natural orifice translumenal endoscopic surgery ( notes ) has developed rapidly in recent years , and transvaginal cholecystectomy has been successfully performed in humans .
researchers are actively evaluating different peritoneal accesses and development of novel instruments and innovative procedures .
there have been few reports , however , on the immunological effects of notes . in the development of laparoscopic surgery
laparoscopic procedures are associated with less host inflammatory response compared with that in open surgery .
in addition , a more favorable cellular and humoral immunological response has been observed in laparoscopic colorectal resection in terms of a lower level of circulating il-6 and tnf-. theoretically , with the lower degree of trauma induced by notes , a lower peri - operative proinflammatory response would be expected . in the current study , we aimed at analyzing the systemic inflammatory responses associated with transvaginal cholecystectomy in a porcine model .
this study , using transvaginal cholecystectomy in the porcine model , was carried out in the animal laboratory , department of surgery , university of hong kong .
the pigs , weighing 30 kg to 35 kg , were cared for by a registered veterinarian working in our laboratory before and after the procedures . before the experiment
, the pigs were housed in a large animal care facility and were maintained in an environment of 201c .
they were quarantined and allowed to acclimatize in the animal facility for a minimum of 1 week before being used .
all animals were given a standard diet and water ad libitum until the time of the experiment .
the study was approved by the committee on the use of live animals in teaching and research of the university of hong kong .
all experiments conducted in pigs followed the animal welfare guidelines for the care and use of laboratory animals .
six female pigs were used for the survival study after transvaginal cholecystectomy performed with endoscopic submucosal dissection ( esd ) instruments and a single - channel endoscope .
preparation was the same as in the pigs used for transvaginal cholecystectomy except that no operation was performed and anesthesia was reversed 180 minutes after induction .
all pigs were fasted overnight before the procedure and were given 0.5 mg / kg ketamine and 1.2 mg atropine sulphate by intramuscular injection as a premedication before anesthesia .
an ear vein was used for injection of 0.5 mg / kg of dormicum , 0.1 mg / kg of pavulon , and 0.01 mg / kg fentanyl for induction .
the trachea was intubated with a cuffed endotracheal tube using an illuminated laryngoscope with an extra - long blade .
periodic injection of pavulon and fentanyl was used to ensure adequate anesthesia and muscle relaxation . on completion of the operation ,
1 mg of neostigmine and 1.2 mg of atropine were used for anesthesia reversal , and the pigs were extubated .
ampicillin in a dose of 500 mg was injected intravenously just before the procedure and continued for 5 days every 8 hours .
buprenorphine 0.05 mg / kg to 0.2 mg / kg was given every 12 hours for 3 days after the operation for pain control .
the pigs were sacrificed with 200 mg / kg of pentobarbital sodium 2 weeks after the experiment .
all operating instruments were sterilized according to the standard autoclave protocol , whereas the endoscope was disinfected by immersing it for 20 minutes in 2.4% glutaraldehyde ( cidex , johnson and johnson , new brunswick , nj ) and then rinsed with sterile water .
the peritoneal access and cholecystectomy were performed with a single - channel endoscope ( gif - q240x , olympus optical co , ltd , tokyo , japan ) .
the skin was prepared with povidone - iodine solution , and the vagina was irrigated with the same solution .
a veress needle was inserted for creation of carbon dioxide pneumoperitoneum and maintenance of intraperitoneal pressure at 12 mm hg .
the urogenital sinus was cannulated with the endoscope , and the vagina cavity was entered .
after the cervix was identified , 2 approaches were used for accessing the peritoneal cavity : ( 1 ) the anterior vaginal wall was dissected with a hook knife ( olympus optical co , ltd , tokyo , japan ) , and air was insufflated during colpotomy to maintain an optimal endoscopic view ; the entrance to the peritoneal cavity was recognized by a sudden increase in monitored intraperitoneal pressure , and the hook knife then served as a guidewire for insertion of the endoscope into the peritoneal cavity : ( 2 ) the endoscope was passed beyond the cervix to enter the retroperitoneal plane , and the peritoneum was incised using a hook - knife to gain access to the peritoneal cavity .
