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ANNALS OF SURGERY
Vol. 217, No. 3, 286-292
© 1993 J. B. Lippincott Company
Effect of Total Parenteral Nutrition
Plus Morphine on Bacterial
Translocation in Rats
Peter M. Kueppers, M.D., Thomas A. Miller, M.D., Chung-Ying K. Chen, M.S.,
Gregory S. Smith, M.S., Liliana F. Rodriguez, B.S., and Frank G. Moody, M.D.
From the Department of Surgery, The University of Texas Medical School, Houston, Texas
Objective
This study tested the hypothesis that gut stasis induced by parenteral morphine sulfate (MS)
leads to enhanced bacterial translocation in rats on total parenteral nutrition (TPN).
Summary Background Data
TPN and MS are common adjuncts in the care of critically ill patients. TPN is known to provoke a
variable degree of translocation. MS induces gut stasis with an accompanying bacterial
overgrowth. The effect of these two treatments in combination on translocation is not known.
Methods
Rats were provided with central and subcutaneous lines for the continuous infusion of nutrients
and drugs, respectively. Intestinal transit was assessed by the caudal movement of a fluorescent
marker intubated into the proximal duodenum. Quantitative bacteriology was carried out from
various segments of the gut and from ileocecal mesenteric lymph nodes (MLN), spleen, liver,
and systemic blood obtained by cardia puncture on sacrifice at 96 hours.
Results
Transit was unchanged by TPN alone but prolonged when given in combination with MS.
Bacterial overgrowth was also enhanced by MS and increased the bacterial translocation to MLN
from 50% of animals with TPN, to 100% in those receiving both TPN and MS; the colony-forming
units per MLN increased from 33 ± 14 with TPN alone to 2079 ± 811 (STD) with TPN plus MS.
Furthermore, no bacteria were found at systemic sites with TPN alone, but in 93.3% of animals
receiving TPN and MS. In a subgroup of rates provided with glutanine in TPN, the TPN plus MS
effects on translocation were not reversed.
Conclusions
These observations demonstrate the important role that morphine plays in promoting
translocation, presumably by disrupting fasting motility and enhancing bacterial overgrowth.
Rats maintained nutritionally on total parenteral nu- organs, also occurred. Bacterial overgrowth, one ofthe
trition (TPN) alone for periods ofat least 14 days have mainfactorspromotingtranslocation,3-6 isusuallyantag-
increased cecal bacterial counts and decreased levels of onized by the clearing effect of small bowel propulsive
intestinal secretory immunoglobulin A.',2 Concomitant motility.7 TPN does not decrease fasting motility, as
with these findings, translocation ofviable bacteria from measuredbysmallintestinal transitofadyeinjectedinto
the gut to mesenteric lymph nodes, but not to distant the duodenum.8 Acute doses ofmorphine, on the other
286
Bacterial Translocation During TPN Plus Morphine 287
hand, are known to suppress small intestinal transit very versity ofTexas Medical School at Houston before com-
effectively in nondependent animals.9"10 When continu- mencement ofstudies.
ously infused at a rate of 3 mg/kg/hr, this suppressive
effect is sustained for 8 hours, while tolerance against the Surgical Procedures
analgesic effect develops more rapidly." There is evi-
dence, however, that tolerance develops against the ef- All animals were equipped with acentral venous and a
fect of repeated morphine applications on gut transit subcutaneous line for continuous infusion of nutrients
within a matter of days.'2"13 In a series of preliminary and drugs, respectively. The central venous line was
experiments, we found that increasing the morphine made from silicone tubing (Silastic, ID 0.020 in, OD
dose 25% per day, beginning on day 1 with 4 mg/kg/hr, 0.037 in, Dow Corning, Midland, MI), and the subcuta-
would slow down transit in the rat by at least 40% over neous line from polyethylene tubing (PE-50, ID 0.023
several days. in, OD0.038 in, Becton Dickinson andCompany, Parsi-
Wehypothesized that chronic depression ofsmall in- panny, NJ). For the central venous line, a venous cut-
testinal transit would allow for increased bacterial over- down was performed on the right jugular vein and the
growth in the gut lumen and that prolonged contact of silastic tube was advanced into a central venous position.
