<em>In vivo</em> Site-Specific Transfection of Naked Plasmid DNA and siRNAs in Mice by Using a Tissue Suction Device

<div><p>We have developed an <em>in vivo</em> transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the <em>in vivo</em> transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.</p> </div

Tissue suction devices used in this study.

<p>Tissue suction devices used in this study.</p

<i>In vivo</i> transfection of naked pDNA by tissue suction.

<p>A) <i>In vivo</i> imaging of luciferase activity in a mouse liver that was suctioned once by the type I device just after intravenous injection of pCMV-Luc. B) <i>Ex vivo</i> imaging of luciferase activity in the liver suctioned by the type I device. C) Bright field image of (B). D) Luciferase levels of various tissues. The right kidney in mice was suctioned once by the type III device. Each value represents means + SD (n  = 4). All mice were alive at the end of the experiment.</p

Effects of tissue suction on hepatic toxicity.

<p>A) Alanine aminotransferase (ALT) in serum. ALT activity was measured at 0, 6, 24, and 48 h after transfection. Type III device was used. Each value represents mean ± SD (n  = 3 [sham operation], or n  = 4 [tissue suction]). *p<0.05 versus sham operation. B) Aspartate aminotransferase (AST) in serum. AST activity was measured at 0, 6, 24, and 48 h after transfection. Type III device was used. Each value represents mean ± SD (n  = 3 [sham operation], or n  = 4 [tissue suction]). *p<0.05 versus sham operation. C) HE staining of the liver section. The suctioned part of the liver (Part I in Fig. 4A) was sampled at 0 and 7 days after tissue suction. Type III device was used. All mice were alive at the end of the experiment.</p

<i>In vivo</i> transfection to various tissues by tissue suction.

<p><i>In vivo</i> transfection by tissue suction was applied to various tissues (including the kidney, heart, spleen, liver, duodenum, muscle, and stomach). A type III device was used for the muscle and stomach. A type IV device was used for the kidney, heart, spleen, liver, and duodenum. Each value represents means + SD (n  = 3 [the kidney, spleen, and muscle], n  = 4 [the liver, duodenum, and stomach], or n  = 5 [the heart]). All mice were alive at the end of the experiment.</p

Luciferase gene expression around the suctioned region of the liver.

<p>A) Schematic illustration of the experiment. The liver was suctioned by the type III device, and the liver was cut into 4 parts (I to IV). B) Luciferase levels in each of the 4 parts. The values were normalized to levels from Part I (n  = 4). C) The luciferase level of <i>in vivo</i> transfection by tissue suction was compared with that by hydrodynamic method and tissue pressure-mediated transfection. *p<0.01 versus tissue pressure. ^#p<0.01 versus tissue suction. Each value represents means + SD (n  = 3 [hydrodynamic method], n  = 8 [tissue pressure], or n  = 5 [tissue suction]). D) Imaging of the top surface of Part I transfected with pCMV-GFP. Scale bar, 200 µm. All mice were alive at the end of the experiment.</p

Tissue suction by using the device.

<p>A) Representative photograph of the tissue suction device (type II). B) Schematic illustration of <i>in vivo</i> transfection by tissue suction. The surface of the target tissues was suctioned by the suction device just after intravenous injection of naked nucleic acids.</p

siRNA transfection to the liver by tissue suction.

<p>GAPDH siRNA and scramble siRNA were transfected by using type II device. mRNA expression of GAPDH after 24 h of transfection was measured. Each value represents means + SD (n  = 3 [N.T.], n  = 4 [GAPDH siRNA and scramble siRNA]). There was a statistically significant difference between 3 groups (ANOVA; F  = 99.72, p<0.0001). Post-hoc analysis (Bonferroni’s test) was performed. *p<0.001 versus N.T. ^#p<0.001 versus scramble siRNA. All mice were alive at the end of the experiment.</p

Effects of the number of tissue suctions on luciferase levels.

<p>A) <i>Ex vivo</i> imaging of luciferase activity in the liver simultaneously suctioned at 2 different parts. Two type III devices were used. B) Bright field image of (A). C) Effects of interval between pCMV-Luc injection and tissue suction on the luciferase levels in liver. The liver was suctioned by type III devices. D) Effects of the number of tissue suctions on luciferase levels were investigated. Liver were serially suctioned 1, 3, and 7 times by using the type III device within 180 s of pCMV-Luc injection. All mice were alive at the end of the experiment.</p

Table_2_Magnetic resonance imaging arterial spin labeling hypoperfusion with diffusion-weighted image hyperintensity is useful for diagnostic imaging of Creutzfeldt–Jakob disease.pdf

Background and objectivesMagnetic resonance imaging with arterial spin labeling (ASL) perfusion imaging is a noninvasive method for quantifying cerebral blood flow (CBF). We aimed to evaluate the clinical utility of ASL perfusion imaging to aid in the diagnosis of Creutzfeldt–Jakob disease (CJD).MethodsThis retrospective study enrolled 10 clinically diagnosed with probable sporadic CJD (sCJD) based on the National CJD Research & Surveillance Unit and EuroCJD criteria and 18 healthy controls (HCs). Diffusion-weighted images (DWIs), CBF images obtained from ASL, N-isopropyl-(123I)-p-iodoamphetamine (123IMP)-single-photon emission computed tomography (SPECT) images, and 18F-fluorodeoxyglucose (18FDG)-positron emission tomography (PET) images were analyzed. First, the cortical values obtained using volume-of-interest (VOI) analysis were normalized using the global mean in each modality. The cortical regions were classified into DWI-High (≥ +1 SD) and DWI-Normal (ResultsThe mean values of ASL-CBF (N = 10), 123IMP-SPECT (N = 8), and 18FDG-PET (N = 3) in DWI-High regions were significantly lower than those in the DWI-Normal regions (p DiscussionPatients with CJD showed ASL hypoperfusion in lesions with DWI hyperintensity, suggesting that ASL-CBF could be beneficial for the diagnostic aid of CJD.</p