the endoscope was then manipulated so that minimal looping would be created and an end - on view of the cystic duct could be achieved . the gallbladder was retracted cranially and laterally .
the covering peritoneum was incised and dissected using a combination of hook knife and hot biopsy forceps .
after the cystic duct and artery were isolated , both were clipped together with endoscopic hemoclip ( olympus optical co , ltd , tokyo , japan ) and divided with endoscopic scissors ( olympus ) .
the gallbladder was then dissected from its liver bed by using a hook knife or it ( insulated tip ) knife , or both , ( olympus optical co , ltd , tokyo , japan ) with pd-60 and endoplasma ( olympus optical co , ltd , tokyo , japan ) where appropriate .
use of the it knife was advantageous as it could protect the liver from diathermy injury and minimize the chance of accidental gallbladder perforation .
after clearing accumulated intraperitoneal fluid by suction via the endoscope , the gallbladder was grasped with either a pair of biopsy forceps or an endoscopic snare under direct vision .
porcine serum was prepared by centrifuging the clotted blood for 10 minutes at 3000 rpm , and the serum was then stored in aliquots at -80c . after collecting all specimens required for experimentation , tnf- and il-6 were quantitatively measured by the elisa method [ r&d systems ( quantikine ) , minneapolis , mn , usa ] .
the colored product formed at the end of assays was measured according to the manufacturer 's instructions , and the concentrations of tnf- and il-6 were subsequently determined from their standard curves .
the differences between operated on and control groups were determined by using the one - way anova test . all statistical analysis was calculated using spss for windows version 11.5 ( chicago , il ) ( tables 1 and 2 ) .
six female pigs were used for the survival study after transvaginal cholecystectomy performed with endoscopic submucosal dissection ( esd ) instruments and a single - channel endoscope .
preparation was the same as in the pigs used for transvaginal cholecystectomy except that no operation was performed and anesthesia was reversed 180 minutes after induction .
six female pigs were used for the survival study after transvaginal cholecystectomy performed with endoscopic submucosal dissection ( esd ) instruments and a single - channel endoscope .
preparation was the same as in the pigs used for transvaginal cholecystectomy except that no operation was performed and anesthesia was reversed 180 minutes after induction .
all pigs were fasted overnight before the procedure and were given 0.5 mg / kg ketamine and 1.2 mg atropine sulphate by intramuscular injection as a premedication before anesthesia .
an ear vein was used for injection of 0.5 mg / kg of dormicum , 0.1 mg / kg of pavulon , and 0.01 mg / kg fentanyl for induction .
the trachea was intubated with a cuffed endotracheal tube using an illuminated laryngoscope with an extra - long blade .
periodic injection of pavulon and fentanyl was used to ensure adequate anesthesia and muscle relaxation .
on completion of the operation , 1 mg of neostigmine and 1.2 mg of atropine were used for anesthesia reversal , and the pigs were extubated .
ampicillin in a dose of 500 mg was injected intravenously just before the procedure and continued for 5 days every 8 hours .
buprenorphine 0.05 mg / kg to 0.2 mg / kg was given every 12 hours for 3 days after the operation for pain control .
the pigs were sacrificed with 200 mg / kg of pentobarbital sodium 2 weeks after the experiment .
all operating instruments were sterilized according to the standard autoclave protocol , whereas the endoscope was disinfected by immersing it for 20 minutes in 2.4% glutaraldehyde ( cidex , johnson and johnson , new brunswick , nj ) and then rinsed with sterile water .
the peritoneal access and cholecystectomy were performed with a single - channel endoscope ( gif - q240x , olympus optical co , ltd , tokyo , japan ) .
the skin was prepared with povidone - iodine solution , and the vagina was irrigated with the same solution .
a veress needle was inserted for creation of carbon dioxide pneumoperitoneum and maintenance of intraperitoneal pressure at 12 mm hg .
