a Division of Cardiovascular Diseases, Department of Biochemistry and Molecular Biology, Molecular Medicine Program, Mayo Clinic and Foundation, Rochester, MN, USA
b Basic Ultrasound Research Laboratory, Mayo Clinic and Foundation, Rochester, MN, USA
* Corresponding author. Robert D. Simari, M.D., Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Tel.: 507-284-3727; fax: 507-284-8566
E-mail address: simari.robert{at}mayo.edu
Received 8 November 2002; revised 13 May 2003; accepted 27 July 2003
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Abstract |
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Methods and Results In vitro, luciferase activity was measured after exposure of primary vascular cells to combinations of luciferase plasmid, contrast agents, and US exposures. US gene transfer was consistently superior to controls. PESDA was better than Optison; there was no significant difference between US modalities. In vivo, luciferase activity in skeletal muscle of rats was measured after injection of plasmid or adenovirus, expressing luciferase with or without US exposure. Diagnostic US was superior to continuous wave. US plasmid gene transfer was highly localized, and was superior to all controls except adenovirus which lacked spatial specificity. To deliver a secreted transgene product, US gene transfer of a plasmid expressing tissue factor pathway inhibitor (TFPI) to skeletal muscle resulted in a dose-related increase in plasma activity for up to 5 days after delivery.
Conclusion US-enhanced plasmid gene transfer is capable of transducing skeletal muscle in vivo either directly or via an intravascular route. This enhanced nonviral method is an alternative to plasmid DNA alone or viral vectors.
Key Words: Ultrasound Gene transfer Contrast agents
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1. Introduction |
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Despite recent advances in the field,815several important aspects remain unexplored, including the relative efficacy of different contrast agents, and the use of commercially available US scanners. While localized expression of a transgene after sonoporation has been demonstrated, the possibility of inducing systemic transgene effects (e.g., by transfer of a gene encoding for secreted protein) has not been previously evaluated. Our hypothesis was that different contrast agents and exposure modalities may be associated with different levels of transfection efficacy. In the present study we have compared the effect of two common echocardiographic contrast agents [Optison and perfluorocarbon exposed sonicated dextrose albumin (PESDA)] and of two US exposure modalities (pulsed US from a commercial scanner vs continuous wave US in a dedicated system) in vitro. Secondly, we tested the feasibility of US gene transfer into skeletal muscle after intramuscular and intravascular delivery of a reporter gene. Lastly, we investigated whether localized sonoporation of a gene encoding for biologically active secreted peptide (tissue pathway factor inhibitor; TFPI) into skeletal muscle results in significant changes in its circulating levels.
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2. Methods |
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2.2. Echocardiographic contrast media and ultrasound exposure modalities
PESDA was freshly prepared before each experiment, by mixing human albumin, 5% glucose, and decafluorbutane in a 1:3:2 ratio, as described elsewhere.17The commercially available contrast agent Optison (Mallinkrodt) was used within 24h after opening. The size distribution of microbubbles was measured from digital images of fresh dilutions (1:100 in 5% glucose), using the ImagePro software. At least 600 bubbles were counted for each measurement (n=4).
Two US systems were used: a commercially available scanner, and a dedicated continuous wave (CW) system. The US scanner (System V, GE Vingmed) was used with a phased-array transducer, in second harmonic mode, at 1.7MHz transmitted and 3.3MHz received frequency, and a mechanical index of 0.9 or 1.7. The dedicated US system was operated in CW mode at 1MHz transmitted frequency and using intensities of 0.5 or 0.75W/cm2. The peak negative pressure of the US exposure using both diagnostic and dedicated systems was measured with a calibrated hydrophone (model Y-33-7611, GEC-Marconi Research Centre, Chelmsford, UK) under mock experiment conditions on the bottom of a six-well plate immersed in a water tank. The peak negative pressures were 0.32 and 0.56MPa with the diagnostic system (at mechanical index of 0.9 and 1.7, respectively) and 0.32 and 0.41MPa (at 0.5 and 0.75W/cm2) with the dedicated system.
