.ku _ Ir. 2%.». ‘1'! r}: , Pry-Nu;- l Wt'QA-x’ .1‘ x , 4.. . . r. h. as in: an“... . ea: , A : iffvfnsiw} . :w ft 3.53.532 .\.\.~.!.v .t. 2-!!Hns.‘ r try? 13. a. 1,. 3.1.3.“ . ‘ ‘ :2. l J .334 I isxln v . .s .1 .¢.L.I.l:l -...‘ 12 hours) in a 90°C gravity convection laboratory oven (Model SW-17TA-1; Blue M, Blue Island, IL), and weighed again (dry weight (mg)). The tissue water content was calculated as: w we' t r—-d wei x 100 wet weight (mg) and expressed as percent of wet tissue weight. 7. Tissue histology: Histologic specimens (lung, liver, intestine) were obtained following exsanguination at 90 minutes post-reperfusion or immediately following death in the survival studies. The intestines were divided into thirds with representative sections of the most severely affected portions retained. Tissues were placed in 10% buffered formalin (Sigma Chemical Company), embedded in paraffin, sectioned at 4 um, and stained with hematoxylin and eosin. All tissues were blind coded and evaluated by a pediatric pathologist who was not present at the time of the initial experiments. Qualitative assessments of the lung and liver were obtained while intestinal specimens were quantitatively graded according to the scale described in Appendix C. In addition, the amount of necrotic bowel on gross observation was measured as: langtn pr grgsaly necrotig snall powel (an) x 100 length of total small bowel (cm) 91 and was recorded as a percent necrosis of total bowel. WWW Peritoneal fluid cultures were obtained for aerobic and anaerobic growth (n=4/group). At the end of the experiment, the peritoneal cavity was exposed to allow access to peritoneal fluid. Cultures were obtained by absorbing peritoneal fluid onto aerobic culturettes (Culturette 110 Dual Swab Culture Collection System; Baxter Scientific Product, McGaw, IL) and anaerobic culturettes (VacutainerO Anaerobic Specimen Collector; Becton Dickinson Microbiological Systems, Cockeysville, MD). Once obtained, the cultures were placed on ice packs and transported to the Clinical Bacteriology Laboratory of the Animal Health Diagnostic Laboratory at Michigan State University for bacterial evaluation. The culture technique has been described in detail previously (Salmon et 31., 1990; Salmon et 31., 1991; Walker et 31., 1983). Primary plating media for the isolation of aerobic bacteria consisted of brain-heart infusion (EBA) agar (supplemented with 5% defibrinated sheep blood, 1% yeast extract and 1% horse serum), phenolethyl alcohol (PEA) agar, MacConkey (MAC) agar and thioglycolate broth supplemented with 1% hemin and 1% vitamin K. For the isolation of anaerobic bacteria, brain-heart infusion agar (supplemented with 5% defibrinated sheep blood, 1% horse serum, 1% yeast extract, 1% hemin and 1% vitamin K) (CDC) and PEA agar plates stored under anaerobic conditions were used as the primary plating media. 92 Submitted samples were inoculated on the appropriate media within one hour of collection. Aerobic cultures were incubated for 24-48 hours in a 5% CO2 environment (EBA and PEA) or atmospheric conditions (MAC and thioglycolate) whereas anaerobic cultures (CDC and PEA) were incubated in an atmosphere of 80% nitrogen, 10% hydrogen, and 10% cor Aerobic bacteria were identified by standard identification procedures including Vitek AMS, API 20E, or standard tube tests. Anaerobic bacteria were identified using the Rapid ANA II System (Innovative Diagnostic Systems, Inc., Atlanta, GA). 2, Serun collacrion: All blood samples were obtained aseptically from normal animals, and from animals at the end of steady state and at the end of the experiment (90 minutes post-reperfusion). Systemic blood samples were drawn from the femoral artery cannula utilizing a 23 gauge needle (Becton-Dickinson) and 3 ml syringe (Becton- Dickinson). Portal blood samples were obtained in parallel from a single portal vein draw using a 23 gauge needle and 1 ml syringe (Becton-Dickinson). Blood was removed until the animal was exsanguinated. The blood samples were immediately transferred to pyrogen-free micro-centrifuge tubes (Sarstedt Inc., Newton, NC) and allowed to clot while on ice packs. When coagulation was complete, the samples were spun for 10 minutes at 16,000 g in a refrigerated (4°C) centrifuge (Micro-Centrifuge Model 235C, Fisher Scientific, Livonia, 93 MI). Serum was then separated, aliquoted (100 ul/aliquot) into new pyrogen-free micro-centrifuge tubes using sterile transfer pipettes (Sarstedt Inc.), immediately frozen (Bio Freezer; Forma Scientific Inc., Marietta, OH), and stored at -80°C until the time of the assays. 0 ~19, Serum chemistry and enzyna analyaia; Serum chemistry and enzyme analyses were performed on systemic blood collected from normal animals, and from animals at the end of steady state and at the end of the experiment (90 minutes post-reperfusion). Since a large volume (approximately 650 pl) of serum was needed for the analysis, pooled samples were required. On the day of the assay, the appropriate number of frozen serum aliquots were randomly chosen (n=6-11/group), thawed, and pooled into pyrogen-free .micro-centrifuge tubes. The pooled samples (n=4-5/group) were blind coded and submitted to the Laboratory of Clinical Medicine, Lansing, MI, for multiple serum chemistry analysis using the Boehringer Mannheim / Hitachi 747 System (Boehringer Mannheim Corp., Indianapolis, IN). l1. Eortal lipopolysacgnaride aaaay; Lipopolysaccharide content was assayed using the quantitative chromogenic Limulus Amoebocyte Lysate assay (Model QCL-1000; Wittaker M.A. Bioproducts, Walkersville, MD). Serum samples were removed from