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Published online before print July 24, 2003, 10.1148/radiol.2283020482
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(Radiology 2003;228:768-775.)
© RSNA, 2003

Experimental Studies

Electrotransfer at MR Imaging: Tool for Optimization of Gene Transfer Protocols—Feasibility Study in Mice1

Marion Paturneau-Jouas, PhD, Elodie Parzy, BSc, Giovanni Vidal, PhD, Pierre G. Carlier, MD, PhD, Claire Wary, PhD, Jean-Thomas Vilquin, PharmD, PhD, Eric de Kerviler, MD, Ketty Schwartz, PhD and Anne Leroy-Willig, MD, PhD

1 From INSERM U 582 (M.P.J., J.T.V., K.S.) and Laboratoire de RMN (AFM-CEA) (E.P., G.V., P.G.C., C.W., A.L.W.), Institut de Myologie, Hôpital de la Pitié-Salpêtrière, 47 Boulevard de l’Hôpital, 75651 Paris, France; and Service de Radiologie, Hôpital Saint-Louis, Paris, France (E.d.K.). Received April 26, 2002; revision requested July 9; final revision received December 4; accepted January 13, 2003. Supported by the Association Française contre les Myopathies, the Institut Fédératif de Recherche 14, the Institut National de la Santé et de la Recherche Médicale, the Commissariat à l’Energie Atomique, and the Centre National de la Recherche Scientifique. E.P. supported by the Ministère de la Recherche. Address correspondence to A.L.W. (e-mail: a.leroywillig@myologie.chups.jussieu.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To test, by using an electrotransfer protocol for the transfection of skeletal muscle with naked plasmid complementary DNA, whether in vivo magnetic resonance (MR) imaging can help delineate either the spatial extent of the electric field when contrast agent is injected intraperitoneally or the transfection area when contrast agent is injected locally.

MATERIALS AND METHODS: Three groups of five mice each were examined at 4 T. Gadopentetate dimeglumine was injected intraperitoneally before electroporation in group 1 and after electroporation in group 2. In group 3, gadopentetate dimeglumine was coinjected in situ with plasmid pCMV-ßGal in the gastrocnemius muscle before electroporation. MR imaging and muscle preparation for histologic examination were performed 3 days later. On T1-weighted images, increase of muscle signal intensity was determined in regions of interest (ROIs) of treated legs and compared with contralateral ROIs. Comparison of signal intensity increase between groups 1 and 2 was performed with Kruskal-Wallis test.

RESULTS: In groups 1 and 3, T1-weighted images of treated muscle showed zones of strongly increased signal intensity. In corresponding ROIs of groups 1, 2, and 3, the mean T1-weighted signal intensity increase at day 3 was 1.64 ± 0.20 (SD), 1.16 ± 0.06, and 1.58 ± 0.17, respectively. The difference between groups 1 and 2 (ie, gadopentetate dimeglumine injected before and after electrotransfer) was significant (P < .001) both without and with correction for T2 variation (1.47 ± 0.19 and 1.04 ± 0.09, respectively). In group 3, after in situ coinjection of gadopentetate dimeglumine and plasmid, the area of increased signal intensity revealed at ex vivo MR imaging of the muscle showed a reasonable concordance with the transfected area revealed with ß-galactosidase on histologic sections.

CONCLUSION: In vivo and ex vivo results indicate that atraumatic visualization of the permeabilized and transfected area is possible.

© RSNA, 2003

Index terms: Animals • Genes and genetics • Magnetic resonance (MR), experimental studies, 45.121411, 45.12143 • Therapeutic radiology, experimental studies


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
In the rapidly developing field of gene therapy, much experimentation is necessary before clinical protocols are determined. Atraumatic imaging techniques are used increasingly to follow every step, from gene delivery (1) to observation of transgene expression (2–5) and therapeutic modifications (6). Molecular imaging, based on highly sophisticated specific contrast agents, gives insights into molecular abnormalities or molecular modification in living cells and directly addresses the possibility of detection of gene delivery (7) and gene expression (8,9). Alternately, very simple contrast agents, which can be used to evidence membrane permeabilization (10), may now provide some hint of the accession of an exogenous gene into cells.

