mice carrying loxp - flanked ( floxed ) alleles of cnb1 ( cnb1 ; backcrossed to a c57bl/6 strain ) were a kind gift from dr gerald crabtree . the cre recombinase estrogen receptor t2 ( ela - creert2 ) mutant line was a kind gift from dr craig logsdon , and it contains a transgenic insertion of a full - length acinar cell - specific mouse pancreatic elastase i ( ela ) promoter that  drives a tamoxifen - inducible creert2 . lox - stop - lox ( lsl)-tdtomato red reporter ( tm ) mice were obtained from the jackson lab ( farmington , ct ) . both male and female genetically engineered mice were used for the in  vivo studies . eight- to 10-week - old wild - type male and female swiss webster mice weighing 25 g were used to assess the efficacy of intraductal administration of fk506 and csa . all mice were housed at 22c with a 12-hour light - dark cycle and maintained on standard laboratory chow with free access to food and water . all animal experiments were performed using a protocol approved by the university of pittsburgh institutional animal care and use committee . cnb1 mice were crossed with ela - creert2 mice to generate homozygous ela - creert2/cnb1 strains . to delete cnb1 in pancreatic acinar cells ( cnb1 ) , creert2/cnb1 mice received a cumulative dose of 56 mg tamoxifen given intraperitoneally either daily or every other day for a total duration of 56 days . cnb1 lines lacking the ela - creert2 insertion served as controls , and they also received tamoxifen . genomic dna was prepared from freshly isolated mouse pancreas and liver tissue , as described . briefly , the tissue was minced on ice and homogenized in sodium chloride tris - edta buffer containing proteinase k. the homogenates were incubated at 55c for 3 hours with intermittent vortexing . after inactivation of proteinase k , the homogenates were centrifuged at 4c , and the supernatants containing genomic dna were precipitated with isopropanol . the precipitated genomic dna was pelleted at 4c , washed with 70% ethanol , air - dried , and dissolved in 200 l of 1    tris - edta buffer for polymerase chain reaction . a schematic of the location and size of the expected amplicons are provided in figure  1 . all sequences were aligned to the national center for biotechnology information database and manually verified to confirm cnb1 deletion and that each component ( eg , ela , cre , and ert2 ) was in frame . briefly , cells were incubated with adenovirus ( titer 2   10 infectious units ) for 30 minutes , and then were exposed to radiocontrast for approximately 6 hours . after stimulation , cells were collected , washed with phosphate - buffered saline once , lysed with 1 lysis buffer ( e397a ; promega , madison , wi ) , and centrifuged at 12,000   g for 5 minutes at 4c . luminescence was measured from the supernatant using the luciferase assay system ( e1483 ; promega ) in a synergy h1 plate reader ( biotek , winooski , vt ) , and total protein , determined by the bca kit ( thermo scientific , rockford , il ) , was used to normalize the data . adeno - associated virus ( aav)6 plasmids were generated by cloning a pela - icre or pcmv - zsgreen control vector into a paav - multiple cloning site plasmid ( vpk-410 ; cell biolabs , san diego , ca ) , as previously described.11 , 12 once cloned , the aav6 plasmid was transfected into hek293 cells along with 2 helper plasmids : ( 1 ) paav - repcap ( 0912 - 06 ; applied viromics , fremont , ca ) , which is a packaging plasmid that carries the serotype 6 rep and cap genes ; and ( 2 ) phelper ( 0913 ; applied viromics ) , which is a plasmid that carries the helper genes . cells were collected 72 hours after transfection and suspended in lysis buffer containing 50 mmol / l tris , 150 mmol / l nacl , and 2 mmol / l mgcl2 . aav6 was purified as previously described.12 , 13 briefly , transfected hek293 cells were freeze - thawed 3 times to release the aav6 virus . cell lysates were treated with benzonase ( 0.05 u ) at 37c for 30 minutes , followed by 1% sodium deoxycholate at 37c for 30 minutes . lysates were spun at 2500   g for 10 minutes , and the supernatant was collected . aav6 was precipitated using a 1:4 mixture of 40% polyethylene glycol ( peg-800 ) and 2.5 mol / l sodium chloride for 2 hours at 0c . the solution was spun at 2500    g for 30 minutes to collect the peg pellet . the pellet was resuspended in hepes buffer ( 50 mmol / l ) , treated with an equal volume of 100% chloroform , spun at 2500    g for 10 minutes , and air - dried for 30 minutes . two - phase partitioning was performed using 50% ammonium sulfate and 40% peg-800 and spun at 2500   g for 15 minutes . the ammonium sulfate phase was collected and dialyzed using a 10-kilodalton molecular weight cut - off slide - a - lyser dialysis cassette ( 66810 ; thermo scientific ) for 4 hours . the aavs were concentrated using a 50-kilodalton centrifugal filter unit ( ufc905024 ; millipore , billerica , ma ) and stored at -80c . the