast agent plus pulse invasion harmonic imaging could be used to assess the size of the focal lesion mentioned above. Further study is to be done on the assessment of the more subtle pathologic changes with ultrasonic contrast agent.PART III Effect of ultrasound contrast agent onexpression of plasmid GFP in Tibialisanterior muscle of Wistar ratsObjectiveTo evaluate effect of ultrasound contrast agent on expression of plasmid green fluorescent protein (GFP) in tibialis anterior muscle of Wistar rats.MethodsForty adult Wistar rats weighted about 200g were studied. The rats were randomly divided into 4 groups. In each group, 10 rats were treated with injection of GFP plasmid in tibialis anterior muscle under ultrasound guidance, injection of the mixtures of GFP plasmid and ultrasound contrast agent, injection of the mixtures of GFP plasmid and ultrasound contrast agent with simultaneous exposure to ultrasound, injection of normal saline individually. Rats were sacrificed 12 days after ultrasound treatment for gene expression assay. The number of GFP-positive skeletal muscle fibers was counted. Hematoxylin-eosin staining was performed on separate tissue specimens for histopathologic evaluation.ResultsMuscle tissue exposed to ultrasound with plasmid and ultrasound contrast agent showed a increase in number of GFP-positive skeletal muscle fibers(345±19) with obvious difference (P<0.01) to other groups(109± 15 and 111 ± 14 respectively). Histologic examination revealed no evidence of inflammation, hemorrhage or necrosis.ConclusionsDiagnostic ultrasound with microbubble contrast agent induced efficient gene transfer to skeletal muscle of rats after local injection of GFP plasmid.