Transient permeabilization of cell membrane by exposure to an external electric field is widely used to introduce foreign DNA into bacteria and eukaryotic cells in vitro and exvivo (11). This method, named electroporation, has recently been applied to targeted tissues in vivo and is increasingly used to improve drug delivery and DNA transfer into specific organs or tumors (12–16). Findings of early experimental studies in animals, mainly rodents, have proved that there is an increase in the efficacy of gene transfer and expression, particularly in skeletal muscle (17–19), and have provided perspectives regarding the correction of genetic muscular diseases (20), secretion of therapeutic factors (21–23), or DNA-mediated vaccination (24). In view of these new therapeutic strategies, optimization of electrotransfer protocols by using different electrode shapes and sizes and different combinations of electric parameters are key variables to the adaptation of the method for targeting of a variety of organs in larger animals and humans.

Global efficiency of gene electrotransfer is appreciated by the level of the reporter gene expression in the homogenate of transfected tissue (12,19,25) or by blood determination of soluble gene products (17,26). Localization and evaluation of the size of the transfected area in muscle can be realized ex vivo with visualization of the reporter gene expression on histologic sections (17,18,20,25). However, this requires animal sacrifice.

We hypothesized that it is possible to monitor the uptake of small-molecular-weight gadolinium chelate with magnetic resonance (MR) imaging as an in vivo atraumatic method to facilitate the evaluation of electrotransfer protocols. Similar molecules, the radioactive tracers chromium 51 (51Cr)-ethylenediaminetetraacetic acid (EDTA) and technetium 99m–gadopentetate dimeglumine, have been used in previous studies to quantify the effect of permeabilization (19) or the spatial distribution of the radiotracer shortly after electrotransfer (27). As was previously shown for naked plasmid complementary DNA (cDNA) or drugs, during a short temporal window, electroporation enables the entrance of 51Cr-EDTA (28) or gadopentetate dimeglumine (10) into the cells. Dependence on the strength of the electric field is similar to that observed for plasmid DNA expression (10). Rapid resealing of the membrane pores (28) triggers the intracellular trapping of these exogenous molecules. After elimination of the contrast agent from the extracellular compartment, its intracellular accumulation can be detected at MR imaging and quantified with T1 measurement.

The purpose of our study, by using a protocol for the transfection of skeletal muscle with naked plasmid cDNA (19,20, 23), was to test whether in vivo MR imaging can help delineate either the spatial extent of the electric field when the contrast agent was injected intraperitoneally or the transfection area when it was locally injected.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Animals
Normal adult C57Bl6J mice (Jackson Laboratory, Bar Harbor, Me) (weight range, 19.5–25.2 g; mean weight, 22.0 g) were bred in the laboratory animal facility. They were housed at room temperature with a 12-hour day and night cycle and were fed standard laboratory chow and water ad libitum. Prior to all procedures, the animals were anesthetized with a subcutaneous injection of 0.01 mL per gram of body weight of ketamine (10 mg/mL solution, Clorketam; Vetoquinol, Lure, France) and xylazine (5 mg/mL, Rompun; Bayer, Leverkusen, Germany). Three groups of five mice each were examined. Studies were conducted according to the recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimentation.

Experimental Protocols
All injections in mice, electric stimulation, histologic processing, and examination of muscle were performed by one author (M.P.J.). MR imaging and processing were performed by another author (A.L.W.).

MR examinations and sacrifice for histologic examinations of animals in groups 1 and 3 were performed 3 days after electrotransfer to ensure a complete elimination of gadopentetate dimeglumine from the extracellular space and a substantial level of ß-galactosidase expression. When required, on day 3, immediately after MR examination and with use of anesthesia, the mice were given a supplementary lethal dose of anesthetics, and the treated gastrocnemius was removed.

For group 1, intraperitoneal injection of 20 µL/g of iso-osmolar gadopentetate dimeglumine solution was administered at time 0. Electroporation was performed 30 minutes later, as described later. MR examination was performed at day 3 and included a T1-weighted multisection acquisition, a single-section T2 measurement, and a T1 map acquisition. The treated gastrocnemius was then quickly removed and frozen for further histologic processing to evaluate tissue modifications.