quicktiter aav quantitation kit ( vpk-145 ; cell biolabs ) was used to measure viral concentrations . the surgical procedure for retrograde pancreatic ductal infusion of briefly , 100 l of purified aav6 ( titer 2   10 plaque - forming units ) was infused into the biliopancreatic duct at a rate of 10 l / min for 10 minutes using a p33 peristaltic syringe pump ( harvard apparatus , holliston , ma ) . surgical anesthesia was achieved by inhaling isoflurane and oxygen . a single injection of the analgesic buprenorphine ( 0.075 mg / kg ) mice recovered on a heating pad for 30 minutes and were housed for 46 weeks with free access to food and water before induction of pep . to verify the efficacy of the aav6 infusion , lsl - tm mice were used . one hundred microliters of purified aav6ela - icre ( titer 2   10 plaque - forming units ) was infused into the pancreatic duct as described earlier . five weeks after the surgery , pancreas tissue , along with the abdominal organs en bloc , was imaged using a fluorescence dissecting microscope , then sectioned and immunostained . polyclonal rabbit anti-amylase ( sigma ) antibody was used for labeling acini , and polyclonal guinea pig anti - insulin ( dako , carpinteria , ca ) antibody was used for islets . ducts were labeled with biotinylated dolichos biflorus agglutinin lectin ( vector labs , burlingame , ca ) . images were acquired using a lsm710 confocal laser scanning microscope ( carl zeiss , jena , germany ) . the percentage of acinar cells showing red fluorescence was quantified using the image analysis software volocity ( perkinelmer inc , santa clara , ca ) , for a total of 18 fields at 200 magnification from 2 independent mouse infusions . briefly , 100  l iohexol ( omnipaque 300 ; ge healthcare , princeton , nj ) was infused retrograde into the biliopancreatic duct at a rate of 20 l / min for 5 minutes . mice from the low - volume normal saline ( lvns ) intraductal infusion group received retrograde infusion of 50 l normal saline into pancreatic duct at a lower rate of 10 l / min for 5 minutes . the experiments with the intraductal administration of cn inhibitors initially were performed in females and then were replicated in both sexes . the 2  complementary conditional knockout experiments each were performed in a single batch , based on the availability of littermates as well as the relatively large volume of aav  required for each mouse infusion . the experiments with the intraductal administration of cn inhibitors were performed in 3 separate batches of mice . serum was prepared by centrifuging at  1500   g for 10 minutes at 4c . serum amylase was measured using a phadebas kit ( amersham pharmacia , rochester , ny ) , and interleukin ( il)6 was measured using a standard enzyme - linked immunosorbent assay ( biolegend , san diego , ca ) . the pancreas , duodenum , and spleen were placed en bloc in a cassette to maintain anatomic orientation . ten systematically selected elds at 200 magnification were graded in a blinded fashion from the head of the pancreas , which was identified by its juxtaposition to the duodenum . the grading score gave equal weight ( from 0 to 3 ) for edema , inflammatory infiltration , and necrosis , as described.14 , 15 edema indices were delineated further objectively by performing intensity thresholding using imagej software ( national institutes of health , bethesda , md ) . were marked as edema , and their surface area was calculated as a percentage of the total parenchymal area . immunohistochemistry for myeloperoxidase ( mpo ) was performed from paraffin - embedded tissue sections using a leica bond - max fully automated immunohistochemistry and in - situ hybridization ( ish ) staining system ( leica , buffalo grove , il ) in a semiautomated manner . all of the products for the immunohistochemistry for mpo were purchased from leica , including the primary antibody . the slides were loaded on the bond system , and the program was set as follows : deparaffinized using bond dewax solution ( # ar9222 ) , dehydrated with alcohol , incubated with mpo ( # pa0491 ; ready - to - use ) primary antibody for 15 minutes and with a bond polymer refine detection kit ( # ds9800 ) . the slides were washed automatically using either bond wash solution ( # ar9590 ) or distilled water between each steps . after systematic optimization of the antibody using positive and negative control tissues , the ideal conditions for mpo were with no pretreatment needed , a 15-minute antibody incubation time , followed by 8 minutes post - primary and 8  minutes diaminobenzidine tetrahydrochloride hydrate incubation . five systematically selected fields at 50  magnification were graded in a blinded fashion from the head of the pancreas . a score from 0 to 3 mice carrying loxp - flanked ( floxed ) alleles of cnb1 ( cnb1 ; backcrossed to a c57bl/6 strain ) were a kind gift from dr gerald crabtree . the cre recombinase estrogen receptor t2 ( ela - creert2 ) mutant line was a kind gift from dr craig logsdon , and it contains a transgenic insertion of a full - length acinar cell - specific mouse pancreatic elastase i ( ela ) promoter that  drives a tamoxifen - inducible creert2 . lox - stop - lox ( lsl)-tdtomato red reporter ( tm ) mice were obtained from the jackson lab ( farmington , ct ) . both male and female genetically engineered mice were used for the in  vivo studies . eight- to 10-week - old wild - type male and female swiss webster mice weighing 25 g were used to assess the efficacy of intraductal administration of fk506 and csa . all mice were housed at 22c with a 12-hour light - dark cycle and maintained on standard laboratory chow with free access to food and water . all animal experiments were performed using a protocol approved by the university of pittsburgh institutional animal care and use committee . cnb1 mice were crossed with ela - creert2 mice to generate homozygous ela - creert2/cnb1 strains . to delete cnb1 in pancreatic acinar cells ( cnb1 ) , creert2/cnb1 mice received a cumulative dose of 56 mg tamoxifen given intraperitoneally either daily or every other day for a total duration of 56 days . cnb1 lines lacking the ela - creert2 insertion served as controls , and they also received tamoxifen . genomic dna was prepared from freshly isolated mouse pancreas and liver tissue , as described . briefly , the tissue was minced on ice and homogenized in sodium chloride tris - edta buffer containing proteinase k. the homogenates were incubated at 55c for 3 hours with intermittent vortexing . after inactivation of proteinase k , the homogenates were centrifuged at 4c , and the supernatants containing genomic dna were precipitated with isopropanol . the precipitated genomic dna was pelleted at 4c , washed with 70% ethanol , air - dried , and dissolved in 200 l of 1    tris - edta buffer for polymerase chain reaction . a schematic of the location and size of the expected amplicons are provided in figure  1 . all sequences were aligned to the national center for biotechnology information database and manually verified to confirm cnb1 deletion and that each component ( eg , ela , cre , and ert2 ) was in frame . briefly , cells were incubated with adenovirus ( titer 2   10 infectious units ) for 30 minutes , and then were exposed to radiocontrast for approximately 6 hours . after stimulation , cells were collected , washed with phosphate - buffered saline once , lysed with 1 lysis buffer ( e397a ; promega , madison , wi ) , and centrifuged at 12,000   g for 5 minutes at 4c . luminescence was measured from the supernatant using the luciferase assay system ( e1483 ; promega ) in a synergy h1 plate reader ( biotek , winooski , vt ) , and total protein , determined by the bca kit ( thermo scientific , rockford , il ) , was used to normalize the data . adeno - associated virus ( aav)6 plasmids were generated by cloning a pela - icre or pcmv - zsgreen control vector into a paav - multiple cloning site plasmid ( vpk-410 ; cell biolabs , san diego , ca ) , as previously described.11 , 12 once cloned , the aav6 plasmid was transfected into hek293 cells along with 2 helper plasmids : ( 1 ) paav - repcap ( 0912 - 06 ; applied viromics , fremont , ca ) , which is a packaging plasmid that carries the serotype 6 rep and cap genes ; and ( 2 ) phelper ( 0913 ; applied viromics ) , which is a plasmid that carries the helper genes . cells were collected 72 hours after transfection and suspended in lysis buffer containing 50 mmol / l tris , 150 mmol / l nacl , and 2 mmol / l mgcl2 . aav6 was purified as previously described.12 , 13 briefly , transfected hek293 cells were freeze - thawed 3 times to release the aav6 virus . cell lysates were treated with benzonase ( 0.05 u ) at 37c for 30 minutes , followed by 1% sodium deoxycholate at 37c for 30 minutes . lysates were spun at 2500   g for 10 minutes , and the supernatant was collected . aav6 was precipitated using a 1:4 mixture of 40% polyethylene glycol ( peg-800 ) and 2.5 mol / l sodium chloride for 2 hours at 0c . the solution was spun at 2500    g for 30 minutes to collect the peg pellet . the pellet was resuspended in hepes buffer ( 50 mmol / l ) , treated with an equal volume of 100% chloroform , spun at 2500    g for 10 minutes , and air - dried for 30 minutes . two - phase partitioning was performed using 50% ammonium sulfate and 40% peg-800 and spun at 2500   g for 15 minutes . the ammonium sulfate phase was collected and dialyzed using a 10-kilodalton molecular weight cut - off slide - a - lyser dialysis cassette ( 66810 ; thermo scientific ) for 4 hours . the aavs were concentrated using a 50-kilodalton centrifugal filter unit ( ufc905024 ; millipore , billerica , ma ) and stored at -80c . the quicktiter aav quantitation kit ( vpk-145 ; cell biolabs ) was used to measure viral concentrations . the surgical procedure for retrograde pancreatic ductal infusion of the aav6 was as previously