A Method to Attenuate Pneumoperitoneum-Induced Reductions in Splanchnic Blood Flow

doi:10.1097/01.sla.0000153034.54128.5e

Journal List > Ann Surg > v.241(2); Feb 2005

Ann Surg. 2005 February; 241(2): 256–261.

doi: 10.1097/01.sla.0000153034.54128.5e.

PMCID: PMC1356910

A Method to Attenuate Pneumoperitoneum-Induced Reductions in Splanchnic Blood Flow

Nishath Athar Ali, MD,^*† W Steve Eubanks, MD,^‡ Jonathan S. Stamler, MD,^§ Andrew J. Gow, PhD,^§ Sandhya A. Lagoo-Deenadayalan, MD, PhD,^† Leonardo Villegas, MD,^† Habib E. El-Moalem, PhD,^* and James D. Reynolds, PhD^*†

From the *Departments of Anesthesiology, †Surgery, and §Medicine, and

Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina; and the ‡Department of Surgery, University of Missouri–Columbia, Columbia, Missouri.

Abstract

Objective:

To determine if increasing nitric oxide bioactivity by inclusion of ethyl nitrite (ENO) in the insufflation admixture would attenuate pneumoperitoneum-induced decreases in splanchnic perfusion.

Summary Background Data:

Organ blood flow is reduced during pneumoperitoneum and can contribute to laparoscopy-associated morbidity and mortality. Previous attempts to control such decreases in flow have been ineffective.

Methods:

Laser-Doppler flow probes were placed on the liver and right kidney of anesthetized pigs. After a baseline recording period, animals were insufflated to a final intraperitoneal pressure of 15 mm Hg. Group one received CO₂ (standard practice), whereas group 2 received CO₂ plus 100 ppm ENO. Insufflation was maintained for 60 minutes and then the abdomen was manually deflated; monitoring was continued for another 60 minutes.

Results:

CO₂ insufflation (n = 5) cut liver blood flow in half; liver flow remained at this level throughout the postinsufflation period. Inclusion of 100 ppm ENO (n = 6) attenuated both the acute and prolonged blood flow decreases. Statistical modeling of the data showed that, on average, liver blood flow was 14.3 U/min higher in the ENO pigs compared with the CO₂ group (P = 0.0454). In contrast, neither treatment significantly altered kidney blood flow (P = 0.6215).

Conclusion:

The data indicate that ENO can effectively attenuate pneumoperitoneum-induced blood flow decreases within the perito-neal cavity. The result suggests a novel therapeutic method of regulating hemodynamic changes during laparoscopic procedures.

Millions of abdominal procedures are conducted in American hospitals annually. Estimates for completed cholecystectomies alone range between 500,000 and 800,000; the figure for appendectomies is even higher (information from the American Society of Abdominal Surgeons). A significant percentage of these surgeries (>90%) is conducted using minimally invasive techniques. By avoiding the large incision and gross manipulations performed during a traditional open procedure, laparoscopic surgery results in quicker postsurgical recovery, including a reduction in time to mobilization and a decreased requirement for postoperative analgesics.^1,2 Technical improvements such as better cameras and light sources along with finer gripping and cutting instruments have greatly enhanced the field so that a laparoscopic procedure is considered a routine event.

Despite its benefits, pneumoperitoneum (the act of insufflating the peritoneal cavity with gas, most often carbon dioxide; CO₂) is not without physiological consequences. In humans, even brief periods of CO₂ insufflation (45–60 minutes) have been shown to significantly reduce blood flow to organs within the peritoneal space. This reduction promotes anaerobic metabolism leading to lactic acidosis³; postoperative manifestations include alteration in liver enzymes,⁴ subclinical hepatic dysfunction,⁵ and the appearance of oxidative stress markers.⁶ As a result, tissue ischemia^7,8 and altered postoperative organ function^4,9,10 are major causes of laparoscopic-associated morbidity and mortality.

The increase in intraabdominal pressure produced by insufflation results in direct mechanical suppression of splanchnic blood flow. This is compounded by the CO₂-stimulated release of various vasoactive substances, including vasopressin, angiotensin, cortisol, and adrenocorticotropin hormone (ACTH).¹¹ At present, an effective method(s) to control the physiological changes produced by the presence of CO₂ in the gut has not been developed.^12,13 Reducing the inflow of CO₂ is not a tenable option because intraabdominal pressures of 12 to 15 mm Hg are typically required to produce enough space within the peritoneal cavity for organ visualization, multiple tool insertions, and surgical manipulations.