For group 2, the electric field was applied at time 0. An intraperitoneal injection of 20 µL/g of iso-osmolar gadopentetate dimeglumine was administered 20 minutes later to verify that no intracellular accumulation of gadopentetate dimeglumine could be detected. This time interval was chosen because resealing of pores is complete at that time (19,28). The MR examination was performed at day 3 and included a T1-weighted sequence and a single-section T2 measurement.

For group 3, 12 µL of an iso-osmolar solution of gadopentetate dimeglumine and 50 µL of a plasmid solution (1 µg/µL of 0.9% NaCl) in the same syringe were injected in the right gastrocnemius to ensure identical delivery of gadopentetate dimeglumine and plasmid. The electric field was applied 1 minute later. In vivo MR examination was performed at day 3 and included a T1-weighted acquisition. The treated gastrocnemius was removed and quickly examined ex vivo with a T1-weighted sequence, and 30 minutes after removal, it was frozen for further histologic detection of ß-galactosidase.

Electrotransfer Protocol
Two external parallel stainless steel plate electrodes were placed on either side of the shaved leg at the level of the gastrocnemius muscle. Electrocardiographic paste was applied to ensure conductivity between the electrodes and the skin. Eight square wave pulses of fixed 20-msec duration each were delivered at a 2-Hz frequency with an electroporator (ECM 830; BTX, San Diego, Calif), as previously described (19). The electric field strength, evaluated as the ratio of the applied voltage to the distance between the electrodes, was 200 V/cm. Each animal was its own control; the treated leg was compared with the contralateral untreated leg.

Plasmids
Supercoiled pCMV-ß-gal plasmids (pCOR plasmid pXL3227, a gift of Gencell, Aventis Pharma, France) containing the Escherichia coli LacZ gene coding for cytoplasmic ß-galactosidase (29) were injected in the gastrocnemius by using a 50-µL syringe (Hamilton, Bonaduz, Switzerland) equipped with a 30-gauge needle. The concentration of plasmids was 1 µg/µL in 0.9% NaCl.

Contrast Agent Injection
The paramagnetic contrast agent gadopentetate dimeglumine (Magnevist; Schering, Berlin, Germany) was used as an iso-osmolar solution after 1.0:7.5 dilution with sterile water. Plasma half-life has been reported to be 20 minutes in rats (30). Preliminary experiments allowed optimization of the gadopentetate dimeglumine dose and of the timing of intraperitoneal or intramuscular injection. After intraperitoneal injection of 20 µL/g, a stable extracellular concentration of approximately 1 mmol/L was obtained during 20–120 minutes. Elimination from the extracellular space was fully completed at day 2.

MR Imaging
MR examinations were performed with a 4-T horizontal magnet (Magnex, Abingdon, United Kingdom) interfaced to a spectrometer (Biospec Avance; Bruker, Karlsruhe, Germany) by using a 60-mm-diameter 60-mm-length home-built coil that housed two mice. The mice were placed on two superimposed flat Plexiglas holders in the prone position and were held with soft tape around the belly and hind limbs that were placed symmetrically. The study of limb muscles was performed in transverse planes by using a T1-weighted spin-echo sequence with the following parameters: 600/11 (repetition time msec/echo time msec), field of view of 7 cm, nine contiguous sections with 2-mm thickness, in-plane spatial resolution of 136 x 273 µm2, and acquisition time of 5 minutes. The section centered on the largest section of the gastrocnemius was chosen for the T2 measurement by using a single-section spin-echo sequence with the following parameters: repetition time of 1,500 msec, eight echoes, first echo at 10 msec, echo spacing of 10 msec, field of view of 7 cm, in-plane spatial resolution of 273 x 273 µm2, section thickness of 2 mm, and acquisition time of 6.4 minutes.