For in vitro experiments, insonation was performed through a water tank. The 6-well plates containing cell cultures were placed with the bottom immersed in 37°C water; the plasmid DNA in different combinations was added to the cell monolayer. In this way, the US beam travelled sequentially through water, plastic, cell monolayer, and media. To maximize cell-contrast microbubble interactions we have used the minimal quantity of media fluid that covered completely the monolayer (1000µl). With the commercial scanner, the phased-array probe was fixed under the water tank, and insonation was performed separately for each well. Focus was set at the bottom of the well. The distance between the probe and the well was 1cm (this was the shortest distance at which the US beam could be focused at 1.7MHz). The dedicated US system was the same as used in previous studies.6Four 35-mm diameter air-backed US transducers were fixed in a frame in such a way that the bottoms of the corner wells on the culture plate were aligned parallel with the top surface of the transducers. The frame was immersed in a water tank; the distance between the top of the transducers and the bottom of the well was 3mm. In this way, four wells could be insonated simultaneously. Total exposure time was 1-180s.
For in vivo experiments, insonation was performed through ultrasound-conducting gel. The targeted muscle was scanned continuously with either the commercial probe or with a dedicated US transducer at peak power (1.7 mechanical index at 1.7MHz with the commercial scanner, and 0.75W/cm2at 1MHz with the dedicated system) for a total of 3min.
2.3. In vitro experiments
Primary porcine vascular smooth muscle cells (VSMC) and human umbilical vein endothelial cells (HUVEC; Clonetics, Inc.) were cultured in supplemented 199 and EmBM growth media, respectively (GIBCO BRL). Cells in the 46th passage grown in 6-well plates at >70% confluence were used for transfection experiments. A dose of 10µg plasmid per well was used throughout the study. The plasmid was diluted in 750µl serum-free media and mixed with 250µl US contrast media. The mixture was added to the culture well, and exposure to US was performed immediately. After 2h, complete media was added and cells were incubated for another 24h prior to assessment of luciferase activity. The experimental regimens included (i) plasmid alone, (ii) plasmid with US, (iii) plasmid with contrast media without US and (iv) plasmid liposomal transfection (with LipofectAmine, GIBCO BRL) as controls. Plasmid liposomal transfection was performed according to the manufacturers protocol. Briefly, after vigorous vortexing for 1min, 20mcg LipofectAmine were suspended in 500µl serum-free growth media. The plasmid DNA was dissolved in separate 500µl media. The two parts were then gently mixed and added to the cell monolayer. Twenty-four hours after transfection, the cells were lysed, and luciferase activity and protein content were measured. Toxicity was assessed in separate experiments by counting the number of cells on ten high power fields in the center of the well before and 24h after US exposure. The number of cells alive at 24h was expressed as a percentage of the cells initially exposed. All experiments were performed in duplicate and reproduced 35 times.
2.4. In-vivo experiments
All animal studies were performed in accordance to the position of the American Heart Association on laboratory animal use and were approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and Foundation. Sprague-Dawley male rats weighing 300400g were anaesthetised with a mixture of ketamine (5mg/kg IM) and xylazine (0.1mg/kg IM). For experiments using IM delivery of tested agent, the skin overlying the triceps brachii or gastrocnemius was clipped, and a commercial depilatory was applied to the site to remove fine hairs. One of four doses of pRL-CMV (20200µg/muscle) was diluted in 100µl phosphate buffered saline (PBS), gently mixed with 50µl fresh PESDA, and injected IM. The needle was held in position for additional 10s to minimize backflow. Insonation was performed 35min after injection as described above. Control injections consisting of the corresponding plasmid dose alone, plasmid with PESDA, or plasmid with LipofectAmine were given into the contralateral triceps and not followed by US exposure. In another group, the gastrocnemius was also injected with pRL-CMV (50200µg) followed or not followed by US exposure. Additional controls were provided by injecting a total dose of 6.6x109plaque forming units AdLux into the triceps; in these animals, the contralateral triceps was used as non-injected control. The total volume for all IM injections was 150µl. In a limited number of experiments, fluorescent microspheres (FluoSphere, diameter 15µm) were injected into the triceps and gastrocnemius to label the injection site.