We reasoned that the deleterious effects of CO₂ insufflation on tissue blood flow would be abrogated by the administration of a vasoactive agent. Because intravenous drug dosing appears to offer limited benefit,¹⁴ we speculated that intraperitoneal administration would be more efficacious. Specifically, we hypothesized that inclusion of a nitric oxide (NO) congener (ie, a compound that contains NO) within the insufflating gas would attenuate the pneumoperitoneum-induced reductions in peritoneal organ blood flow. For the actual agent, we selected ethyl nitrite (ENO) because it is highly volatile and readily decomposes in biologic medium to produce S-nitrosothiols (SNOs; endogenous NO donors).¹⁵ We tested our hypothesis by measuring liver and kidney blood flows in swine during CO₂ pneumoperitoneum with and without ENO in the insufflating gas.

MATERIALS AND METHODS

All procedures described in this report were reviewed and approved by the Duke University Institutional Animal Care and Use Committee before their inception. The study consisted of 3 parts: 1) a preliminary dose response assessment to select a concentration of ENO that appeared to produce minimal cardiovascular effects; 2) use of ENO during a 60-minute period of insufflation; and 3) a laparoscopic survival surgery to determine if ENO produced any unexpected intra- or postoperative complications.

Surgery

Young adult pigs (25–35 kg) were sedated with 0.8 mg/kg intramuscular acepromazine and 20 mg/kg ketamine; 0.04 mg/kg atropine was given to reduce mucosal secretions. Surgical anesthesia was induced with intravenous propofol (3–6 mg/kg to effect), the lungs were then intubated, and surgical anesthesia was maintained with isoflurane (1.5–2.25%) in oxygen administered by positive pressure. All animals were actively ventilated to maintain end-tidal CO₂ at or below 32 mm Hg. Paralytic agents were not administered. Femoral artery and venous catheters were inserted to measure heart rate and arterial pressure and to obtain blood samples; other standard operative monitoring devices (eg, pulse oximetry) were also used. Arterial and venous blood gas status was determined at regular intervals during each study (Instrumentation Laboratories, Lexington, MA). A small ventral midline laparotomy was performed to permit placement of a laser-Doppler probe (Vasamedics, St. Paul, MN) in the fissure between the right medial and right lateral lobes of the liver. Using a retroperitoneal approach, a second probe was placed on the right kidney. After ensuring strong blood flow signals from both probes, the 2 incision sites were closed in layers. Finally, 10-mm Versaport trocars (U.S. Surgical Corp., Norwalk, CT) were inserted under direct vision into the 4 quadrants of the peritoneal cavity using an open technique for each placement (ie, we did not preinsufflate with a Veress needle). The entire procedure took approximately 30 minutes to complete.

Ethyl Nitrite Dose Response

When cardiovascular parameters and liver and kidney blood flows were stable, the pigs (n = 3) were insufflated with a high-flow clinical insufflator (Stryker Endoscopy, Santa Clara, CA) at a rate of 1.0 L/min to a final intraperitoneal pressure of 15 mm Hg. After 35 minutes, ENO (Sigma Chemical Co., St. Louis MO) was introduced into the peritoneal space by passing the CO₂ gas exiting the insufflator through a Fisher Milligan gas washer containing a fixed amount of ENO in ethanol. The actual amount of ENO was adjusted to produce serial increases in ENO concentration (from 50–300 ppm) at 30-minute increments. With each increase, the ENO concentration was confirmed by gas chromatography/mass spectrometry analysis of the gas entering the peritoneal space (Hewlett-Packard, Palo Alto, CA). To ensure continual peritoneal exposure to the NO congener, we took advantage of the 3-way stopcock that is part of each Versaport trocar and is used to regulate the inflow and outflow of the insufflating gas. In the present case, the stopcock of the trocar perpendicular to the trocar connected to the insufflator was turned to deflate. This allowed gas to exit the area and, by extension, necessitated the insufflator to pump more ENO-containing CO₂ gas into the peritoneum to maintain the intraabdominal pressure at 15 mm Hg. ENO was undetectable in the gas samples obtained from this exit port. At the completion of the study, the anesthetized pigs were euthanized with an injection of saturated potassium chloride.