the freezer, thawed, vigorously vortexed (Vortex-GenieO; Scientific Industries Inc., Bohemia, NY), pipetted (Certified Low Endotoxin® pipet tips; USA/Scientific Plastics, Ocala, FL) into pyrogen-free 94 polypropylene tubes (Falcon 2063; Becton-Dickinson Labware, Lincoln Park, NJ), and diluted 1:10 with pyrogen-free water (Abbott Laboratories). The tubes were heated at 100°C for 10 minutes in a heating block (Thermolyne Dri-Bath; Barnstead/Thermolyne, Dubuque, IA) to remove non-specific inhibitors, and then placed on ice packs. The diluted- samples were then aliquoted (20 pl) in duplicate into a pyrogen-free cell culture plate (Falcon 3072; Becton- Dickinson Labware). Escherichia coli lipopolysaccharide standard was reconstituted with 1.0 ml pyrogen-free water and vigorously vortexed. This solution was then diluted to 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, and 0.01 Endotoxin Units (EU)/ml according to the lipopolysaccharide activity described in that particular E. coli LPS standard lot. The tissue culture plate that contained the diluted samples was placed in the heating block (37°C) and the standards added. The Limulus amoebocyte lysate was reconstituted in 3.0 ml pyrogen-free water, aliquoted (50 pl/well), and allowed to incubate for 10 minutes. Chromogenic substrate (100 pl/well) was added and incubated for an additional 6 minutes. Acetic acid (25%) was then added to each well (100 pl) to stop the chromogenic reaction. The optical density was assessed at 405-410 nm using the Microplate Bioreader (Model EL311; Bio- Tek Instruments Inc., Winooski, VT). All samples were corrected for color with the appropriate serum blanks. 95 At 405-410 nm absorbance, the standard curve dilutions of 0.01-1.0 EU/ml are linear. Portal serum lipopolysaccharide concentrations (EU/ml) were extrapolated from the standard curve and transformed to pg/ml using the Difco standard (1 EU/ml = 100 pg/ml; adjustable according to the specific activity of the particular E. coli LPS lot). 12, gall-line maintenanga; All cell-lines were maintained in disposable sterile tissue culture flasks (Corning Glass Works, Corning, NY) and incubated (Steri-Cult 200 Incubator; Forma Scientific Inc., Marietta, OH) at 37%: in 90% humidity and 5% carbon dioxide. The tumor necrosis factor-sensitive WEHI 164 clone 13 cell-line was maintained as previously described (Espevik and Nissen-Meyer, 1986). Briefly, the mouse fibrosarcoma cells were cultured in growth media consisting of 10% heat-inactivated fetal calf serum in RPMI 1640 tissue culture media (See Appendix D for composition; Whittaker M.A. Bioproducts, Walkersville, MD) supplemented with 0.1 mM glutamine (GIBCO BRL, Grand Island, NY) and 30 pg/ml gentamicin (GIBCO BRL). The interleukin-6-sensitive 7TD1 murine B-cell hybridoma was also maintained as previously described (Van Snick et 31., 1989). Cells were cultured in 10% fetal calf serum/RPMI 1640 tissue culture medium supplemented with glutamine, gentamicin, and 0.1 mM sodium hypoxanthine / 16 pM thymidine (American Type Culture Collection, Rockville, MD). 96 memteruxtekinuseam WW Tumor necrosis factor activity was determined by assessing duplicate sera Isamples for WEHI-164 clone 13 cytotoxicity (Ayala et 31., 1992). One hundred fifty microliters of murine tumor necrosis factor standard (200 U/ml; Amgen Corp., Thousand Oaks, CA) were added to each of three wells (row A) of a 96- well tissue culture plate (Corning Glass Works). Rows B-H received 100 pl of media (10% heat-inactivated fetal calf serum/RPMI 1640). Fifty microliters of standard from row A was removed and added to row B, mixed with row B, 50 pl removed from row B and added to row C, etc., with three-fold serial dilution (1:1, 1:3, 1:9, 1:27, etc.) continued until row H. The 50 pl removed from row H was then discarded. Serum samples were thawed and diluted 1:20 (sera:media) in 10% fetal calf serum/RPMI 1640, and 200 pl pipetted in duplicate into row A. The samples in row A then underwent two-fold serial dilutions (1:20, 1:40, 1:80, 1:160, etc.) by removing 100 pl of row A and adding to row B, etc., until row H had been diluted. At the completion, 100 pl of diluted standard or sample were in each well. WEHI 164 clone 13 cells were harvested from cell line cultures by adding 7-10 ml trypsin (0.25%)-EDTA (0.02%) in Hank’s Balanced Salt solution (See Appendix E for composition; GIBCO BRL) for 45 seconds to dissociate cells from the tissue culture flask. Cells in solution were resuspended in 10% fetal calf serum/RPMI 1640 to obtain a 97 cell concentration of 5 x 105 cells/ml. Actinomycin (0.5 pg/ml; Calbiochem, San Diego, CA) was diluted 1:100 per volume of cells, 100 pl of cells were pipetted into each well of the standard/sample-filled 96-well plate, and the plate incubated for 20-24 hours at 37%:. At the end of the incubation period, 150 pl of supernatant were aspirated from the wells, and 20 pl of 5 mg/ml MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; Sigma Chemical Company, St. Louis, MO) added for an additional 4 hour, 37°C incubation period. At the end of this, 150 pl of supernatant were aspirated from the wells, and 150 pl of 10% sodium dodecyl sulfate (Sigma Chemical Company) in phosphate buffered saline (See Appendix E for composition; GIBCO BRL) were added. The wells were wrapped in foil to protect from light, and left at room temperature overnight in a moist area until the purple MTT crystals dissolved. Optical density was then assessed at 620 nm using a Microplate Bioreader. Since the WEHI-164 clone 13 cell line is sensitive to the cytotoxic effects of tumor necrosis factor and MTT is only absorbed by viable cells, the concentration of tumor necrosis factor and the concentration of viable cells are inversely proportional; the lower the number of viable cells as indicated by a lower optical density indicates higher concentrations of tumor necrosis factor in the sera supernatants. 