For intra-arterial delivery, animals were premedicated with ketorolac (0.3mg/kg IM) and dexamethasone (1mg/kg IM) to minimise adverse reactions to PESDA. The left carotid artery was dissected free and a thin silicon tube attached to a 30G needle was carefully advanced into the femoral artery. The left carotid artery was used, as its angle with the aortic arch favors advancing the silicon tube down the descending aorta into its femoral position. We were not able to guide the silicon catheter into a pre-defined artery (i.e., right or left femoral artery). We have instead advanced the silicon tube to a predefined depth (estimated at the beginning of the experiment as the distance between the neck incision and the groin), and verified the position of the catheter into the left or right hindlimb by imaging the limbs during injection of a minimal volume of diluted contrast (50% PESDA in PBS) via the silicon tube. A dose of 400µg pRL-CMV was diluted up to a total volume of 600µl with PBS and then gently mixed with 200µl PESDA. The mixture was then slowly injected intra-arterially over a 35min period. Insonation with the diagnostic scanner was performed during contrast injection and for additional 2min (to allow destruction of microbubbles that escaped during first passage). The contralateral gastrocnemius was used as a control non-injected, non-exposed muscle. All animals undergoing intraarterial delivery received also triceps IM injections of pRL-CMV as controls. The catheter was removed, the wound closed with standard surgical techniques, and the animals allowed to recover. Three days after US exposure the animals were sacrificed with a sodium pentobarbital overdose and the targeted and control muscles were excised in block. Samples were also taken from remote, non-injected muscles, as well as from liver, kidney, lung and heart. All animal samples were ground in luciferase lysis buffer (Promega).
Additional experiments were performed with IM injections of pCMV-TFPI plasmid in combination with PESDA into the triceps brachii. In these animals, blood samples were obtained at baseline, after 3 days, and at sacrifice.
2.5. Luciferase and TFPI assays
Cell cultures or tissue were lysed and exposed to one freeze-thaw cycle. Protein content was measured with a Coomassie blue colorimetric assay (Bio-Rad). Luciferase activity was measured with a commercially available kit (Promega) using a standard luminometer (Turner Designs). The results were expressed in light units (LU) and corrected for protein content. Plasma TFPI activity was measured with a chromogenic assay (Actichrome, American Diagnostica). The experiment was not randomized, but data analysis of the in vivo study was performed blinded to the allocated treatment by assigning a sample code that was broken only upon completion of the various assays.
2.6. Histological analysis
Serial sections from muscles injected with 100µg pRL-CMV plus PESDA and exposed to US and from contralateral muscles injected with PBS were stained with haematoxylin and eosin, and analysed for the presence of inflammation (46 sections per muscle). The inflammatory reaction was graded on a 3-point scale (0: normal tissue; 1: presence of inflammatory infiltrate; 2: presence of necrosis).
2.7. Detection of Apoptosis
Serial sections from muscles injected with 100µg pRL-CMV plus PESDA and exposed to US and from contralateral muscles injected with PBS were obtained at 46 separate levels of the muscle to be analysed. To detect presence of apoptotic nuclei, we have used the DeadEnd Fluorometric TUNEL System Promega kit (Promega, Madison WI). The kit measures the fragmented DNA of apoptotic cells by catalytically incorporating fluorescein-12-dUTP (a) at 3'-OH DNA ends using the enzyme Terminal Deoxynucleotidyl Transferase (TdT). TdT forms a polymeric tail using the principle of the TUNEL (TdT-mediated dUTP Nick-End Labelling) assay. The fluorescein-12-dUTP-labelled DNA can then be visualized directly by fluorescence microscopy. A negative control was obtained by omitting the TdT enzyme during incubation with the buffer. DNA labelling was performed by co-staining the sample with propidium iodide. Tissue sections were examined using a Nikon®fluorescent microscope equipped with an epi-illumination single band emitter filter cassette for the separate illumination of green (FITC) and blue (ultraviolet) fluorescence. Fluorescein-12-dUTP, once conjugated to the 3'-OH ends of fragmented DNA, stains the nuclei of apoptotic cells green, while propidium-iodide stained nuclei appear red. Each sample was inspected for presence of apoptotic nuclei; five representative low power field images (200x) were captured for further analysis.
