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Xiaoming Yang, MD, PhD Digital Optical Imaging of Hong Liu, PhD Dechun Li, MD, PhD Green Fluorescent Proteins for Xianzheng Zhou, MD, PhD William C. Jung, MS Tracking Vascular Gene Abby E. Deans, BS Yan Cui, PhD Expression: Feasibility Study Linzhao Cheng, PhD in Rabbit and Human Cell Index terms: 1 Animals Models Carotid arteries, 942.1299, 942.91, 942.99 Experimental studies Genes and genetics PURPOSE: To investigate the feasibility of using a sensitive digital optical imaging Molecular analysis technique to detect green fluorescent protein (GFP) expressed in rabbit vasculature Proteins and human arterial smooth muscle cells. Radiology 2001; 219:171–175 MATERIALS AND METHODS: A GFP plasmid was transfected into human arterial smooth muscle cells to obtain a GFP–smooth muscle cell solution. This solution was Abbreviations: GFP ϭ green fluorescent protein imaged in cell phantoms by using a prototype digital optical imaging system. For in SMC ϭ smooth muscle cell vivo validation, a GFP-lentivirus vector was transfected during surgery into the carotid arteries of two rabbits, and GFP-targeted vessels were harvested for digital optical imaging ex vivo. 1 From the Departments of Radiology (X.Y., H.L.), Anesthesiology and Criti- RESULTS: Optical imaging of cell phantoms resulted in a spatial resolution of 25 cal Care Medicine (D.L.), and Oncol- ogy (X.Z., Y.C., L.C.) and the Center ␮m/pixel. Fluorescent signals were detected as diffusely distributed bright spots. At for Medical Optics and Electronic Im- ex vivo optical imaging of arterial tissues, the average fluorescent signal was signif- aging (H.L., W.C.J., A.E.D.), the Johns icantly higher (P Ͻ .05) in GFP-targeted tissues (mean Ϯ SD, 9,357.3 absolute units Hopkins University School of Medi- cine, Outpatient Center, Rm 4243, of density Ϯ 1,001.3) than in control tissues (5,633.7 absolute units of density Ϯ 601 N Caroline St, Baltimore, MD 985.2). Both fluorescence microscopic and immunohistochemical findings con- 21287-0845. Received March 27, firmed these differences between GFP-targeted and control vessels. 2000; revision requested May 13; re- vision received July 27; accepted Au- CONCLUSION: The digital optical imaging system was sensitive to GFPs and may gust 29. Supported in part by the potentially provide an in vivo imaging tool to monitor and track vascular gene Cardiovascular and Interventional Radi- ology Research and Education Foun- transfer and expression in experimental investigations. dation (CIRREF) and a National Insti- tutes of Health grant CA 70209. Ad- dress correspondence to X.Y. (e-mail: [email protected]). © RSNA, 2001 Atherosclerotic cardiovascular disease remains the leading cause of mortality in the United States (1). Gene therapy is a rapidly expanding field with great potential for the treatment of cardiovascular diseases. Many genes have been shown to be useful for preventing acute thrombosis, blocking postoperative restenosis, and stimulating growth of new blood vessels (angiogenesis) (2–5). However, precise monitoring of gene delivery into and ade- quate follow-up of gene expression in targeted atherosclerotic plaques are two challenging tasks. To date, most investigations about imaging of gene therapy have been focused Author contributions: primarily on noncardiovascular systems, and, to our knowledge, no in vivo imaging Guarantors of integrity of entire study, modalities are currently available for vascular gene therapy (6). X.Y., H.L.; study concepts and design, The recent emergence of a viable marker, green fluorescent protein (GFP), has opened X.Y., H.L.; literature research, X.Y., H.L.; experimental studies, all authors; the door for the convenient use of intact living cells and organisms as experimental data acquisition and analysis, all au- systems in fields ranging from cell biology to biomedicine (7,8). GFP has been widely used thors; statistical analysis, X.Y., H.L.; as a sensitive marker that can be detected with fluorescence microscopy or flow cytometry manuscript preparation and editing, (9,10). The native protein of GFP is fluorescent in living cells, which allows in situ X.Y., H.L.; manuscript definition of in- tellectual content, revision/review, detection in the living animal. Moreover, the fluorescent signal emitted from GFP can be and final version approval, all authors. detected with optical imaging. This property of GFP has been widely used in in vitro investigations with living cells and has been experimentally tested in vivo to track the 171 distribution of gene transfer in the eye Animal Health, Fort Dodge, Iowa), ace- (11), tumors (12), and the airway (13). promazine maleate (1.1 mg/kg; Fermenta These results demonstrate the potential Animal Health, Kansas City, Mo), and at- application of optical imaging of GFP to ropine sulfate (0.05 mg/kg; American Re- monitor gene transfer and track gene ex- gent Laboratories, Shirley, NY). An ear pression. To our knowledge, however, vein was cannulated to permit mainte- the ability to optically image GFP in the nance of anesthesia. Pentobarbital so- cardiovascular system has not yet been dium (20 mg/kg; Abbott Laboratories, explored. North Chicago, Ill) was later adminis- The objective of this study was to in- tered intravenously to bring the animal vestigate the feasibility of using a highly to a surgical plane of anesthesia. Animals sensitive digital optical imaging tech- were intubated and mechanically venti- nique to detect GFPs expressed from the lated by using a respirator (model 55- Figure 1. Surgery-based gene delivery into vasculature. 