98 pl__lnrarlanrin;§; Interleukin-6 activity in serum was determined by assessing the ability of the sera supernatant to induce the proliferation of the 7TD1 B-cell hybridoma (Ayala et 31., 1992). One hundred microliters of media (10% fetal calf serum/RPMI 1640) were pipetted into each well of a 96-well tissue culture plate. The recombinant human interleukin-6 standard (200 U/ml; Amgen Corp.) was added (100 pl) to three wells of a 96-well tissue culture plate (row A), while a second aliquot of thawed serum was diluted 1:15 (sera:media) in RPMI 1640 and added (100 pl/well in row A) in duplicate to row A. Standards and samples underwent three-fold serial dilutions thereafter. The interleukin-6-sensitive cell-line, 7TD1 B-cell hybridoma, was harvested and resuspended at a concentration of 4 x 10‘ cells/ml in 10% fetal calf serum/RPMI 1640 media containing 10 pg/ml Polymyxin B (Sigma Chemical Company) to bind lipopolysaccharide. One hundred microliters of cells were added to each well, and the plate incubated at 37°C for 72 hours. At the end of the incubation period, 20 pl of MTT were added to each well and the plate incubated for an additional 4 hours at 37%L At the end of the incubation period, 150 pl of supernatant were aspirated from each cell _ and discarded. One hundred microliters of 10% sodium dodecyl sulfate in phosphate buffered saline were added, the plates wrapped in foil, and left at room temperature overnight. Optical density of the dissolved MTT crystals in solution was assessed at 620 nm. 99 The relative units of monokine (TNF, IL-6) activity per milliliter of sera were determined by comparison of the curves produced from experimental sera supernatants to that of standard curves produced from a murine tumor necrosis factor standard or a recombinant human interleukin-6 standard according to previously described methods (Mizel, 1981). E, Sparisrical analysis: The appropriate statistical tests (Sokal and Rohlf, 1981) were performed and significance was recognized when p<0.05. For parametric data, a One-way Analysis of Variance (ANOVA) or Randomized Block ANOVA was used followed by the Student-Newman-Keul’s aposteriori post-hoc test for multiple comparisons. Non- parametric percentage data were transformed by the arcsine squareroot percentage method and then tested as with the parametric data. The bowel histology data were assessed for significance with the Mann-Whitney U non-parametric test. Overall group survival significance was evaluated with Fisher's Test for 2x2 tables. RESULTS . 'o e ch e ' e u a t ' ' ° Table 1 shows the characteristics of each group prior to experimentation. There were no significant differences between groups with respect to fasted weight or age at the time of the experiment. Table 1: Experimental group characteristics. normal saw 131; 9.8.1: LAM Number of animals: 22 47 46 37 37 Fasted weight: 71.6:1.3 73.7io.8 73.5io.8 73.210.9 74.2io.7 (grams) Age: 28.0:0.0 27.6:0.1 27.810.1 27.7:0.1 27.8¢O.1 (Gaye) All values are means 1 Standard Error of the Mean (SEM) . Normal{ Normal animals; SH15, Sham animals receiving 15 ml kg“ hr'; IR15, Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65, Sham animals receiving 15 ml kg“ hr“; IR15, Ischemia/Reperfusion animals receiving 15 ml kg“ hr“. 100 101 . "v s a and emato ' su ° 1, Maan arterial pressure: Steady state mean arterial pressure was the same for all groups (Figure 12, top). Following superior mesenteric artery occlusion, mean arterial pressure increased significantly in both ischemia groups (IR15 and IR65) compared to their equivalent sham groups (SH15 and SH65, respectively). In addition, the IR15 group displayed a significantly higher mean arterial pressure than in the IR65 group during superior mesenteric artery occlusion. This elevated mean arterial pressure in the ischemic groups remained significantly higher than their respective sham groups during the first half of the ischemic period. Following clamp removal after 90 minutes of superior mesenteric artery occlusion, both ischemia/reperfusion groups (IR15 and IR65) displayed a decrease in mean arterial pressure. The IR65 group decreased to and maintained a A pressure of approximately 78-80 mmHg, which was significantly lower than its equivalent sham group (SH65). The IR15 group became profoundly hypotensive, however, decreasing to and maintaining a pressure of approximately 60-62 mmHg for the entire 90 minute reperfusion period. This hypotension in the IR15 group was not only significantly lower than its respective sham group (SH15), but was also significantly lower than the IR65 group. 102 90 min 90 min Steady ischemia reperfusion State I H 110 , , , I . . . . f . MAP #v* # * 100 ~ .. I I 90 — 3 A 80 i- —1 OD II: E 70 _ _ E i l T . v / .....v T m 60 _. - SH15 : i *\f _ o: * a V IR15 # # # # E 50 ” o SH65 — (E * V 6 ‘ c1. / IR 5 / o / / O . . S 4 - _ CD . 3 L _ 2 ..- _. 1 '- 1 O _ — ..1 . J , 1 l . 1 1 TIME (hours) Figure 12: Mean arterial pressure (MAP, top) and central venous pressure (CVP, bottom) over the course of the, experiment (time) . ' All values are means 1; SEM. SH15 (n=47), Sham animals receiving 15 ml kg“ hr“; IR15 (n=46), Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=37), Sham animals receiving 15 ml kg“ hr“; IR15 (n=37), Ischemia/Reperfusion animals receiving 15 ml kg“ hr“. (* = p<0.05 vs Sham group at respective fluid resuscitation rate)(f = p<0.05 vs IR65). 