2.8. Statistical analysis
Statistical analysis was performed with the SAS software package. Two-way ANOVA was used to test the effect of the following parameters: exposure time and contrast agent (in vitro), US type and intensity (in vitro), dose and US or no US (in vivo). The null hypothesis were: no time effect, no contrast agent effect, no time-contrast agent interaction (in vitro); no US modality effect, no US intensity effect (in vitro); no plasmid dose effect, no US exposure effect (in vivo); no TFPI plasmid dose effect, no time effect, and no dose-time interaction (TFPI experiments). One way ANOVA was used to test the differences between treatment and controls in vivo; Scheffes t-test was used for direct pairwise comparisons. Finally, repeated measures ANOVA was used when analyzing plasma TFPI activity data. The normal distribution was tested with the Shapiro-Wilk statistic and logarithmic transformations were performed when appropriate. A P-value less than 0.05 was considered significant (two-sided). All data are presented as mean±SEM.
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3. Results |
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3.3. In vivo luciferase experiments
A total of 41 rats were assigned to IM injections of pRL-CMV (n=26), intra-arterial (gastrocnemius) and IM (triceps) injections of pRL-CMV (n=9), and AdLux IM injections (n=6). The effects of diagnostic and dedicated US systems on gene transfer after intramuscular injection of a 100µg-dose of pRL-CMV into the triceps brachii were compared. Ultrasound exposure in the presence of PESDA resulted in higher luciferase activities than injection of plasmid alone, plasmid plus PESDA (without insonation), or plasmid lipofection (Fig. 3A). Luciferase activity was higher with diagnostic US than with the dedicated CW system (P<0.05). In the absence of US exposure, there were no significant differences between injection of plasmid DNA with and without PESDA. There was virtually no luciferase activity in non-treated muscle beds or in the liver, kidney, heart and lungs after US-mediated transfection (<500LU/mg for all). Luciferase activity after adenoviral gene transfer was 2-fold higher than after sonoporation. However, these results were obtained at the expense of a lack of specificity, with significant luciferase activity in the liver after AdLux (Fig. 3D).
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Of the nine animals that underwent intra-arterial injection of pRL-CMV (into the superficial femoral artery), due to technical difficulties in selective engagement only six showed contrast enhancement of the muscle bed at US during the intra-arterial injection. In these animals, luciferase activity in the corresponding gastrocnemius was comparable with that achieved after IM delivery into the triceps brachii, while the contralateral, non-injected hindlimb as well as the liver, kidney, heart and lung showed no luciferase activity. Animals in which no contrast enhancement of the muscle bed was observed (n=3) showed very low or absent luciferase activity.
3.4. Gene transfer of a secreted protein
The potential of enhancing gene transfer of a secreted protein was tested by injecting IM a plasmid expressing a secreted peptide (pCMV-TFPI) together with PESDA, followed by insonation with the diagnostic system. Plasma TFPI activity was measured up to 5 days post delivery. Both triceps brachii were injected in order to maximize a possible systemic effect. A total of nine rats were injected pCMV-TFPI at different doses: 200µg followed by US (n=3), 400µg followed by US (n=3) and 400µg not followed by US (n=3). Additional negative controls were obtained from rats used in luciferase experiments. Animals receiving a combination of plasmid with PESDA and US showed a significant dose-dependent increase of plasma TFPI activity 5 days after delivery (P<0.05) fig 4. No change in plasma TFPI activity was detected after IM injection of pCMV-TFPI and PESDA in the absence of US exposure.
3.5. Detection of inflammation and apoptosis
Detection of inflammation and apoptosis was performed in a limited number of experiments (n=3). A total of 52 hematoxylineosin stained sections from 3 animals were analysed (46/muscle, from both triceps and gastrocnemius). The vast majority of the sections showed presence of inflammatory infiltrate, mostly along the needle track, in both placebo and plasmid injected muscle. The average inflammation score from muscles injected with plasmid and PESDA and exposed to ultrasound was 1.05±0.33, with focal necrosis in 2 of 36 sections analysed. Muscles injected with saline had a score of 0.94±0.44 with focal necrosis in one of 16 sectionsanalysed. These differences were not statisticallysignificant.
Apoptosis was detected in all muscles injected with plasmid and PESDA and exposed to ultrasound, but not in those injected with saline. However the number of apoptotic nuclei was minimal (less than 1%) in all cases.