bacteria with the intestinal wall would potentiate bacte- The distal end ofthe tube was tunneled subcutaneously
rial translocation induced by total parenteral feed to the nape ofthe neck where it exited the skin. A 14 G
ing.'4-6To test this hypothesis, rats were exposed to over-the-needle IV catheter (Angiocath, Becton Dickin-
combined treatment with TPN and increasing doses of son and Company, Sandy, UT) was inserted in the nape
morphine for 4 days. Glutamine was added to the TPN of the neck and advanced in a caudal direction in the
solution in an additional group of animals to assess its subcutaneous layer. The PE-50 tube was placed subcuta-
protective effect under these conditions.'17"8 Our results neously through this catheter and the over-the-needle
indicated that while morphine produced a dramatic in- catheter was removed. Both lines, IV and SC, were then
crease in translocation to mesenteric lymph nodes and placed in a spring-coil apparatus, secured to a harness
distal sites in animals on TPN, glutamine treatment did and connected to a dual channel swivel mechanism.
not alter these morphine-induced effects. Such an arrangement allowed animals to move freely
throughout their cages.
Animals used for intestinal transit studies were
METHODS equipped with an additional duodenal catheter, made
from the same material as the IV line, at least 4 days
Animals before the implantation ofthe central venousand subcu-
taneous lines. Their duodenum was carefully exposed
Male Sprague-Dawley rats (Harlan Lab, Houston, through a 2-cm midline laparotomy. The tube was intro-
TX), weighing between 250 and 350 g, were housed in duced into the duodenum 1 cm distal to the pylorus on
metabolic cages in rooms with regulated temperature, the antimesenteric border, advanced into the duodenum
humidity, and light/dark cycles. They were provided for 2 cm, and secured with a 6-0 silk purse string suture.
standard rat chow (Ralston Purina, St. Louis, MO) and Thedistal end ofthis catheter was also tunneled subcuta-
water adlibitum during a minimum stabilization period neously to the nape ofthe neck. This catheter was sealed
of4 days. Animals that received oral feedings during the with a paper clip and covered under the harness until 30
experimentwerefastedovernightbeforesacrifice. Opera- minutes before sacrifice.
tive procedures were performed under sterile conditions
using methoxyflurane (Metofane, Pitman-Moore, Mun- Basic Experimental Protocol
delein, IL) anesthesia. All experimental protocols were After the initial surgical procedures described above,
approved by the Animal Welfare Committee ofthe Uni- all animals were randomly assigned to one ofa variety of
treatment groups detailed in Table 1.
The various treatments rendered were administered
Supported by the National Institutes ofHealth grant GM38529 and by for 4 days. All IV infusions ran at 2 mL/hr, SC infusions
the Deutsche Forschungsgemeinschaft grant KU782/1-1. at 0.6 mL/hr. The TPN solution (Parenteral Nutrition
Presented in part at the Annual Meeting ofthe American Gastroenter- Kit, Baxter, Deerfield, IL) contained 25% dextrose,
ological Association, San Francisco, California, May 9-13, 1992. 4.25% aminoacid mixture, electrolytes and 1 mL/Lvita-
Address reprint requests to Frank G. Moody, M.D., Department of min solution (M.V.I.-12, Rorer, Fort Washington, PA).
Surgery, The University of Texas Medical School, 6431 Fannin, Morphine sulfate (Mallinckrodt, St. Louis, MO) wasdis-
Houston, TX 77030.