103 21__Central_xenou§_eressurei Central venous pressure (Figure 12, bottom) remained essentially unchanged for the aggressive fluid resuscitation groups (SH65 and IR65) while decreasing as time progressed for the conventional fluid resuscitation groups (SH15 and IR15). This depression in central venous pressure became significant for the IR15 group during the later ischemic period and the early reperfusion period (Time = 2 hours 15 minutes through 3 hours 30 minutes). 3. Hamatgcrit: Initial hematocrit was the same for all groups and similar to normal animals (Figure 13). At the end of steady state, there appears to be a trend for hematocrit to increase in the conventional fluid resuscitation groups (SH15 and IR15). By the end of the ischemic period, this trend became notable; the hematocrit of the IR15 group was significantly higher than that of the IR65 group (42.7:0.7 vs 35.9io.7 percent, respectively), and the SH15 group was significantly greater than the SH65 group (40.8:1.0 vs 35.410.8 percent, respectively). At 90 minutes post-reperfusion (End of Experiment), hematocrit was again significantly higher in both conventional fluid resuscitation groups (SH15 = 41.8¢o.9 percent; IR15 = 48.710.9 percent) versus their equivalent aggressive fluid resuscitation group (SH65 = 33.611.1 percent; IR65 = 32.9:0.8 percent). In addition, the IR15 group was significantly more hemoconcentrated than its respective sham (SH15) group (48.711.2 vs 41.8:0.9 percent, 104 course of the experiment. 70 _ E SH15 m SH65 g3 IR15 - IR65 60 — — m Normal # rs =* +5 50 — — 02 5'4 2 - ** t4 . . v 9 o. 40 — N N - \I 0’ ‘0 T ’o‘ ’0‘ E: ’ * "' 7‘38 }{ ' ‘.R% ‘.ss a: tfies §§§$ U 3 O - } 4:43:31 F (3:15: - O 962:5: 94 E* Owe Owe e . 82:22:; 82:32 - 2 82.525; 82322222 m 20- am 5% — :3 Edit figfié _ >438 >183 _ <0me 4.33 82:25:32 his; 10- we 8% 8325252 8322323 - 83.2555: 855255: 54:22:22 543.255: 0 }{3n Zm$§ Initial Steady End of‘ End of State Ischemia Experiment . Figure 13: Systemic arterial hematocrit (percent) over the All values are means i SEM. (n=17), Sham animals receiving 15 ml kg“ hr“; Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; (n=18), Sham animals receiving 65 ml kg“ hr“; Normal, Normal animals; IR15 (n=18), Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. (* = p<0.05 vs respective group receiving 65 ml kg“ hr“) (f = p<0.05 vs SH15). SH15 SH65 IR65 (n=l8), 105 respectively). A, Arrarial pg, pigarbpnate, and plppd gaaaa; The initial blood pH, bicarbonate (HCO;), and blood gas parameters (Table 2) were not different among groups. The IR15 group displayed a profound metabolic acidosis by the end of the experiment (final). This was reflected by the significantly lower arterial pH and bicarbonate levels versus its initial value and its respective sham (SH15) group at the end of the experiment. As a result of the metabolic acidosis, there was significant respiratory compensation in the IR15 group, as indicated by the decreased Pco2 versus its initial value and its respective sham (SH65) group at the end of the experiment. The metabolic acidosis was significantly less severe in the aggressively resuscitated ischemia/reperfusion (IR65) group (Table 2). Respiratory compensation occurred, as reflected by the significantly decreased Pooh. Even though the arterial bicarbonate concentration of the IR65 group was significantly higher than the IR15 group at the end of the experiment, the final bicarbonate concentration in the IR65 group was significantly lower than its initial value and the SH65 final value. In this instance, aggressive fluid resuscitation in the IR65 animals allowed for sufficient respiratory and metabolic compensation to prevent the acidosis as seen in the IR15 group at the end of the experiment. 106 Table 2: Arterial pH, bicarbonate (ncog), and blood gases (Pco,, P0,) following steady state (initial) and at the end of the experiment (90 minutes post-reperfusion, final). GROUP poo2 po2 Hco; :T183 3 __28___ iterrl ltorrl. innelLLl SH15 * . -initial 7 7.38:0.01 47.612.l. 62.715.1 28.311.3 -final 6 7.41:0.04 42.4:3.7 77.118.o 27.7:3.2 IR15 -initial 9 7.39:0.01 42.5:3.3 75.2is.7 25.7il.8 -final 8 7.27:0.02'” 24.7il.7"’° 98.7:3.7“' 11.9:1-1‘” SH65 -initial 9 7.37:0.01 43.812.o_ 82.4:3.9 26.3il.2 -final 6 7.3610.03 46.012.1 79.7:3.2 26.0io.8 IR65 -initial 5 7.35:0.02 50.1:o.9 76.912.8 28.eil.o -final 5 7.32:0.01 32.7iz.3'le 95.713.4'“ 16.8i1.0“ All values are means 3 SEM. Normal, Normal animals; SH15, Sham animals receiving 15 ml kg“ hr“; IR15, Ischemia/ Reperfusion animals receiving 15 ml kg“ hr“; SH65, Sham animals receiving 65 ml kg“ hr“; IR65, Ischemia/ Reperfusion animals receiving 65 ml kg“ hr“. (a = p<0.05 vs IR15 - initial) (b = p<0.05 vs SH15 - final) (8 p<0.05 vs IR65 - final) (d = p<0.05 vs IR65 - initial) (e p<0.05 vs SH65 - final). 