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4. Discussion |
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Many investigators consider inertial cavitation as the mechanism responsible for sonoporation.1,2,18However, knowledge of possible involvement of other mechanisms and of the relative role of different contrast agents remains limited. Ward et al.19have previously compared Albunex (an albumin-shell air-containing agent) and Optison. Their results have demonstrated that contrast agents are not equally effective in enhancing sonoporation. The differences were attributed mainly to the gas component of microbubbles, with air being less likely to be associated with cell membrane lysis. Taniyama and colleagues recently determined that Optison-enhanced gene transfer of hepatocyte growth factor increased angiogenesis in a rabbit model.14In our study we extended the results of these prior studies, by directly comparing Optison and PESDA. Our initial in vitro experiments have shown that the two contrast agents are not equally effective in enhancing US-mediated gene transfer. This finding is intriguing, considering the exceptionally similar physical properties. Thus, differences in the levels of gene transfer efficacy observed with the two agents cannot be directly explained by their composition and size. Considering that cavitation depends primarily on ultrasound field parameters (which were equal for the two agents) and on microbubble properties (gas compressibility, bubble diameter, shell properties, etc.), one could infer that Optison and PESDA should be equally effective in generating cavitation upon US exposure and, implicitly, induce membrane permeabilization to similar extent. Our findings suggest that mechanisms beyond cavitation could be also implied in sonoporation. Our results concur with the recent observation of Xie et al that higher negative electrostatic charge of the microbubble shell may play a role in sonoporation.20Further studies of the binding kinetics to the albumin shell, of electrical charge, and of the mechanism of DNA uptake upon insonation seem warranted.
When comparing the two US exposure systems, the effects in vitro were not significantly different. However, the diagnostic system was more efficient in vivo (P<0.05). Although we do not have a definitive explanation for the different efficacy of CW system in vitro and in vivo, one could speculate on the role played by standing waves and focusing. Although we were not able to identify the factors responsible for the noticed differences, our results demonstrate that optimal US conditions for sonoporation differ for in vitro and in vivo experiments, and thus in-vivo optimization should be considered for each gene delivery condition. Furthermore, optimal conditions are probably different for CW and diagnostic US, even when using similar peak negative pressures.
Our study demonstrates that sonoporation can be used to enhance gene transfer into skeletal muscle. Luciferase activities were on average 10 fold higher with a combination of plasmid, PESDA, and diagnostic US than with IM injections of plasmid alone. These differences were observed at all doses and in all muscle beds evaluated. Sonoporation-mediated gene transfer is robust, with US enhancement of luciferase activity observed in all animals tested.
We also compared in our experiments the efficacy of sonoporation in different muscle beds. Very large differences were unexpectedly observed in luciferase activity in the triceps brachii and gastrocnemius muscles. Danko et al. have also described inhomogeneity in luciferase activity after IM injection of a luciferase plasmid into various muscle beds.21To further evaluate the responsible mechanisms, in a small number of experiments (n=4) we injected fluorescent tracer microspheres into the triceps and gastrocnemius. Despite efforts to inject the total volume in different locations into the muscle (by positioning the needle at various angles during injection), the location of labelled microspheres was essentially restricted to the needle track. Furthermore, areas of fluorescence were also observed on the fascia separating the various hindlimb muscles. These observations suggest that the lower transfection efficacy achieved in the gastrocnemius may be at least in part due to flaws inherent to IM injection. Also the relative doses per volume in the two muscle beds may have been different. On the other hand, multiple injections of smaller doses of plasmid will probably allow larger distribution and implicitly larger effects than a single injection of a larger dose. Indeed, we took advantage of this observation in the TFPI studies, in which the total dose was divided into four separate injections given into the triceps brachii (two per triceps).
Sonoporation significantly enhanced gene transfer in comparison to plasmid alone or plasmid lipofection, and was approximately 50% of adenoviral gene transfer. However, the high luciferase activity obtained after viral transfection was obtained at the expense of decreased spatial specificity, with small, but measurable transfection into the contralateral limb, and higher levels of expression in the liver. These findings underscore once again one of the major limitations of adenoviral gene transfer, i.e. generalised effects and hence increased potential for adverse reactions and the potential for sonoporation of plasmid DNA.