In group 1 animals, a T1 measurement was performed at the same level by using a dedicated single-section multishot saturation-recovery fast spin-echo sequence combined with residual longitudinal magnetization saturation, further abbreviated as T1 map sequence. Eight images were obtained in 4.3 minutes with eight shots, recovery delays ranged from 0.3 to 10 seconds, first echo at 2.4 msec, interecho spacing at 2.4 msec, effective echo time at 19.2 msec, field of view of 8.4 x 4.2 cm, in-plane spatial resolution of 650 x 650 µm2, and 2-mm section thickness.

For the mice in group 3, the ex vivo study of the removed and mounted gastrocnemius was performed before freezing, within 10 minutes after death, by using a 28-mm-diameter birdcage coil (Rapid, Wurzburg, Germany) with a T1-weighted spin-echo sequence (500/9, field of view of 5.1 cm, 12 sections with 1-mm thickness, in-plane spatial resolution of 100 x 200 µm2, acquisition time of 4 minutes).

MR Imaging Processing
For in vivo examinations in group 1 and 2 animals, muscle signal intensity increase on T1-weighted images was determined as the ratio of the mean signal intensity in the region of interest (ROI) of the treated leg to the mean signal intensity in the reference ROI of a homogeneous muscle in the contralateral limb. In each leg, two circular ROIs with area of 76 pixels (3 mm2) were positioned in the gastrocnemius at the location of electrotransfer plates on the treated leg 1 mm from the middle of either side of the leg to avoid superficial fat contamination.

In the plane in which the T2 measurement was performed, the ROIs were pasted onto the eight-echo images, and the eight mean signal intensities of each ROI were fitted to an exponential decay with echo time. To calculate the T2 value at that location, the following equation was used: SIn = SI0 x exp(-TEn/T2), where SIn is the signal intensity from image n, SI0 is the signal intensity extrapolated at echo time of 0 msec, and TEn is echo time of n x 10 msec. This enabled further correction of signal intensity variations of the corresponding T1-weighted image for the contribution caused by T2 increase.

In group 3 animals, ROIs were drawn on the T1-weighted images of treated legs according to the topography of signal intensity increase. The ROIs on images of untreated legs were drawn as indicated previously.

In group 1 animals, T1 quantification from the T1 map sequence was determined from smaller ROIs to avoid contamination by fat or bone, the T1 values of which are markedly different. These ROIs were drawn on the higher spatial resolution T1-weighted image previously obtained and pasted onto the eight images acquired at different recovery delays by fitting these eight signals according to a three-parameter exponential model. The relaxivity, R1 = 1/T1, was compared with muscle relaxivity, R10, without use of gadopentetate dimeglumine; the same sequence was used as for previously examined control mice. The difference, R1 - R10, was converted into a mean tissue gadopentetate dimeglumine concentration, C, according to the equation R1 = R10 + K x C, where K is the relaxivity of the gadopentetate dimeglumine in muscle, which was determined as 3 mmol-1 · sec-1. This value was derived from T1 values determined with the same method in a phantom with gadopentetate dimeglumine concentrations in water that ranged from 0.05 to 0.5 mmol and by assuming the relaxivity of the contrast agent in muscle to be 0.9 times the value in water, as determined at 6.3 T (31).

The ex vivo MR image obtained at the middle of each removed treated muscle was compared visually with the image of the corresponding histologic section that was stained for ß-galactosidase, after same-scale printing and an in-plane rotation to set in coincidence the contours of muscle in both images. This histologic section was determined according to the best concordance between external contours of muscle, since one 1-mm-thick MR section corresponds to the volume sampled with multiple histologic 8-µm-thick cryostat sections.

Histologic Processing
Muscle morphology was examined 3 days after injection. The gastrocnemius was removed, mounted in tragacanth gum, frozen in isopentane that was cooled in liquid nitrogen, and stored at -80°C until it was processed into 8-µm-thick cryostat sections. Serial sections were either stained with hematoxylin and eosin to visualize the cells and nuclei or processed for ß-galactosidase expression. After a slight fixation in 0.25% glutaraldehyde and two rinses in phosphate-buffered saline, the sections were stained with X-Gal reagent (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; Boehringer, Roche, France) and incubated for 2 days at room temperature. Sections selected at light microscopy were digitized with a microscope (Leica, Wetzlar, Germany) connected to a color video camera (CCD; Sony, Berlin, Germany) and software (1.2.0.6; Thunder Software, Westerville, Ohio). The images were computerized at full size and processed (Adobe Photoshop; Adobe Systems, Seattle, Wash) to allow comparison with MR images.