107 Q, §nryival srudias: All sham animals, regardless of fluid resuscitation rate, survived the protocol and the 72 hour observation period (Table 3). The IR15 group had no survivors at 72 hours, while the IR65 group displayed a significantly greater survival of 27 percent at 72 hours. Moreover, the survival time of non-surviving animals was increased with aggressive fluid resuscitation from 4.3:1.8 hours from the end of the experiment in the IR15 group to 11.3:2.4 hours in the IR65 group. D, Iissue microvascular blood flow: There was no change in renal or hepatic blood flow over the course of the experiment with one exception; renal cortical blood flow in the IR15 group at the end of the experiment was significantly lower than its respective sham (SH15) group (Table 4). Ascending colon blood flow in both ischemia/reperfusion groups was significantly lower at the end of ischemia than either of their respective sham groups (Table 4). Following reperfusion, however, blood flow to the colon was returned to normal and maintained throughout the experiment. The remainder of the splanchnic circulation, however, was significantly affected by superior mesenteric artery occlusion (Table 5). Duodenal, jejunal, and ileal blood flows were significantly reduced immediately following superior mesenteric artery occlusion in both ischemia/reperfusion groups. In these groups, blood flow remained significantly lower than their respective sham 108 Table 3: Overall group survival (percent) and survival time of non-surviving animals (hours). GROUP SURVIVAL (72 hrs) Ferrel 8813 1813 889: 1893 Overall number: N/A (15/15) (0/15) (15/15) (4/15) Percent: N/A 100 o' 100 27* SURVIVAL TIME (non-survivors only) ormal 881; 181; 8893 1893 Time (hrs): N/A N/A 4.3il.8 N/A 11312.4b All values are means 1 Standard Error of the Mean (SEM). N/A, not applicable; SH15, Sham animals receiving 15 ml kg“ hr'; IR15, Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65, Sham animals receiving 65 ml kg“ hr“; IR65, Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. (a = p<0.05 vs Sham group at respective fluid resuscitation rate) (b = p<0.05 vs IR15). 109 Table 4: Laser Doppler blood flow of the kidney, liver, and ascending colon over the course of the experiment. £311 131; §§£§ 152; Kidne - 5 m n ' ischemia 100.014.0 94.3:3.2 100.9:8.1 92.3:3.7 - 85 min iSChemla 108.3:5.6 97.413.0 97.7:9.1 98.6:3.2 - 5 min reperfusion 98.0iS.7 88.5:5.8 90.8:3.7 93.314.4 - 85 min reperfusion 101.5:8.1 73.3:7.8‘ 96.7:7.6 86.3:3.8 Liver - 5 min ischemia 100.014.0 102.1i4.2 102.517.? 92.3:3.7 - 85 min ischemia 108.3:5.6 94.5i6.9 99.4:2.2 98.4i7.0 - 5 min ‘ reperfusion 98.015.7 101.1i6.8 101.2:3.4 104.3:6.2 - 85 min ‘ reperfusion lOl.5¢8.1 93.7:7.0 99.7:3.8 105.9:8.5 Ascending colon - 5 min ischemia 96.4112.4 75.1:13.l 95.6:14.8 81.816.9 - 85 min ischemia 102.2116.4 59.4:8.7‘ 103.6i16.0 55.735.4' - 5 min reperfusion 103.314.3 91.5112.9 118.5:12.3 99.1:10.2 - 85 min reperfusion 93.4ill.7 114.9:24.1 95.815.4 96.5113.3 Data are presented as percent (%) of steady state values (ml min“ 100 9“ tissue). All values are means 1 SEM. 8815 (n=3-6), Sham animals receiving 15 ml kg“ hr“; IR15 (n=5-6) , Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=3-6), Sham animals receiving 65 ml kg‘ hr“; IR65 (n=6), Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. (a = p<0.05 vs respective Sham group). 110 Table 5: Laser Doppler blood flow of the duodenum, jejunum, and ileum over the course of the experiment. £31; 1315 fifiéi 1355 Duodenum - 5 min ischemia 105.912.1 73.2:6.6‘ 106.5:5.0 69.719.8‘ - 85 min - ischemia 102.6i7.7 85.616.5‘ lll.7i4.0 83.1:4.8' - 5 min reperfusion 99.5:11.3 88.4:11.4 104.4:8.1 100-2i9-4 - 85 min reperfusion 97.3:9.5 69.0:2.5“’ 94.3:8.3 85.8:7.9 Jejunum - 5 min ischemia 100.3:7.8 58.6:7.8' 126.7:28.5 63.5:7.2‘ - 85 min ischemia lOl.4il7.5 69.8:6.2 107.8i14.3 102.1i13.8 - 5 min reperfusion 97.516.2 86.6:10.3 110.3:11.8 124.5:13.0 - 85 min reperfusion 89.6:8.l 81.7ill.8 89.7:8.2 101.7il4.2 Ileum . - 5 min ischemia 104.014.6 44.4:10.2' 86.1:5.3 45.5:3.4' - 85 min ischemia 116.3i15.3 63.9113.3' 105.5113.3 56.117.1‘ - 5 min reperfusion 102.8:7.3 66.6:8.9‘ 103.7il4.3 94.4:12.7 - 85 min reperfusion 108.0:15.5 61.319.6' 126.3:48.5 82.419.8 Data are presented as percent (%) of steady state values (ml min“ 100 9“ tissue). All values are means i SEM. SH15 (n=3-6), Sham animals receiving 15 ml kg“ hr“; IR15 (n=5-6) , Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=3-6), Sham animals receiving 65 ml kg“ hr“; IR65 (n=6) , Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. (a = p<0.05 vs respective Sham group) (b = p<0.05 vs IR65). 111 groups throughout the ischemic period. Jejunal blood flow in either ischemia/reperfusion groups at the end of ischemia, however, was not significantly different than their corresponding sham groups. Following reperfusion, jejunal and duodenal blood flow increased in the ischemia/reperfusion groups, but still displayed a trend toward reduced flow (Table 5). This reduction in blood flow became significant in the duodenum of the IR15 group at the end of the experiment. The ileum of the IR15 group, however, did not return to steady state blood flow levels following reperfusion and remained persistently hypoperfused for the duration of the experiment. E. Eluid gainszlosses during the experiment: The aggressively resuscitated groups (IR65 and SH65) had a significantly elevated post-experimental body weight and net weight gain as compared to the conventional resuscitation groups (IR15 and SH15) at the end of the experiment (Table 6). Similarly, total fluid administered and fluid loss were higher in the IR65 and SH65 groups; however, net fluid loss (%) appeared to be greater in the conventional fluid resuscitation (SH15 and IR15) groups as compared to the aggressively resuscitated groups (SH65 and IR65). This difference, however, was not statistically significant. 