Accepted for publication July 7, 1992. solved in normal saline. Rats received 4 mg/kg/hr mor-
288 Kueppers and Others
Bacteriologic Studies
..... ..-..... ..... Animals in which bacterial translocation
Control (I) Saline IV, saline SC, free access was studied
to lab chow. were subjected to laparotomy under sterile conditions
TPN alone (11) TPN solution IV, saline SC, using methoxyflurane anesthesia. Upon entrance into
nothing by mouth. the abdomen a peritoneal swab was taken, following
TPN + MS (Ill) TPN solution IV, morphine whichthespleen, one lobeoftheliverandthe mesenteric
sulfate (MS) SC, nothing by lymph node complex from the ileocecal area to the root
mouth. ofthe mesentery were excised for quantitative bacterial
TPN/Gln + MS (IV) Glutamine (Gin) supplemented culture. Further, a blood sample was obtained by cardiac
TPN solution IV, morphine and the thorax was
sulfate (MS) SC, nothing by puncture opened and the left lung as
mouth. well as the tip of the central venous catheter were har-
* All treatments rendered for 4 days. vested for culture. For bacterial counts in the intestine, 5
cm segments ofproximal duodenum, midjejunum, ter-
minal ileum and the entire cecum were removed.
phine on day 1, 5 mg/kg/hr on day 2, 6.25 mg/kg/hr on All sampleswereassignedacodesothatthebacteriolo-
day 3, and 7.8 mg/kg/hr on day 4. The dose was always gist was unaware oftreatment. Peritoneal swabs and the
increased at the beginning ofa 24-hour period. All ani- tip of the central venous catheter were cultured in
mals were killed at the end ofthe fourth 24-hour period. Schaedler's broth (Adams Scientific, West Warwick, RI)
For experiments with glutamine supplementation, the at 35°C for 5 days. Blood was cultured in 20 mL brain
TPN solution specified above was altered to contain 2% heart infusion (BHI) bottles (Adams) and incubated in a
L-glutamine plus 2.25% ofthe original amino-acid mix- CO2atmosphere incubator for up to 7 days. Gram stains
ture. The L-glutamine solution used was sterile. and blind subcultures on chocolate agar plates (Adams)
were made on days 2 and 7. Spleen, liver, lung, MLN,
and the intestinal samples were collected into sterile
plastic bags (Tekmar, Cincinnati, OH), weighed and ho-
Small Intestinal Transit Study mogenized with a stomacher (Tekmar) in 2 mL ofsterile
Twenty-five minutes before sacrifice, all animals in BHI broth; 9 mL were used for cecum. Serial tenfold
the intestinal transit study were given 0.1 mL of5 mmol/ dilutions of the intestinal homogenates were made in
Lfluorescein isothiocyanate-dextran (FITC, average mo- BHI, and at least three from each were plated onto blood
lecular weight 10,000 dalton, Sigma, St. Louis, MO) so- agar, MacConkey agar and Columbia colistin-nalidixic
lution in normal saline through their duodenal catheter, acid agar plates and incubated for 48 hours at 35°C in an
which was then flushed with 0.1 mL normal saline. Care air and CO2 incubator. The inoculum (0.1 mL) was
was taken not to agitate the animals during administra- spread evenly on the agar surface with a sterile glass rod.
tion ofthe dye and forthe following 25 minutes. The rats The solid organ homogenates were processed similarly,
were then anesthetized and their entire small intestine, but without serial dilutions. The intestinal bacterial
from the proximal duodenum to the distal ileum, was counts were expressed as the logl0 number of colony-
removed and divided into ten segments ofequal length. forming units (CFU) per gram oftissue. Gram-negative
Each segment was flushed with 3 mL Tris-buffered sa- rods were identified by the API 20E System (Analytab
line (pH 10.5). The resulting mixture was briefly vor- products, Plainview, NY). Gram-positive cocci and rods
texed and then centrifuged at 1200 rpm for 5 minutes. were identified using standard bacteriologic tech-
The fluorescent activity of the supernatant was mea- niques.20
sured using a Perkin-Elmer LS-3 fluorescence spectro-
photometer (Oak Brook Instrument Division, Oak
Brook, IL) at an excitation wavelength of 490 nm and Data Analysis
emission wavelength of 520 nm. Using the result from
this measurement the absolute amount ofFITC recov- Results amongthe variousgroupswere analyzed using
ered from each segment was calculated from a standard chi-square analysis for nonparametric data and ANOVA
curve prepared on the same day. The amount ofmarker followed by Student-Newman-Keuls post-hoc test for
per segment was expressed as a fraction of the total parametric data. Criteria for exclusion ofdata from anal-
amount of marker recovered. The geometric center of ysis were (a) positive cultures from the central venous
markerdistribution represents the sum ofall fractions of catheter tip and (b) technical failure ofthe infusion sys-
marker per segment times their segment number.'9 tems.