Ethyl Nitrite and Blood Flow During Pneumoperitoneum

After instrumentation, baseline liver and kidney blood flows were recorded for between 35 and 40 minutes before insufflation. Two cohorts of pigs were then insufflated to 15 mm Hg using the previously described parameters. Group 1 (n = 6) received plain CO₂, whereas group 2 (n = 6) received CO₂ enriched with 100 ppm ENO. Group sizes and ENO concentration were based on the data from the dose-response experiment. Group assignments were randomly determined before starting the study. As before, a valve was used to vent off the gas ensuring continual peritoneal exposure. ENO concentration in the inflowing gas and the absence of ENO in the outflowing gas were again confirmed by gas chromatographic analysis. Animals were insufflated for 60 minutes and then monitored for an additional 60 minutes after termination of insufflation and manual deflation of the peritoneum. Arterial and venous blood samples were obtained at regular intervals to assess blood gas status. As a positive control, recording was continued after potassium chloride euthanization to demonstrate a cessation of kidney and liver blood flow.

Laparoscopic Survival Surgery With Ethyl Nitrite

Two pigs were anesthetized as described and then underwent a laparoscopic cholecystectomy. The procedure was conducted by a skilled laparoscopic surgeon and used standard clinical techniques and tools. For the insufflating gas, we chose to use a 3-fold higher dose of ENO because we felt a concentration of 300 ppm would be sufficient to identify any complications, either intraoperative (eg, flammability, camera visualization) or postoperative (increased bleeding, impaired wound healing). During the surgery, abdominal pressure was maintained at 15 mm Hg. After the gallbladder was removed and the cystic duct and cystic artery ligated, the ports were removed, the abdomen was manually deflated, and the skin incisions were closed with pursestring stitches. Bupivacaine was infused at each port insertion site. The pigs were recovered from anesthesia and then euthanized 3 days later with an intravenous injection of sodium pentobarbital–phenytoin (Euthasaol®) to allow for qualitative assessment of the surgical area.

Statistical Analysis

Physiological data are presented as group means ± standard deviation (SD). Statistical analysis focused on the primary end point, ie, change in liver and kidney blood flow. Laser Doppler flows in the liver and kidney were recorded 3 times per second before, during, and after insufflation with either CO₂ or CO₂ plus 100 ppm ENO. The data obtained before insufflation were averaged to determine basal blood flow for each organ. Subsequent data (ie, during and after pneumoperitoneum) were averaged in 10-minute increments. These data were then analyzed to test for differences in tissue blood flow (either liver or kidney) between the CO₂ group and the CO₂ plus ENO group. A mixed statistical model was created that related tissue blood flow to time, basal tissue blood flow, treatment, and to time-by-treatment interaction. The between-pig variation was captured by assuming a random linear trend for flow over time for each pig around the population line relating flow to time. The modeling was accomplished using a statistical software package (version 8.2; SAS Institute, Cary, NC). A statistically significant difference between treatment groups in either liver or kidney blood flow was assumed at P <0.05.

RESULTS

The time courses of heart rate and mean arterial pressure changes for the 3 animals used in the ENO dose-finding study are presented in Figure 1. By itself, 30 minutes of insufflation had little effect on the 2 parameters. Once ENO was included in the insufflating gas, it appeared to increase heart rate and decrease pressure in a dose-related manner. Overall, these changes were modest with the highest concentration (300 ppm) producing less than a 20% alteration in heart rate or mean arterial pressure. Qualitative assessment of these data and the liver and kidney blood flow numbers (data not shown) led us to select 100 ppm as the concentration of ENO to be used in the subsequent study. At this dose, blood pressure was unchanged from baseline and heart rate was only increased by approximately 10 beats/min.

FIGURE 1. Time course of heart rate and mean arterial pressure changes during the ethyl nitrite (ENO) dose-response study. The abdomen was kept at a pressure of 15 mm Hg during insufflation with CO₂ and the serial increases in the concentration of ENO. (more ...)