the urogenital sinus was cannulated with the endoscope , and the vagina cavity was entered .
after the cervix was identified , 2 approaches were used for accessing the peritoneal cavity : ( 1 ) the anterior vaginal wall was dissected with a hook knife ( olympus optical co , ltd , tokyo , japan ) , and air was insufflated during colpotomy to maintain an optimal endoscopic view ; the entrance to the peritoneal cavity was recognized by a sudden increase in monitored intraperitoneal pressure , and the hook knife then served as a guidewire for insertion of the endoscope into the peritoneal cavity : ( 2 ) the endoscope was passed beyond the cervix to enter the retroperitoneal plane , and the peritoneum was incised using a hook - knife to gain access to the peritoneal cavity .
the endoscope was then manipulated so that minimal looping would be created and an end - on view of the cystic duct could be achieved . the gallbladder was retracted cranially and laterally . the covering peritoneum was incised and dissected using a combination of hook knife and hot biopsy forceps .
after the cystic duct and artery were isolated , both were clipped together with endoscopic hemoclip ( olympus optical co , ltd , tokyo , japan ) and divided with endoscopic scissors ( olympus ) .
the gallbladder was then dissected from its liver bed by using a hook knife or it ( insulated tip ) knife , or both , ( olympus optical co , ltd , tokyo , japan ) with pd-60 and endoplasma ( olympus optical co , ltd , tokyo , japan ) where appropriate .
use of the it knife was advantageous as it could protect the liver from diathermy injury and minimize the chance of accidental gallbladder perforation .
after clearing accumulated intraperitoneal fluid by suction via the endoscope , the gallbladder was grasped with either a pair of biopsy forceps or an endoscopic snare under direct vision .
porcine serum was prepared by centrifuging the clotted blood for 10 minutes at 3000 rpm , and the serum was then stored in aliquots at -80c . after collecting all specimens required for experimentation , tnf- and il-6 were quantitatively measured by the elisa method [ r&d systems ( quantikine ) , minneapolis , mn , usa ] . the colored product formed at the end of assays was measured according to the manufacturer 's instructions , and the concentrations of tnf- and il-6 were subsequently determined from their standard curves .
the differences between operated on and control groups were determined by using the one - way anova test .
all statistical analysis was calculated using spss for windows version 11.5 ( chicago , il ) ( tables 1 and 2 ) .
twelve female pigs were used for the present study in which 4 were assigned to the control group without intervention after general anesthesia with the same protocol previously mentioned ( c ) .
two pigs underwent laparoscopic cholecystectomy ( l ) , and 6 pigs underwent notes transvaginal cholecystectomy with a single - channel endoscope and esd instruments ( n ) . in all 6 pigs in the treatment group , satisfactory control of the cystic complex was achieved , and no major intraoperative complications occurred .
the mean operating time for the notes group was 123 minutes ( range , 92 to 175 ) .
the mean preoperative il-6 was 59.54 pg / ml for the control group ( c ) , 63.5 pg / ml for the laparoscopic group ( l ) , and 50.47 for the notes group ( n ) ( p=0.897 ) .
day 1 postoperative il-6 levels were 67.31 pg / ml ( c ) , 68.68 pg / ml ( l ) , and 57.37 pg / ml ( n ) , respectively ( p=0.790 ) .
postoperative day 2 il-6 levels were 59.67 pg / ml ( c ) , 54.07 pg / ml ( l ) , and 57.54 pg / ml ( n ) ( p=0.945 ) .
similarly , no significant difference was noted in mean preoperative tnf- : 79.94pg / ml for the control group ( c ) , 82.68 pg / ml for the laparoscopic group ( l ) , and 53.99 for the notes group ( n ) ( p=0.349 ) .
mean postoperative day 2 tnf- levels were 69.19 pg / ml ( c ) , 105.12 pg / ml ( l ) , and 63.29 pg / ml ( n ) ( p=0.11 ) .