Statistical Analysis
Comparison of signal increase in the treated leg normalized to that of the control leg with and without correction for T2 modification was performed between groups 1 and 2 by using nonparametric Kruskal-Wallis tests. Comparison of T2 values between groups 1 and 2 was performed by using a one-way analysis of variance. Probabilities of P less than .05 were considered to indicate a statistically significant difference. All statistical tests were performed with software (Vassar Stats; Vassar College, Poughkeepsie, NY).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
In Vivo MR Visualization of the Area Permeabilized to the Contrast Agent
On T1-weighted images sensitized to the tissue concentration of gadopentetate dimeglumine, areas with a clear signal intensity increase were observed in muscles of the limb that underwent electroporation in animals of groups 1 and 3. In group 1 animals, which received intraperitoneal gadopentetate dimeglumine injection, the zones of increased signal intensity were located on both sides of the animal leg facing the electrodes. The signal intensity increase was still visible at day 6 in two animals and in one animal at day 21 but no longer by day 60.

In group 3 animals, in which the gadopentetate dimeglumine was coinjected in the same syringe with the plasmid cDNA and delivered intramuscularly to a restricted leg territory in the gastrocnemius, the topography of signal intensity increase was different, covering a smaller surface. In group 2 animals, zones of weaker signal intensity variation were observed, with a topography similar to that observed in group 1 animals. Typical images of control and electroporated legs in the three groups are shown in Figure 1.



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  		Figure 1. In vivo MR images at day 3 after electroporation in animals of groups 1-3. Transverse spin-echo T1-weighted images (700/11) of control and electroporated legs of animals of groups 1 (A, B), 2 (C, D), and 3 (E, F). A, C, E, Images of contralateral control legs of the animals do not exhibit signal intensity variation. B, D, F, Images of electroporated treated legs show areas of increased signal intensity that mostly reflect the accumulation of gadopentetate dimeglumine. B, Electroporation after intraperitoneal injection of the contrast agent. In the presence of extracellular gadopentetate dimeglumine, muscle signal intensity is increased, with a complex pattern through a large fraction of leg section. Intermuscular aponeurosis and a central zone show lower signal intensity. D, Electroporation before intraperitoneal injection of gadopentetate dimeglumine. Muscle signal intensity is very faintly increased. F, Electroporation after intramuscular injection of gadopentetate dimeglumine. Muscle signal intensity increase is strong but distributed into a smaller volume compared with that in B.

 
From ROIs drawn on these T1-weighted images, the values of muscle signal intensity increase determined in each treated limb with comparison to the contralateral limb are shown in Figure 2 before and after correction for T2 variations, when applicable, for the 15 animals of all groups.



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  		Figure 2. Graph depicts signal intensity variations measured in vivo on T1-weighted images of electroporated treated legs, normalized to that of the control legs, in groups 1-3. In groups 1 and 3, a strong signal intensity increase ({bullet}) is measured in ROIs of treated legs normalized to corresponding ROIs of control legs (mean value, 1.64 ± 0.2 and 1.58 ± 0.17, respectively). In group 2, the mean signal intensity increase is much weaker (mean value, 1.16 ± 0.06). It is related to the contamination of T1-weighted images by T2 variation. Signal intensity increase corrected for T2 values ({circ}) is measured in the same ROIs of treated legs and control legs (mean value, 1.47 ± 0.19 in group 1 and 1.04 ± 0.09 in group 2). Bars indicate mean ± SD of signal intensity increase.

 
Mean values of signal intensity increase uncorrected for T2 variation in ROIs selected for animals in groups 1, 2, and 3 at day 3 after electroporation were 1.64 ± 0.02 (SD), 1.16 ± 0.08, and 1.58 ± 0.17, respectively. Mean values corrected for T2 variation were 1.47 ± 0.19 for group 1 and 1.04 ± 0.09 for group 2.

