Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence

Orabi, Abrahim I.; Sah, Swati; Javed, Tanveer A.; Lemon, Kathryn; Good, Misty; Guo, Ping; Xiao, Xianmin; Prasadan, Krishna; Gittes, George K.; Jin, Shunqian; Husain, Sohail Z.

doi:10.1074/jbc.m115.647933

Cited by 14 publications

(13 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Live, dynamic imaging of pancreatic NF-κB was achieved through a novel AAV6-mediated gene delivery of NF-κB-luciferase into the mouse pancreas by infusing AAV6-NF-κB-luciferase into the pancreatic duct, as detailed in the Methods 39 . Compared to a sham-operated mouse that received intraductal normal saline, RC-infused mice had a 13-fold peak in NF-κB luciferase 4 hr after surgery, within the region of the pancreas (Figure 5D).…”

Section: Resultsmentioning

confidence: 99%

“…An AAV6-NF-κB-luciferase reporter was constructed, expanded in HEK293 cells, and purified as previously published 18–20, 39 . A volume of 100 μl of virus (10 12 GCP/ml) was infused into the pancreaticobiliary duct at a rate of 10 μl/min using the same technique of RC infusion described above and recently published 21, 39 .…”

Section: Methodsmentioning

confidence: 99%

“…A volume of 100 μl of virus (10 12 GCP/ml) was infused into the pancreaticobiliary duct at a rate of 10 μl/min using the same technique of RC infusion described above and recently published 21, 39 . After a 5-week recovery from the surgery, mice underwent PEP induction with or without FK506 treatment.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

Jin

Orabi

et al. 2015

Gastroenterology

Self Cite

View full text Add to dashboard Cite

Background & Aims Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice. Methods We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque) and measured intracellular release of Ca2+, calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAβ−/− mice were also used. This experiment was also performed in mice given infusions of AAV6-NF-κB-luciferase, to assess activation of this transcription factor in vivo. Results Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca2+ signaling, along with activation of the transcription factors NF-κB and NFAT. Suppressing Ca2+ signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aβ-deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice; this was observed by live imaging of mice given infusions of AAV6- NF-kB-luciferase. Conclusions Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca2+ signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis in patients.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

Jin

Orabi

et al. 2015

Gastroenterology

Self Cite

View full text Add to dashboard Cite

show abstract

“…AAV6 plasmids were generated by cloning a pEla-iCre or pCMV-ZsGreen control vector into a pAAV-MCS plasmid (Cell Biolab #VPK-410, San Diego, CA), as previously described 11, 12 . Once cloned, the AAV6 plasmid was transfected into HEK293 cells along with two helper plasmids: (1) pAAV-RepCap (Applied Viromics #0912-06, Fremont, CA), which is a packaging plasmid that carries the serotype 6 rep and cap genes; and (2) pHelper (Applied Viromics # 0913, Fremont, CA), which is a plasmid that carries the helper genes.…”

Section: Methodsmentioning

confidence: 99%

Targeted Inhibition of Pancreatic Acinar Cell Calcineurin Is a Novel Strategy to Prevent Post-ERCP Pancreatitis

Orabi

Wen

Javed

et al. 2017

Cellular and Molecular Gastroenterology and Hepatology

Self Cite

View full text Add to dashboard Cite

Background and Aims There is a pressing need to develop effective preventative therapies for post-ERCP pancreatitis (PEP). We demonstrated that early PEP events are induced through the calcium-activated phosphatase calcineurin and that global calcineurin deletion abolishes PEP in mice. A crucial question is whether acinar cell calcineurin controls the initiation of PEP in vivo. Methods We used a mouse model of PEP and examined the effects of in vivo acinar cell-specific calcineurin deletion by either generating a conditional knockout line or infusing a novel AAV-Ela-iCre into the pancreatic duct of a calcineurin floxed line. Results We found that PEP is dependent on acinar cell calcineurin in vivo, and this led us to determine that calcineurin inhibitors, infused within the radiocontrast, can largely prevent PEP. Conclusions These results provide impetus for launching clinical trials to test the efficacy of intraductal calcineurin inhibitors to prevent PEP.

show abstract

“…[4,19–22] Despite the ability of the L342A luciferase to emit light given sufficient quantities of D-luciferin and ATP in vitro, in the context of the live mouse brain we do not detect a signal. On the other hand, strong brain bioluminescence is observed from the L342A luciferase when using the aminoluciferins, but not with the double mutant R218K/L342A, despite its higher peak performance in vitro.…”

mentioning

confidence: 99%

Firefly Luciferase Mutants Allow Substrate‐Selective Bioluminescence Imaging in the Mouse Brain

et al. 2016

View full text Add to dashboard Cite

Bioluminescence imaging is a powerful approach to visualize specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Here we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. Using adeno-associated viral (AAV) vectors to express luciferases in the brain, we find that mutant luciferases that are inactive or weakly active with D-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.

show abstract

Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence

Cited by 14 publications

References 54 publications

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

Targeted Inhibition of Pancreatic Acinar Cell Calcineurin Is a Novel Strategy to Prevent Post-ERCP Pancreatitis

Firefly Luciferase Mutants Allow Substrate‐Selective Bioluminescence Imaging in the Mouse Brain

Contact Info

Product

Resources

About