Bacterial Translocation During TPN Plus Morphine 289
RESULTS 60
E- 50 Control
Datafromthreeanimalshadtobeexcludedfromanal- 40 I GC=6.0±0.3
ysis. One IV line in an animal from the control group ° 40
was leaking at its connection to the swivel mechanism, *= 30
making it unclear how much fluid the rat had received. .0
' 20
TworatsintheTPN + MSgroupwereexcludedbecause *W
ofpositive cultures from the central venous catheter tip a 10
that caused insecurity as to whether iatrogenic contami-
nation or bacterial translocation was responsible for the o 1 2 3 4 5 6 7 8 9 10
bacterial sepsis observed. 60 Intestinal segment
cJ
60 L TPN alone
E 50
Small Intestinal Transit La: GC=5.8±0.3
o 40
C I
Intestinal transit in animals infused with morphine 0
* 30
was significantly slower than in all other groups (Fig. 1). .0
The bulk of the marker in morphine-treated rats was 3, 20
located in segment 3 or 4 twenty-five minutes after injec- 10
tion into the proximal duodenum. Less than 1% ofthe 0 aD 1 2 3 4 5 6 7 8 9 10
dye reached segment 5, except in two ratsthat had athird
of the marker recovered from this location. While all Intestinal segment
other morphine rats showed a narrow peak in either seg- 60
ment 3 or 4, these two were characterized by a wide C.)
LA. 50 - T
spread of the marker over several segments. In one, I- TPN+MS
20.9% of the marker reached segment 6 and 1.7% seg- 0 40 - GC=3.6±0.2a
C
ment 7. These two segments were completely free ofany 0
30 -
detectable dye in all other animals of this treatment 20 -
.0
group. a
In controls only one animal had marker left in seg- 10 _
ment 4 (20.4%), while all others showed a narrow peak
around segment 6. Segments proximal to the peak, that 0 1 2 3 4 5 6 7 8 9 10I
had been passed by the bolus ofdye, were almost com- Intestinal segment
pletely cleared of any fluorescent activity. Treatment Figure 1. Effects of various treatments on gut transit. Small intestinal
with TPN alone did not cause significant changes in transit was not changed by treatment with TPN alone as compared with
small intestinal transit. Like controls, these rats pro- the control group. The addition of morphine resulted in significant depres-
pelled the whole amount ofdye as a tight bolus that was sion of small intestinal transit. Transit was measured as distance travelled
recovered from segment 5, 6, or 7, or a combination of by a fluorescent marker (FITC) over the length of the small intestine within
two segments. 25 minutes after application to the proximal duodenum (segment 1
= duodenum, 10 = ileum). GC = geometric center of marker distribution.
Data expressed as mean ± SEM with n = 6 for all groups.
a p < 0.05 versus all other groups.
Bacterial Intestinal Overgrowth
Feeding by TPN resulted in a significant increase over significant in this group when compared with rats receiv-
control in the total number of aerobic plus facultative ing TPN alone. Also, after the addition of morphine,
bacteria in the ileum but not elsewhere in the gut. The Gram-negative bacteria in the duodenum were signifi-
number of gram-negative enteric rods alone was in- cantly elevated over treatment with TPN alone. These
creased in ileum and cecum. In contrast rats that were findings are shown in Figures 2 and 3.
treatedwith morphineinaddition toTPNshowedsignifi- The use ofglutamine supplemented TPN solution in
cantly increased bacterial counts when compared with combination with morphine did not protect against bac-
controls in duodenum, ileum and cecum for the total terial overgrowth. The numbers of bacteria actually
numberofaerobicplusfacultative bacteria, aswell as for tended to be higher than in the group that received stan-
enteric Gram-negative rods alone. In duodenum and dard TPN solution. However, significance was not
ileum the increase in the total bacterial count was also achieved in any part ofthe gut when thisgroup wascom-
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