described . briefly , 100 l of purified aav6 ( titer 2   10 plaque - forming units ) was infused into the biliopancreatic duct at a rate of 10 l / min for 10 minutes using a p33 peristaltic syringe pump ( harvard apparatus , holliston , ma ) . surgical anesthesia was achieved by inhaling isoflurane and oxygen . a single injection of the analgesic buprenorphine ( 0.075 mg / kg ) mice recovered on a heating pad for 30 minutes and were housed for 46 weeks with free access to food and water before induction of pep . to verify the efficacy of the aav6 infusion , one hundred microliters of purified aav6ela - icre ( titer 2   10 plaque - forming units ) was infused into the pancreatic duct as described earlier . five weeks after the surgery , pancreas tissue , along with the abdominal organs en bloc , was imaged using a fluorescence dissecting microscope , then sectioned and immunostained . polyclonal rabbit anti-amylase ( sigma ) antibody was used for labeling acini , and polyclonal guinea pig anti - insulin ( dako , carpinteria , ca ) antibody was used for islets . ducts were labeled with biotinylated dolichos biflorus agglutinin lectin ( vector labs , burlingame , ca ) . images were acquired using a lsm710 confocal laser scanning microscope ( carl zeiss , jena , germany ) . the percentage of acinar cells showing red fluorescence was quantified using the image analysis software volocity ( perkinelmer inc , santa clara , ca ) , for a total of 18 fields at 200 magnification from 2 independent mouse infusions . pep was induced as previously described . briefly , 100  l iohexol ( omnipaque 300 ; ge healthcare , princeton , nj ) was infused retrograde into the biliopancreatic duct at a rate of 20 l / min for 5 minutes . mice from the low - volume normal saline ( lvns ) intraductal infusion group received retrograde infusion of 50 l normal saline into pancreatic duct at a lower rate of 10 l / min for 5 minutes . the experiments with the intraductal administration of cn inhibitors initially were performed in females and then were replicated in both sexes . the 2  complementary conditional knockout experiments each were performed in a single batch , based on the availability of littermates as well as the relatively large volume of aav  required for each mouse infusion . the experiments with the intraductal administration of cn inhibitors were performed in 3 separate batches of mice . serum was prepared by centrifuging at  1500   g for 10 minutes at 4c . serum amylase was measured using a phadebas kit ( amersham pharmacia , rochester , ny ) , and interleukin ( il)6 was measured using a standard enzyme - linked immunosorbent assay ( biolegend , san diego , ca ) . the pancreas , duodenum , and spleen were placed en bloc in a cassette to maintain anatomic orientation . ten systematically selected elds at 200 magnification were graded in a blinded fashion from the head of the pancreas , which was identified by its juxtaposition to the duodenum . the grading score gave equal weight ( from 0 to 3 ) for edema , inflammatory infiltration , and necrosis , as described.14 , 15 edema indices were delineated further objectively by performing intensity thresholding using imagej software ( national institutes of health , bethesda , md ) . labeled areas within the parenchyma were marked as edema , and their surface area was calculated as a percentage of the total parenchymal area . immunohistochemistry for myeloperoxidase ( mpo ) was performed from paraffin - embedded tissue sections using a leica bond - max fully automated immunohistochemistry and in - situ hybridization ( ish ) staining system ( leica , buffalo grove , il ) in a semiautomated manner . all of the products for the immunohistochemistry for mpo were purchased from leica , including the primary antibody . the slides were loaded on the bond system , and the program was set as follows : deparaffinized using bond dewax solution ( # ar9222 ) , dehydrated with alcohol , incubated with mpo ( # pa0491 ; ready - to - use ) primary antibody for 15 minutes and with a bond polymer refine detection kit ( # ds9800 ) . the slides were washed automatically using either bond wash solution ( # ar9590 ) or distilled water between each steps . after systematic optimization of the antibody using positive and negative control tissues , the ideal conditions for mpo were with no pretreatment needed , a 15-minute antibody incubation time , followed by 8 minutes post - primary and 8  minutes diaminobenzidine tetrahydrochloride hydrate incubation . five systematically selected fields at 50  magnification were graded in a blinded fashion from the head of the pancreas . a score from 0 to 3 was used to grade the extent of brown color in each field . to delete cn selectively in pancreatic acinar cells , we crossed a mouse line containing floxed alleles for the critical regulatory subunit b1 ( cnb1 ) with a tamoxifen - inducible cre line driven by a full - length acinar - specific mouse