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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 ...

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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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...Annals of surgery vol no j b lippincott company effect total parenteral nutrition plus morphine on bacterial translocation in rats peter m kueppers d thomas a miller chung ying k chen s gregory smith liliana f rodriguez and frank g moody from the department university texas medical school houston objective this study tested hypothesis that gut stasis induced by sulfate ms leads to enhanced tpn summary background data are common adjuncts care critically ill patients is known provoke variable degree induces with an accompanying overgrowth these two treatments combination not methods were provided central subcutaneous lines for continuous infusion nutrients drugs respectively intestinal transit was assessed caudal movement fluorescent marker intubated into proximal duodenum quantitative bacteriology carried out various segments ileocecal mesenteric lymph nodes mln spleen liver systemic blood obtained cardia puncture sacrifice at hours results unchanged alone but prolonged when given also ...

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