Kruskal-Wallis nonparametric statistical analysis showed significant differences between uncorrected signal intensity increase values in groups 1 and 2 (P < .001), as well as between corrected signal intensity increase values in groups 1 and 2 (P < .001). The mean signal intensity increase after correction was different from unity in group 1 (P < .001, t = 7.8) but not in group 2 (P > .99). Thus, we observed significant signal intensity variations in the three groups of animals between the treated and control leg, which resulted both from gadopentetate dimeglumine uptake and from T2 modification. In group 2 after correction for T2 variation, no residual effect of gadopentetate dimeglumine uptake was observed.

The mean tissue gadopentetate dimeglumine concentration in selected zones, derived from T1 values obtained from the T1 map sequence in the animals of group 1, was 137 µmol/L ± 88. From this value, the osmolarity added intracellularly with use of gadopentetate dimeglumine may be calculated to be approximately 800 µosm/L. This concentration was also evaluated from the mean signal intensity increase in group 1: By assuming values of the intracellular and extracellular water volumes to be 70% and 7%, respectively (32), we calculated a concentration of 150 µmol/L, which is consistent with the evaluation from the T1 map sequence.

Tissue Modifications on T2-weighted Images
MR signal intensity variations on T2-weighted images were observed in all animals of groups 1 and 2. The topography was similar for all animals, with two lateral zones in the leg facing both sides where electrodes were applied (Fig 3). In ROIs selected on T1-weighted images and pasted on T2-weighted images, as detailed previously for group 1 and 2 animals, the mean T2 values in treated legs at day 3 were 51 msec ± 9 in group 1 and 58.6 msec ± 8 in group 2 animals. The mean value in control legs was 35.8 msec ± 2. No difference in T2 values between group 1 and 2 was observed. T2 values were still elevated on day 6 in two animals.



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  		Figure 3. MR images at day 3 in two mice of groups 1 and 2. Transverse spin-echo T2-weighted images (1,100/50) of control and electroporated legs of one animal of group 1 (A, B) and one animal of group 2 (C, D). A, C, Control legs of the animals do not exhibit signal intensity variation. B, D, MR signal intensity increase is observed in electroporated areas. It reflects both the accumulation of gadopentetate dimeglumine, which increases signal intensity, and tissue modification, which increases T2 and also signal intensity at high echo time. B, In group 1 animal, topography of signal intensity increase is similar to that observed in Figure 1, B. D, In group 2 animal, signal variation exhibits similar topography but weaker intensity, reflecting that only T2 variation is involved.

 
In Vivo and ex Vivo Visualization of the Cellular Uptake of Intramuscularly Injected Gadopentetate Dimeglumine
Variation in the size and shape of the T1-weighted signal intensity increase, which reflected the injection protocol, was observed among the animals in group 3 on both the in vivo and ex vivo images. Direct comparison between in vivo and ex vivo images was not possible, since the shape and orientation of the gastrocnemius were modified with dissection and freezing.

Results from Histologic Examination and Comparison with MR Images
In group 3 animals, images of the X-Gal-stained histologic sections selected with light microscopy were compared with the corresponding T1-weighted ex vivo images. Despite the deformation of these small muscles due to dissection, freezing, and slicing that occurred between MR imaging and X-Gal staining, it appeared that areas of increased MR signal intensity matched well with areas containing X-Gal-stained fibers (Fig 4).



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  		Figure 4. Comparison of ex vivo T1-weighted images with the corresponding histologic images of group 3. MR image of the central slice through the removed gastrocnemius muscle and the corresponding histologic section are displayed for the treated legs of the five mice (1-5) of group 3. Magnification is the same for all images. Differences between animals in shape and size of the labeled territories are obvious with both techniques. For each animal, a clear similarity is observed between the area where muscle MR signal intensity is increased through intracellular trapping of gadopentetate dimeglumine and the area where expression of the reporter gene is revealed with staining. (X-Gal blue stain; original magnification, x6.8.)