The 12 animals in second portion of the investigation tolerated the procedure well with the exception of 1 pig in the CO₂ group. Shortly after induction, this animal started to exhibit significant and prolonged tachycardia and hypertension. The cause was presumably malignant hyperthermia,¹⁶ and it necessitated excluding this animal from the study. Arterial and venous blood gas data for the remaining 11 pigs are presented in Table 1. Overall, the animals in each group were well ventilated with the initial values (time 0) consistent with previous data indicating pigs are alkalotic compared with humans.¹⁷ Sixty minutes of pneumoperitoneum produced the anticipated increase in pCO₂ and decrease in pH, which resolved after deflation.

TABLE 1. Arterial and Venous Blood Gas Data Before, During, and After Pneumoperitoneum

Mean liver and kidney blood flow data (n = 5 in the CO₂ group and n = 6 in the ENO group), expressed in 10-minute increments, along with mean heart rates are presented in Figure 2. As expected from the dose-response study, heart rate was essentially unaffected by either treatment. The effect of insufflation alone to decrease liver blood flow occurred almost immediately; 20 minutes into the procedure; flow was less than half of the starting level. Furthermore, liver blood flow remained significantly depressed even 60 minutes after insufflation had ceased. Inclusion of 100 ppm ENO significantly abrogated this decrease in blood flow; at no time point did liver flow decline to levels produced by CO₂ alone and, toward the end of the insufflation period, flow was similar to baseline. In addition, unlike the CO₂ group, no postinsufflation changes in flow were observed. Incorporation of these data into the statistical model (adjusted for baseline differences in flow) showed that on average, liver blood flow was 14.3 U/min higher in the ENO pigs compared with the pigs in the CO₂ group (P = 0.0454). In contrast, neither treatment significantly altered kidney blood flow (P = 0.6215).

FIGURE 2. Time course of heart rate and liver and kidney blood flow during and after insufflation with CO₂ (n = 5) or CO₂ plus 100 ppm ethyl nitrite (ENO) (n = 6). Intraabdominal pressure was kept at 15 mm Hg during the 60-minute period (more ...)

For the 2 animals that underwent laparoscopic cholecystectomy in the presence of 300 ppm ENO, the surgery was unremarkable. There was no interference with camera visualization and there was no problem with bleeding. Tissue cauterization proceeded smoothly indicating that the presence of ENO within the peritoneal cavity was not a combustion risk. Recovery from the procedure was uneventful and internal wound healing, when assessed 3 days later, was excellent.

DISCUSSION

There is widespread recognition that pneumoperitoneum-induced reductions in tissue perfusion are a major cause of laparoscopic morbidity and mortality. In this study, we present a novel method for attenuating such untoward effects: inclusion of the nitric oxide congener ENO in the insufflating gas.

ENO is a relatively low-molecular-weight (75.07) colorless organic nitrite with a density of 0.9. It can be stored as a liquid but with a low boiling point (16.5–17°C); it is extremely volatile at room temperature. In its pure form, ENO is quite combustible. However, when combined with CO₂ up to several hundred ppm (>300), it is nonflammable and poses no combustion risk. Our utilization of ENO is based on the vasoactive properties of SNOs, which constitute the majority of NO bioactivity in blood and tissues.¹⁸ In addition to acting locally, NO can alter blood flow distal from its site of formation.¹⁹ It appears to do this through the transnitrosation of reduced sulfhydryl groups of low- (eg, cysteine, glutathione) and high- (eg, albumin, hemoglobin) molecular-weight thiols.²⁰ The end result is a circulating reservoir of NO bioactivity. This aspect of SNOs has therapeutic implications for various respiratory and circulatory conditions.²¹

For the present situation, we reasoned that elevating the levels of SNOs would combat the pneumoperitoneum-induced decrease in tissue perfusion. One method to produce such an increase would have been to include NO gas in the insufflation admixture. However, administration of NO itself is an inefficient means of generating SNOs, and it has a predilection to produce tissue damaging higher-order nitrogen oxides along with methemoglobinemia.²² Instead, the consummate NO agent would preferentially react with thiols, resist decomposition in a gaseous medium, have a limited ability to oxidize hemoglobin, and yet be highly volatile so that it readily mixes with the insufflating gas. Recently, ENO was identified as not only meeting these criteria, but also as being able to provide superior activity compared with NO.¹⁵ Specifically, this was determined in an experimental animal model of pulmonary hypertension in which ENO exhibited a decreased propensity for hypertensive “rebound” and improved cardiac function compared with inhaled NO. Preliminary neonatal human data further support ENO's therapeutic potential and superior SNO-generating capacity.²³ Combined with the results of the present study, it appears that inclusion of ENO in the insufflating gas offers a safe and relatively simple therapy to attenuate the unwanted side effects of CO₂ pneumoperitoneum.