but there was a significant increase in postoperative day 1 tnf- level in the laparoscopic group compared with the control and notes groups : 63.53 pg / ml ( c ) , 93.83 pg / ml ( l ) , and 50.59 pg / ml ( n ) ( p=0.049 ) .
for the past 2 decades , laparoscopic surgery has been shown to be associated with less postoperative pain and faster postoperative recovery .
this is usually attributed to the small incision , less manipulation , and avoidance of exposure of internal viscera during laparoscopic surgery .
together with the improved operative outcomes , the minimally invasive approach is merited with triggering fewer inflammatory responses in the patient .
inflammatory cytokines , as an indirect measurement of host acute inflammatory response , are usually used in the laboratory and clinically to assess the magnitude of the acute - phase reaction .
significantly lower levels of tnf- , il-1 , and il-6 and other inflammatory markers were released in the laparoscopic surgery group compared with markers released in the open surgery group . with the recent increased interest in notes , the impact of transvaginal cholecystectomy on host response is being studied . in our experiment , we compared the postoperative inflammatory response between laparoscopic , notes , and control groups in a porcine model . circulating cytokines , being the most sensitive indicator for host trauma , objectively reflect host immunological response .
the differences in host immune responses between open and laparoscopic cholecystectomy have been well validated in the literature , and therefore we did not include an open cholecystectomy control group .
we demonstrated no significant difference in terms of tnf- and il-6 levels 48 hours after surgery .
however , a significant increase in tnf- levels was detected in the laparoscopic group compared with control and notes subjects on postoperative day 1 .
but the rise is not significant when comparing laparoscopic and notes groups alone ( p=0.059 ) .
it is difficult to predict whether it was due to a transient inflammatory host response to the skin incision , surgical procedures , or purely due to chance alone .
because only 2 subjects were included in the laparoscopic group , it is difficult to draw a conclusion about the significance of the greater cytokine response in laparoscopic surgery compared with that in notes .
however , with smaller and fewer abdominal incisions , less inflammatory response should be triggered compared with that in laparoscopic or open surgery .
although the sample size is small for the whole study , it is logical to conclude that notes is safe in terms of the microcellular level in pigs .
this finding needs to be further validated in randomized control trials in humans . besides surgical access
consistent or repeatedly high intraabdominal pressure causes abdominal compartment syndromes , with the potential danger of hemodynamic disturbances , impairing end - organ perfusions , or even pulmonary embolism .
however , it is not an easy task to detect all these subtle changes in the porcine model unless invasive monitoring is adopted perioperatively . therefore , pigs survival does not necessarily equal clinical safety .
notes is safe in animal models on the anatomical and cellular levels with minimal systemic inflammatory host responses elicited .
further study needs to be carried out in humans before notes can become part of routine practice .
 objective : we analyzed circulating tnf- and il-6 to determine systemic inflammatory responses associated with transvaginal cholecystectomy in a porcine model.methods:six female pigs were used for a survival study after transvaginal cholecystectomy ( notes group ) using endoscopic submucosal dissection ( esd ) instruments and a single - channel endoscope . 
 blood was drawn preoperatively and 24 hours and 48 hours postoperatively . 
 four pigs were used as controls . 
 in addition , laparoscopic cholecystectomy was performed in 2 pigs for laparoscopic control.results:in all 6 pigs in the notes group , no major intraoperative complications occurred . 
 no significant differences were found between control , laparoscopic , and notes groups in terms of preoperative il-6 level ( p=0.897 ) and at 24 hours ( p=0.790 ) , and 48 hours postoperatively ( p=0.945 ) . similarly , there was no significant difference in mean preoperative ( p=0.349 ) and mean day 2 postoperative tnf- levels ( p=0.11 ) . but a significant increase in day 1 postoperative tnf- levels in the laparoscopic group compared with that in the control and notes groups was observed ( p=0.049 ) . 
 one limitation of our study is that the sample size was relatively small.conclusion:notes is safe in animal models in terms of anatomical and cellular level changes with minimal systemic inflammatory host responses elicited . 
 further study needs to be carried out in humans before notes can be recommended for routine use . 