 
In group 1 animals, the percentage of regenerating cells in the muscles, either treated or untreated, was lower than 1% as quantified at visual counting of centronucleated fibers, which indicated that neither the electrotransfer protocol nor the injection of gadopentetate dimeglumine induced significant muscle cell necrosis before day 3. A large number of inflammatory cells were observed in the cross-sectional area of the electroporated gastrocnemius, with no obvious relation to the increased signal intensity territories.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
Electrotransfer of genes or drugs has a wide potential for treatment of various clinical disorders and for DNA vaccination. However, progress in experimental studies and in application to human therapy may be hampered by the difficulties involved in tailoring the electrical protocol and delivery system to the needs of the different situations. The results presented here provide evidence that MR imaging with use of intracellular uptake of a contrast agent allowed noninvasive in vivo visualization of the permeabilized area obtained transiently after application of an electric field. A significant T1-weighted signal intensity increase, approximately 1.6, is observed in the electrically stimulated muscle in the presence of a homogeneous extracellular gadopentetate dimeglumine concentration (group 1). This increase results from the persistence of a significant concentration of gadopentetate dimeglumine in muscle at a time when its clearance out of the extracellular space is completed. The zones where electroporation allowed the cellular uptake of gadopentetate dimeglumine are thus revealed in locations where the electric field was delivered above the threshold value for membrane permeabilization (19). Conversely, after adequate correction for T2 variation, no significant variation in signal intensity is observed in muscles of group 2 animals electroporated before gadopentetate dimeglumine injection.

In animals of groups 1 and 2, the topography of the signal intensity increase, both on T1-weighted and T2-weighted images, is clearly related to the position of the plate electrodes, which determine the electrical field distribution inside the interelectrode space, and to the slight leg deformation due to the electrodes. Some anatomic structures such as bone and aponeurosis appear with lower signal intensity increase due to high collagen and low cellular fraction and consequently decreased access to gadopentetate dimeglumine. Indeed, the method described here allowed in vivo visualization of the spatial distribution of the efficient electric field evidenced by transient membrane permeabilization to gadopentetate dimeglumine.

The large T2 increase observed in all animals of groups 1 and 2 may result from cell necrosis (33) or edema. The low level of regenerating fibers determined in animals of group 1 does not argue in favor of a significant contribution of cell damage and regeneration to T2 variation. Edema may result from the intracellular penetration of osmotically active molecules during electroporation (34). Significant T2 modifications are known to result from weak variations of muscle volume induced by intense exercise, with osmotic flux driven by lactate accumulation (35). The measurement of local T2 variations is important in our experiments for correction of this effect.

Mathematic modeling has been proposed for calculation of the electric field distribution (36). Prediction of the magnitude and orientation of the electric field can be provided with three-dimensional computation of the electric field. However, this requires a precise knowledge of electrical constants in the biologic system and a mathematic description of interfaces between both tissues and the biologic system and the electrodes. In a rabbit liver (37), the predicted area of the electric field was confirmed with the experimental observation of the necrotic area submitted to the electric field intensity above the irreversible threshold. However, such a model, suitable for an isotropic and homogeneous organ such as the liver, would be extremely difficult to construct for any heterogeneous or complex-shaped organ. The results presented here show that MR imaging offers a new and atraumatic way to map the spatial distribution of an efficient electric field by using intracellular trapping of a contrast agent, thus avoiding difficulties of both modeling and invasiveness of ex vivo studies.

The efficiency and spatial extent of gene electrotransfer depend not only on the choice of the electric field parameters applied but also on other experimental design optimizations. Various experimental conditions (eg, volume and concentration of the injected plasmid cDNA, sites of delivery, depth of injection, delay between injection and electroporation, and temperature) can modify the results, as well as the ability of the plasmid to diffuse inside the extracellular space around the injection site. However, none of these previously described methods allowed in vivo visualization of the cellular uptake in complex conditions.