2, East-experimental weight gain: Figure 14 displays weight gain for surviving animals over the 72 hour observation period. Both sham groups, irrespective of the 112 Table 6: Net fluid gains/losses during the experiment. ggis IR15 sac; IRG§ Fasted weight (grams) 75.0il.7 75.711.5 77.5il.4 78.8il.o Post-experimental weight (grams) 79.3il.6 80.3:1.7 97.7:2.1' 100.2:1.3b Net weight gain (grams) 4.7io.7 4.6:0.3 20.210.9' 21310.7b Total fluid administered 6.5io.2 6.5:O.l 26.310.4‘ 26.7io.3b (grams) Fluid loss 2.2:o.6 2.0io.3 6.2:0.8' 5.4io.7b (grams) Net fluid loss (%) 33.7:9.4 30.3:4.o 23.513.o 20.112.5 All values are means‘: SEM; SH15 (n=7), Sham animal receiving 15 ml kg“ hr“; IR15 (n=7) , Ischemia/reperfusion animal receiving 15 ml kg“ hr“; SH65 (n=6) , Sham animal receiving 65 ml kg“ hr“; IR65 (n=6), Ischemia/reperfusion animal receiving 65 ml kg“ hr“. (a = p<0.05 vs SH15) (b = p<0.05 vs IR15). 113 100 u I T I F e SH15 SH65 95 _ V IR15(N0 Survivors) IR65 " <10 90— — 85— 80— '75— 70~ BODY WEIGHT (grams) 65— — \T‘ 60/ O 1 1 1 l O 24 48 72 TIME (hours) Figure 14: Post-experimental weight gain (grams) over the 72 hour observation period. All values are means -_+_ SEM. SH15 (n=11), Sham animals receiving 15 ml kg“ hr“; IR15 (n=0), Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=9), Sham animals receiving 65 ml kg“ hr“; IR65 (n=3) , Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. (* = p<0.05 vs SH65) . 114 fluid resuscitation rate, gained weight at similar rates during the observation period following the experiment. The IR65 group weighed significantly less than the SH65 group at 72 hours (73.5:1.5 vs 85.6il.7 grams, respectively). There was no weight gain data for the IR15 group since none survived (Table 3). . . Q, Egtiphetgl organ tissue water content; There was no difference in water content in any of the tissues during steady state as compared to normal animals (Table 7). Moreover, no significant changes in tissue water content were seen in the liver or the thymus of any experimental group at the end of the experiment. The splenic tissue "water content of animals in the IR15 group was significantly greater than normal animals by the end of the experiment. Likewise, the kidneys of animals in the IR15, SH65, and IR65 groups at the end of the experiment contained significantly more tissue water than normal. Tissue water content changes were most pronounced in the lung and intestine. At the end of the experiment, the IR15 group contained significantly less lung tissue water than normal, steady state, or SH15 animals. The SH65 group had significantly more lung tissue water than normal. The IR65 group displayed values similar to normal with one exception; there was significantly less lung tissue water in the IR65 group as compared to its respective sham (SH65) group. 115 Table 7: Peripheral organ tissue water content (8) of normal animals, and of animals at steady state, and at the end of the experiment (90 minutes post-reperfusion). Normal "“I§£§§g¥"§£§£%EF" 9:923 125121 132121 = Liver 71.8:0.1 72310.2 72.9io.2 Kidney 76.53503 77410.3 77.4io.2 Lung 79.5:0.4 79.810.2 80.3:0.2 Spleen 76.6io.6 77.110.2 77.1:0.2 Thymus 79.8:0.2 79.5io.2 79.5io.2 Duodenum 78.2:0.2 78310.2 79.3104 Jejunum 78.3:0.2 78.4iO.2 79.8:O.2 Ileum 79.2105 78.8:0.4 78.9io.3 90 minutes post-reperfusion SH15 IR15 SH65 IR65 thgn (n=;0) (n=10) (n=6) (n=7) Liver 73.2105 73.1io.3 72.9io.2 74.7:1.1 Kidney 77.810.23 78.710.4' 80.410.1' 80.710.6‘ Lung 79.7:o.2 78.0io.2“" 81.1:0.2' 79.710.6‘ Spleen 78.0:0.1 78.2iO.1' 77510.1 77.71043 Thymus 79.7io.1 78.8:O.2 80.8io.2 79.8104 Duodenum 80.1io.2"’ 86.310.5‘5‘ 80.9iO.3‘ 89.6io.5""° Jejunum 79.4:0.5 37.91045:be 82.210.4" 90.1io.3""° Ileum 78.8iO.S 37.30.?“ 81.5104 90.3i1.:3Me All values are means 1 SEM. Steady State 15, animals at the end of Steady State receiving 15 ml kg“ hr“; Steady State 65, animals at the end of Steady State receiving 65 ml kg“ hr“; SH15, Sham animals receiving 15 ml kg“ hr“; IR15, Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65, Sham animals receiving 65 ml kg“ hr“; IR65, Ischemia] Reperfusion animals receiving 65 ml kg“ hr“. (a = p<0.05 vs Normal) (b = p<0.05 vs Steady State at respective fluid resuscitation rate) (c = p<0.05 vs SH15) (d = p<0.05 vs IR15) (e = p<0.05 vs SH65). 116 ‘Tissue water content in the intestine changed most drastically of all (Table 7). Except for the ileum in the SH65 group, all groups had significantly greater tissue water content than normal animals. The IR15 group also contained more tissue water in all parts of the intestine (including duodenum) than the SH15 group. The IR65 group contained significantly more tissue water than any of the other groups in all areas of the intestine. W931i 1, Intestines; Histologic and gross bowel necrosis data are displayed in Table 8. There was a graded increase in intestinal injury from duodenum to ileum in both ischemia/reperfusion groups. Although the ischemia/reperfusion groups displayed significantly greater histologic injury to all the areas of small bowel than their respective shams, the duodenum of the aggressively resuscitated group (IR65) was spared from injury. In contrast, the duodenum in the IR15 group showed a significantly greater degree of injury compared to its respective sham (SH15) group. Both ischemic groups suffered larger areas of grossly necrotic bowel than their respective shams and, in addition, the IR15 group grossly displayed an approximately 23% greater amount of gross intestinal injury than the IR65 group. zt__2g;ipngt§;_gtggn§; Regardless of the group, the kidney, spleen, and lung showed no significant histological change in the survival experiments. There was, 117 Table 8: Histologic scores for duodenum, jejunum, and ileum at the end of the experiment (90 minutes post-reperfusion) and the percentage of grossly necrotic bowel observed. NECROTIC QBQQE Q QUODENQM JEJUNQM ILEQfl BQWEL (3) SH15 6 0.5i0.4 _ 0.7:0.5 1.8iO.4 0.0i0.0 IR15 6 2.73:0.