Increasing the amount of SNOs can produce sustained vasorelaxation,²⁴ but this may not be the only beneficial action of ENO. Although the pneumoperitoneum-induced increase in intraabdominal pressure produces a direct mechanical suppression of splanchnic blood flow,²⁵ it also elicits a humoral stress response. This systemic effect includes elevations in the blood concentrations of catecholamines,^26,27 ACTH, and cortisol.^28,29 Moreover, blood levels of these vasoactive substances remain elevated into the postoperative period,³⁰ which likely accounts for the persistent depression of liver blood flow observed in our CO₂-treatment group (Fig. 2). Such a response explains why infusion of vasoactive agents such as dobutamine are ineffective at improving splanchnic blood flow during pneumoperitoneum.¹⁴ It also provides a second potential site of action for ENO. Various studies indicate that NO plays a role in modulating the release of stress hormones.³¹ Exposure of organs involved in the stress response (specifically the adrenals) to inhibitors of NO synthase can increase the efflux of catecholamines, aldosterone, and so on,^32,33 whereas exposure to an NO donor decreases such secretions.³² Combining these observations with the previously cited surgery studies leads us to propose that ENO insufflation may attenuate the decrease in liver blood flow through an NO-mediated inhibition of the stress response in addition to its presumed vasodilatory actions.

This study is not without its limitations. At present, our ability to predict additional actions of ENO (be they beneficial or deleterious) is limited because the full physiological spectrum of effects produced by CO₂ insufflation alone has not been determined. Nonetheless, it appears reasonable to suggest that ENO would have a beneficial effect on other organ systems where blood flow is reduced during surgery. One such site is the gastrointestinal (GI) tract. Increasing NO bioactivity with ENO could attenuate the impaired gastric motility that can result from insufflation-induced reductions in GI flow as well as having a direct positive effect on the intestines as NO appears to play a role in maintaining intestinal motility.³⁴ Another limitation is that the dose of ENO we selected did not completely abolish (at least early on) the induced decrease in liver blood flow. This observation suggests that the effective concentration may actually be somewhat higher than 100 ppm. The fact that 2 animals tolerated the survival surgery with 300 ppm supports the use of a higher ENO concentration, although we will need to conduct additional survival procedures to confirm ENO's safety.

Overall, we are encouraged by the initial findings and we are continuing to investigate the therapeutic potential of ENO. Research currently underway is aimed at delineating the acute and postoperative effects of ENO/CO₂ pneumoperitoneum on various organ systems, both local and distal to the site of insufflation. As a technical advance, we have eliminated the need for the somewhat cumbersome Fisher Milligan gas washer setup by having pressurized CO₂ tanks prepared with fixed concentrations of ENO. Within the tanks, ENO remains stable for extended periods of time. If our current studies reaffirm the benefits of including ENO in the insufflating gas, this method of ENO delivery could be readily incorporated into the clinical setting.

In summary, the inclusion of a vasodilator gas ENO with the CO₂ gas used to inflate the peritoneal cavity provides a unique, and apparently sound, method for controlling a major source of pneumoperitoneum-related morbidity and mortality.

ACKNOWLEDGMENTS

The authors thank Dr. Bruce Klitzman in the Department of Surgery at Duke University Medical Center for his technical expertise with the Vasamedics Laser Doppler equipment.

Footnotes

This work was support in part by National Institutes of Health grant HD042471 (JDR) and an unrestricted educational grant from United States Surgical Corporation (WSE). JSS is an associate investigator with the Howard Hughes Medical Institute.

Reprints: James Dixon Reynolds, PhD, Assistant Professor, Departments of Anesthesiology and Surgery, Room 119, Research Park Building 4, Box 3094, Duke University Medical Center, Durham, NC 27710. E-mail: reyno010/at/mc.duke.edu.

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