Here in mice, in which gadopentetate dimeglumine was coinjected in the gastrocnemius prior to electroporation with the plasmid containing the ß-galactosidase reporter gene, the T1-weighted signal intensity increase reflected the intracellular uptake of gadopentetate dimeglumine in a region of both the available extracellular gadopentetate dimeglumine and the efficiently applied electric field. The extent of this region in the treated gastrocnemius differed in shape and size among the animals of group 3 according to the site of delivery. For each animal, after X-Gal staining of histologic sections, the corresponding images evidenced a similarity between the area of plasmid uptake and the regions of MR signal intensity increase. The variation of gadopentetate dimeglumine uptake with increasing electric field strength is similar to the variation of cDNA expression reported by Mir et al (19), though gadopentetate dimeglumine is a much smaller molecule (700 Da) than the plasmid (32.105 kDa) and less negatively charged. Indeed, in this efficient protocol, which is widely used in mice, ex vivo MR images with use of intracellular gadopentetate dimeglumine demonstrated a useful representation of the area of cDNA uptake and expression. However, in some protocols there could be a dissociation between permeabilization and cDNA uptake. Control studies with histologic detection of gene expression should be performed before wider use of MR imaging.

Taken together, the in vivo and ex vivo results show that gadopentetate dimeglumine injection alone may help to set up experimental or therapeutic electrotransfer protocols. Furthermore, by assuming prior experimental verification that in the chosen conditions the contrast agent and the gene expression areas colocalized in muscle, gadopentetate dimeglumine coinjection with plasmid cDNA may be used to follow up the efficiency of the chosen procedure. MR imaging could then be used to guide control biopsies toward zones where a contrast agent has been trapped.

The issue of gadolinium (Gd) toxicity must be addressed. Gadopentetate dimeglumine residence time within muscle cells seems long, more than 1 week, which is consistent with the fact that no specific mechanism for its egress is known. Clearance of the contrast agent can only result from a very slow dissociation of the chelate, with elimination of the resulting low concentration of Gd3+ out of the cell. From the intracellular concentration of gadopentetate dimeglumine that we determined and from its stability constant (38), free Gd3+ ion concentration would be about 10-10 mol/L in this animal experiment performed with a high-dose contrast agent and thus would be of negligible toxicity.

Practical applications: High-field-strength MR imaging is used increasingly for animal physiologic studies (39), particularly in the field of muscle disorders (6,40,41), and is now available in many experimental centers. In this study, we show that MR imaging, coupled with an injection of a simple, inexpensive, and well-tolerated contrast agent, offers the opportunity to optimize muscle electrotransfer protocols without animal sacrifice. The intracellular trapping of paramagnetic contrast agents reveals the formation of transient pores in a given anatomic region. MR imaging can also be used as a control of the injection protocol, either shortly after injection without combination with electric pulses, or later, as in the previous studies, after electroporation. The paramagnetic contrast agent can be used either alone or mixed with a plasmid cDNA. In the latter case, interventional imaging may facilitate implementation in difficult injection protocols, with interactive guidance of the injection tool or with real-time control of the location of the contrast agent (1,42).

In vivo electroporation with use of cytotoxic drugs has already been used in humans in phase II trials (43–45) as a means for electrochemotherapy of tumors. In view of future human clinical applications for gene therapy, the method described here, which can be performed in a clinical environment, may provide a tool with which effectiveness of electrotransfer protocols is maximized while safety risks are minimized.


   ACKNOWLEDGMENTS
 
We thank Philippe Bozin for technical assistance and Dr Marc Fiszman for critically reading the manuscript and for discussions.


   FOOTNOTES
 
Abbreviations: cDNA = complementary DNA, EDTA = ethylenediaminetetraacetic acid, ROI = region of interest

Author contributions: Guarantors of integrity of entire study, A.L.W., K.S.; study concepts, A.L.W., M.P.J., P.G.C., J.T.V.; study design, M.P.J., P.G.C., A.L.W.; literature research, M.P.J., J.T.V., E.P., E.d.K.; experimental studies, M.P.J., A.L.W., E.P., C.W.; data acquisition, M.P.J., A.L.W., E.P.; data analysis/interpretation, M.P.J., A.L.W., G.V., C.W., E.d.K.; statistical analysis, P.G.C.; manuscript preparation, A.L.W., M.P.J., E.d.K.; manuscript definition of intellectual content, A.L.W., M.P.J., P.G.C.; manuscript editing, A.L.W., M.P.J., G.V., C.W.; manuscript revision/review and final version approval, all authors


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
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