‘7‘l 4.5:0.6' 5.3:0.7' 31.1i6.4¢ SH65 6 O.8:O.4 0.8iO.6 1.2i0.4 0.0i0.0 IR65 6 l.5i0.4 4.7:1.0' 5.7:0.8' 8.4:3.0' Appendix D defines the grading scale. All values are means 1 SEM. SH15 (n=6), Sham animals receiving 15 ml kg“ hr“; IR15 (n=6-9) , Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=6), Sham animals receiving 65 ml kg“ hr“; IR65 (n=6- 7), Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. (a = p<0.05 vs Sham at respective fluid rate) (b = p<0.05 vs IR65). 118 however, occasional polymorphonuclear granulocyte (neutrophil; PMN) cell margination in the pulmonary pre- capillary vascular beds. This was most pronounced in the IR15 group (5 of 6 rats), while it still occurred in the SH15 group (1 of 6), the IR65 group (1 of 3) and the SH65 group (1 of 7) to’a lesser extent. I, Eezitongal fluid gultutes: Peritoneal fluid cultures (n=4/group) displayed small amounts (5-50 Colony Forming Units) of Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium in all animals, suggesting mild contamination from skin flora. Clostridium (<100 Colony Forming Units) was evident in one animal in the IR15 group (25%), while nOne of the other groups displayed any anaerobic growth. J. ch 'st and e z e an 5's: 1, Elggttolytes and glucgse: Normal and steady state serum chemistry data are presented in Table 9. During steady state, fluid resuscitation at either rate (15 or 65 ml kg“ hr“) significantly lowered serum sodium, calcium, and phosphorus levels, while significantly elevating serum potassium and glucose levels when compared to normal. Serum glucose was significantly higher and bicarbonate significantly lower in the 8865 group as compared to the 8815 group. Although the differences have statistical significance, all values (except glucose) are within normal ranges for 5 week old rats (Harkness and Wagner, 1983; Loeb and Quimby, 1989). 119 Table 9: :Normal and steady state levels of serum.electrolytes and glucose. 0 Ra 0 Otflll .§§1§ §§§§ Sodium (mEq/L) 137-155 140:0 135:1' 134:1' Potassium (mEq/L) 4.9-9.9 6.9:0.1 9.1:0.4' 8.3:0.3' Calcium (mg/dL) 5.0-13.0 10.0:0.1 9.5:0.1' 9.6:0.0‘ Phosphorus (mg/dL) 7.1-13.1 11.2:0.1 10.1:0.1"’ 8.4:0.1' Chloride (mEq/L) 97-109 10610 10610 106:0 Glucose (mg/dL) 128-170 133:6 213:5“ 280:7' All values are means : SEM. Normal ranges obtained from the literature (Harkness and Wagner, 1983; Loeb and Quimby, 1989). Normal (n=10), Normal animals; $815 (n=8), animals at the end of Steady State receiving 15 ml kg“ hr“; SS65 (n=lO) , animals at the end of Steady State receiving 65 ml kg“ hr“. (a = p<0.05 vs Normal) (b = p<0.05 vs 8865). 120 Table 10: Experimental group levels of serum electrolytes and glucose at the end of the experiment (90 minutes post- reperfusion). £311 IR15 5365 1365 Sodium (mEq/L) 143:4 134:0' 147:7 133:1' Potassium (mEq/L) 9.1:0.2 11.2:0.4¢ 7.7:0.4 6.6:0.4 Calcium (mg/dL) 9.4:0.3 9.3:0.1b 9.3:0.s 3.0:0.1' Phosphorus (mg/dL) 9.1:0.4 13.3:0.5'ID 8.1:0.3 8.9:0.2 Chloride (mEq/L) 113:2 108:0 118:5 111:1 Glucose (mg/dL) 189:7c 233:4* 435:23 6413:13'b All values are means : SEM. SH15 (n=11), Sham animals receiving 15 ml kg“ hr“; IR15 (n=10), Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=7), Sham animals receiving 65 ml kg“ hr“; IR65 (n=6), Ischemia/Reperfusion animals receiving 65 ml kg“.hr“. (a = p<0.05 vs Sham group at respective fluid resuscitation rate) (b = p<0.05 vs IR65) (c = p<0.05 vs SH65). 121 By the end of the experiment (90 minutes post- reperfusion), serum chemistries were significantly altered (Table 10). When compared to its respective sham group (SH65), the IR65 group displayed a significantly lower serum sodium and calcium concentrations, while having a significantly elevated glucose. The IR15 group displayed a similar hyponatremia while demonstrating a significantly elevated potassium, phosphorus, and glucose, when compared to its respective sham (SH15) group. More importantly, the IR15 group as compared to the IR65 group displayed significant hyperkalemia, hypocalcemia, and hyperphosphatemia while having a significantly lower serum glucose concentration. The sham (SH15 and SH65) groups were similar in all parameters, except the SH65 group was significantly more hyperglycemic than the SH15 group. 2, Enzymgg and metabglig ygstgs: Normal and steady state serum levels of cellular enzymes and metabolic waste products are presented in Table 11. Fluid administration per se significantly (p<0.05) altered alkaline phosphatase, serum glutamic pyruvate transaminase, serum glutamic oxalacetic transaminase, creatine phosphokinase, lactic dehydrogenase, blood urea nitrogen, and bilirubin; however, these differences were within normal limits (Harkness and Wagner, 1983; Loeb and Quimby, 1989). By the end of the experiment (90 minutes post- reperfusion), serum glutamic pyruvate transaminase, serum glutamic oxalacetic transaminase, creatine phosphokinase, Table 11: 122 ensymes and waste products. 321151.81292 HQIEAI Alkaline Phosphatase (U/L) SGPT (U/L) SGOT (U/L) Creatine Phosphokinase (U/L) Lactic Dehydrogenase (U/L) Blood Urea Nitrogen (mg/dL) Creatinine (mg/dL) Bilirubin (mg/dL) 121-407 24-50 67-104 237-1490 12-20 0.4-0.5 0.1-0.5 24914 4011 88:1 250:5 294318 8811 235:4' 41:1b 104:4“ 419:36“ 725:98“ Normal and steady state levels of serum cellular fifiéi 232:4' 3412‘ 8310 310:6 454:30 0.3:0.1* 0.1:0.0 All values are means : SEM. literature (Harkness and Wagner, (n.a., not available). 