ela promoter ( figure  1 ) . a mild model of pep was first induced by infusing lvns into the biliopancreatic duct . in this model , we found that there was a near - complete reduction in histologic damage among the acinar cell - specific cnb1-deficient mice ( cnb1 ) ( figure  2 ) . furthermore , we developed a more severe model of injury that mimics pep by infusing radiocontrast at double the volume and rate of infusion within the same time frame as  the mild model . in this model , we observed that the cnb1 mice also had a marked reduction in histologic damage by 75% , down to the level of the sham - operated negative control arm . each parameter of the overall histologic score was diminished , including edema , inflammatory infiltrate ( additionally examined by mpo staining ) , and necrosis . in addition to the mild and moderate models of pep , we observed that the acinar cell - specific cn deletion also protected against a disparate model of acute pancreatitis induced by infusion of the bile acid taurocholate ( figure  3 ) . the findings lend further support to the broad importance of acinar cell cn in mediating pancreatic injury . we complemented the breeding strategy for acinar cell cn knockouts by generating an aav6 vector , which houses an enhanced version of cre ( icre ) that is driven by a shorter , independently constructed rat ela promoter ( figure  4 ) . among serotypes , aav6 , along with aav8 , offer the highest infection efficiency into acinar cells.12 , 18 as proof of principle for targeting acinar cells , the aav6-ela - icre induced acinar cell fluorescence in lsl - tm mice . immunostaining confirmed that the activation of icre is restricted largely to acinar cells , but not ducts and islets . the percentage of acinar cells showing red fluorescence was 37%  3.88% and western blot from the head of the pancreas showed that cnb1 expression was reduced by 33% ( figure  4 ) . despite the modest reduction in cn systemic inhibition of cn with administration of multiple doses of cn inhibitors before and after pep induction was shown previously to protect against pep . however , the current findings , with the 2 genetic cn deletion models , that acinar cell cn in  vivo is necessary for pep prompted us to question whether selectively targeting acinar cell cn activity by administering a single , acute dose of cn inhibitor , along with the radiocontrast infusion , could mitigate pep . this unique compartmentalized method of delivery of a small amount of drug would additionally reduce the toxicity profile of the inhibitors . mol / l ) each were dissolved easily in the ready - to - use iohexol formulation , and the concentrations of each were chosen based on our previous ex  vivo data . in contrast to the finding with the cnb1 conditional knockouts ( ie , the cnb1 line ) , intraductal fk506 or csa therapy did not affect the mild histologic damage seen with lvns ( figure  6 ) . this differential response likely represents incomplete cn blockade with the current dosing schema . however , this pharmacologic intervention reduced the severity in the moderate model of pep by 61% and 37% down to sham levels , respectively . both serum il6 and amylase levels in summary , using 2 complementary genetic approaches to delete acinar cell cn in  vivo and in 2 severity models of pep in mice , as well as a bile infusion model of pancreatitis , we show that pep and pancreatitis can be largely prevented by acinar cell cn deletion . notably , even a partial deletion of acinar cn ( by approximately one third with the intraductal aav strategy ) led to marked protection against pep . the translational corollary to these significant findings is that intraductal delivery of cn inhibitors , to target acinar cell cn in  vivo , also was shown to reduce pep . these novel findings reconcile the paradox that chronic and systemic administration of cn inhibitors could predispose to pancreatitis and pancreatic fibrosis,19 , 20 whereas acute and targeted delivery to the pancreas protects against pancreatitis . this work provides the impetus for launching clinical trials to test the efficacy of a novel ercp infusion formulation containing cn inhibitors to prevent pep .
background & aimsthere is a pressing need to develop effective preventative therapies for post  endoscopic retrograde cholangiopancreatography pancreatitis ( pep ) . 
 we showed 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.methodswe 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 adeno - associated virus 
 pancreatic elastase improved cre ( i  icre ) into the pancreatic duct of a calcineurin floxed line.resultswe found that pep is dependent on acinar cell calcineurin in  vivo , and this led us to determine that calcineurin inhibitors , infused within the radiocontrast , largely can prevent pep.conclusionsthese results provide the impetus for launching clinical trials to test the efficacy of intraductal calcineurin inhibitors to prevent pep .