In the next step, we evaluated sonoporation after selective injection of pRL-CMV plasmid and PESDA into the artery supplying the hindlimb muscles in rats. In these experiments we mimicked the situation where gene transfer to a muscle not directly accessible to IM delivery is contemplated, such as the heart, or when more diffuse delivery of genetic material into a particular target is required. We were able to induce luciferase activity at levels comparable to those obtained after direct IM injections of the triceps. Although sonoporation after IV injection has been demonstrated in the heart, the study was done in the context of a highly efficient adenoviral vector.9In our experiments, we were able to increase transgene expression in the skeletal muscle by exposure to US during intravascular delivery of a plasmid. These effects were obtained in the absence of gene transfer to the contralateral muscle or to internal organs, showing that highly localized gene therapy can be obtained by sonoporation even after systemic delivery of the genetic material, an important advantage over viral vectors with regard to safety concerns.
We have used a larger plasmid dose for intraarterial delivery. Indeed, after intraarterial injection, one can presume that during the first passage only a fraction of the plasmid will be retained in the muscle, while a significant portion will escape into the systemic circulation. On the contrary, when delivering a plasmid by IM injection, one presumes that the bulk of the product will remain in the muscle for a longer period of time. To the best of our knowledge, there is currently no published study that quantifies the amount of plasmid retained in the muscle after intraarterial delivery. We have therefore arbitrarily selected a double dose for intraarterial delivery to compensate for these differences.
Many human diseases could be treated with circulating biologically active peptides. Therefore, in the final step we have evaluated whether local gene transfer of TFPI into skeletal muscle by sonoporation is capable of inducing detectable changes in circulating levels of the corresponding peptides. In the case of TFPI, a relatively modest, but significant dose-related effect was observed after bilateral sonoporation of the triceps brachii. The effects persisted for at least five days. Whether these changes are associated with a significant biological effect and whether such an effect reaches the therapeutic level remains to be established.
Our results demonstrate that gene transfer by US exposure is feasible and robust. This method allows highly efficient tissue targeting that could be used when localised effects such as angiogenesis or antitumoral therapy are needed. The technique is straightforward; experiments were conducted with widely available equipment and reagents: commercially available US scanner, known echocardiographic contrast agents, plasmid DNA. Moreover, sonoporation into skeletal muscle is able to induce detectable changes in circulating levels of biologically active peptides, suggesting also the possibility of treating system diseases such as hypertension, heart failure, and coagulopathies.
Although contrast ultrasound examination is considered in general to be safe, interaction of tissue with ultrasound in the presence of gas-containing bubbles has potentially detrimental effects. Indeed, apoptosis was detected in as many as 17% of exposed cells in an in-vivo model.22A recent report by Feril et al shows apoptosis in as many as 30% of surviving cells, but this occurred at 4W/cm2under cell culture conditions. The same study showed apoptosis in less than 10% of the cells at levels of 1W/cm2and below.23In our experiments, apoptosis occurred in all animals exposed to ultrasound in the presence of PESDA and in none of the controls, but its extent was minimal. Furthermore, the presence of few isolated apoptotic nuclei in a polynucleated cell such as the striated muscle is of doubtful biological significance. We believe that our findings do not contradict existing data in the literature, but merely show that different conditions of exposure (power, duration, targeted tissue, in-vivo vs in-vitro) have different effect with regard to apoptosis.
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5. Study limitations |
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Transgene expression was assessed at a limited number of time points (three days for the luciferase experiments, and up to five days for the TFPI experiments). Therefore, the evolution in time of transgene expression in the targeted muscle cannot be assessed.
A dose-related response was observed with intramuscular pRL-CMV gene transfer up to 100 microgram. The 200-µg dose was less effective than the 100-µg dose, although differences were not statistically significant. We believe we have probably reached a plateau of gene expression, but we do not have data to confirm, as higher doses of plasmid were not tested. We did not test higher doses, as the volume to be injected was too large to be accommodated by the triceps.
Finally, blood samples for liver and kidney function tests were not obtained. This was due to the difficulty of obtaining a significant amount of blood at various time intervals in small rodents.
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Acknowledgments |
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Footnotes |
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References |
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