0798; Harvard Apparatus, South Natick, the rabbit carotid artery (solid arrow). After Mass). The animals were also adminis- isolation of the artery by means of two Senti- MATERIALS AND METHODS tered heparin (100 IU per kilogram body nel loops (arrowheads), the GFP–lentivirus vec- weight). Anesthesia was monitored for tor solution (open arrow) is injected into the In Vitro Experiments the duration of the experiment by using carotid artery. We used an N-terminal–enhanced GFP regular tests of the eyelid reflex and mild plasmid vector (pEGFP-N1; Clontech, paw compression. Surgery-based GFP vector delivery.—Us- Palo Alto, Calif). Human pulmonary ar- optical imaging and another for immedi- ing an arteriotomy approach (14), we terial smooth muscle cells (SMCs) (Clo- ate frozen sections and subsequent fluo- first exposed an approximately 1.5-cm- netics, San Diego, Calif) were cultured in rescence microscopic examination, as long portion of the bilateral carotid arter- SMC growth medium (SmGM-2; Clonet- well as immunohistochemical confirma- ies. The exposed arterial portion on the ics, Walkersville, Md). They were then tion. seeded into a six-well plate at a concen- right side was isolated with sutures and 5 then harvested (prior to administration tration of 10 cells per well and were in- Digital Optical Imaging of any GFP vector in the left side) to serve cubated at 37°C (with 5% CO2) for 24 hours. Then, GFP plasmid vector (1 ␮g) as control. The exposed arterial portion Digital optical imaging system.—To im- was transfected for 30 minutes into SMCs on the left side was isolated by tempo- age SMC-GFPs in vitro and GFP-targeted with a transfection reagent (lipofect- rarily tightening the target vessel proxi- vessel tissues ex vivo, a digital optical AMINE PLUS Reagent; Life Technologies, mally and distally with two Sentinel imaging prototype was developed in our Rockville, Md). Subsequently, the non- loops (Sherwood Medical, St Louis, Mo) laboratory. The system consists of an ex- transfected plasmid was removed, and (Fig 1). By inserting a 24-gauge catheter ternal light source, a fiberoptic light the plate was further incubated in the (Quik-Cath; Baxter, Marion, NC) into the guide, wavelength selective optics, a sam- 37°C incubator for an additional 2 days isolated segment of the left carotid artery, ple holder, a custom relay lens, and a to allow sufficient GFP expression. After we drew off the blood from the vessel highly sensitive charge-coupled–device this, the cells were harvested by means of lumen and thereafter directly injected a detector (Fig 2) (15). trypsinization and washing and were an- GFP-containing lentiviral vector solution The detector module uses a mechani- alyzed by using flow cytometry (Decton (developed and produced in our labora- cal shutter. An optical hood is used to Dickinson, Immunocytometry Systems, tory in January 2000) to fill the isolated shield the detector from ambient light. San Jose, Calif) to quantify the transfec- vessel portion for 1 hour. A silk tie was The charge-coupled–device arrays con- tion rates of the cell phantom. placed in the adjacent tissue to identify sist of 1,024 ϫ 1,024 pixels, and each the vessel segment to be harvested. After pixel measures 0.024 ϫ 0.024 mm with a the transfection, we drew off the GFP 0.999 fill factor. The spatial resolution of In Vivo and ex Vivo Experiments vector solution, removed the catheter, the overall digital optical imaging system Animals.—We used the bilateral ca- closed the puncture point with cyanoac- is 20 line pairs per millimeter (16). The rotid arteries from two New Zealand rylate glue (Superglue; Elmer’s, Colum- camera is operated at a temperature of white rabbits (Robinson Services, Clem- bus, Ohio), loosened the Sentinel loops, Ϫ25°C to reduce thermal electron noise. mons, NC), each approximately 5 kg in closed the arteriotomy incision with su- The low temperature is achieved with a weight. All animals were treated accord- tures, and kept the rabbit alive for 4 days. compact thermoelectric cooler. This pro- ing to the “Principles of Laboratory Ani- After the surgery-based gene delivery, we totype provides 14-bit digitization. mal Care” of the National Society for administered a postoperative analgesic During image acquisition, the cell Medical Research and the Guide for the (0.01 mg/kg buprenorphine, Buprenex; phantoms or tissue samples were placed Care and Use of Laboratory Animals (Na- Reckitt & Coleman Pharmaceuticals, on top of the holder.

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