1989). Transaminase; Normal (n=10) , $315 (n=8) , ssss (n=10), Normal ranges obtained from the 1983; Loeb and Quimby , (I = p<0.05 vs Normal) (b = p<0.05 vs 8865). __-_ SGPT, Serum Glutamic Pyruvate SGOT, Serum Glutamic Oxalacetic Transaminase. Normal animals; animal receiving 15 ml kg“ hr“; animal receiving 65 ml kg“ hr“. Steady State Steady State 123 Table 12: Experimental group levels of serum cellular ensymes and waste products at the end of the experiment (90 minutes post-reperfusion). £31; 131; 8865 13;; Alkaline Phosphatase (U/L) 196:8c 228:6“ 160:8 150:4 sop'r (U/L) 55:3c 196:8“ 36:2 180:3' sso'r (U/L) 190:12c 325:5“ 86:4 178:2‘ Creatine Phosphokinase (U/L) 1944:190c 2819:88“ 439:21 1171:67' Lactic Dehydrogenase (U/L) 893:70c 2559:113“ 437:20 1252:114' Blood Urea Nitrogen (mg/dL) 9:1c 25:1“ 4:0 11:0' Creatinine (mg/dL) 0.5:0.0 0.7:0.0“ 0.4:0.0 0.5:0.0 Bilirubin (mg/dL) 0.2:0.0° 0.6:0.0“ 0.1:0.0 0.2:0.0 All values are means : SEM. SGPT, Serum Glutamic Pyruvate Transaminase; SGOT, Serum Glutamic Oxalacetic Transaminase; BUN, Blood.Urea.Nitrogenu SH15 (n=11), Sham animals receiving 15 ml kg“ hr“; IR15 (n=10), Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=7), Sham animals receiving 65 ml kg“ hr“; IR65 (n=6), Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. * (a = p<0.05 vs Sham group at respective fluid resuscitation rate) (b = p<0.05 vs IR65) (c = p<0.05 vs SH65). _—— r i 124 lactic dehydrogenase, and blood urea nitrogen were all elevated in the IR65 group versus its respective sham (SH65) group (Table 12). These same parameters were also significantly elevated in the IR15 group versus its respective sham (SH15). In addition, alkaline phosphatase, creatinine, and bilirubin were significantly elevated in the IR15 group as compared to its sham (SH15). Strikingly, all measured enzyme levels (except SGPT) were significantly greater in the conventionally resuscitated IR15 group versus the aggressively resuscitated IR65 group. K, Eortal lipopolysaccharide concentratlgngg Portal serum lipopolysaccharide (LPS) concentrations were not significantly elevated in normal animals (13.1:0.7 pg/ml), at any time during steady state (8815 = 23.4:6.9 pg/ml; 8865 = 15.3:1.3 pg/ml) or in any group at the end of the experiment (Figure 15). There appears to be a trend toward elevated portal LPS in the IR65 group (76.9:32.0 pg/ml) at the end of the experiment; however, this increase was not significantly different (p<0.09) from either the IR15 group (20.0:2.1 pg/ml) or the SH65 group (19.6:5.6 pg/ml). L. Inflammatory cytokine relegse: l. Tuna; necrosis factor: Tumor necrosis factor was not detected by bioassay in the systemic circulation of any group at any measured time point. z:__lntgtlgnkin;§: In contrast (Figure 16), interleukin-6 (IL-6) was significantly elevated by the end of the experiment in the IR15 group versus the SH15 group 125 200 3 [22] 3815 E3 SH15 SH65 : 175 3 M 8865 IR15 IR65 : ‘3 150 E- 3 ° - ‘: S '- .. 2 : : “a 125 :- j 0) h .. O A : : S g 100 _— ': U\ _ _ m 3.0 1 : 3" 75 T —_ E i : 50 - 8 ~ 1 0. : : 25 } -§ 0 ~ H E SE _ ~ Normal Steady 90 minutes State post-reperfusion Figure 15: Portal serum lipopolysaccharide (LPS; pg/ml) concentration over the course of the experiment. All values are means : SEM. Normal (n=7); Normal animals; 8815 (n=7) , animals at the end of Steady State receiving 15 ml kg“ hr“; 8865 (n=7) , animals at the end of Steady State receiving 65 ml kg“ hr“; SH15 (n=7) , Sham animals receiving 15 ml kg“ hr“; IR15 (n=7), Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=5) , Sham animals receiving 65 ml kg“ hr“; IR65 (n=7) , Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. No significant differences among any groups at any time. 126 1360 ~ SH15 SH65 : ZZI IR15 IR65 * # c: _ .9. 200 —. J _ +3 7- o L: t E ’ / 0 L / 2 150 ~ / _ 0 2 b / U E _ / (O \\ . / .5 3 ~ / '— 100 e j _ E * / E ’ / * h / 9’2 - / 50 —— _ é .. I @/ * / L NLD. NID. / O /:3 Normal Steady 90 minutes State post-reperfusion Figure L6: Serum Interleukin-6 (IL-6; U/ml) concentration over the course of the experiment. All values are means : SEM. N.D., Not Detectable; Normal (n=10), Normal animals; Steady State, Steady State animals (n=8/group) receiving either 15 or 65 ml kg“ hr“; SH15 (n=9) , Sham animals receiving 15 ml kg“ hr“; IR15 (n=10), Ischemia/Reperfusion animals receiving 15 ml kg“ hr“; SH65 (n=10), Sham animals receiving 65 ml kg“ hr“; IR65 (n=9), Ischemia/Reperfusion animals receiving 65 ml kg“ hr“. (* = p<0.05 vs respective Sham group) (i = p<0.05 vs IR65) (O = p<0.05 vs SH65). 127 (174.4:39.2 vs 18.8:7.4 U/ml, p<0.05) and the IR65 group versus the SH65 group (59.1:8.7 vs 4.2:0.4 U/ml, p<0.05). Strikingly, the IR15 group had a significantly greater concentration of circulating interleukin-6 than the IR65 group (174.4:39.2 vs 59.1:8.7 U/ml, p<0.05). DISCUSSION Using a model of acute small intestinal ischemia followed by reperfusion in adult rats, a recent study has reported difficulty in reproducing published mortality rates (Megison et a1., 1990). The authors concluded that I individual anatomical differences in collateral circulation may have provided differential mesenteric perfusion during and after ischemia. This resulted in widely variable bowel injury and, ultimately, the nonreproducibility of previously published mortality rates (Boorstein et a1., 1988; Dalsing et 61., 1983). Our rationale for undertaking the present study was stimulated by a our similar observation of nonreproducibility in immature rat models of intestinal ischemia/reperfusion. For example, we also were unable to duplicate reported survival rates. Moreover, we noted that a circulatory shock-like state occurred following reperfusion which was characterized by significant hypotension and hemoconcentration. These observations led us to hypothesize that concurrent cardiovascular instability may influence the secondary consequences of intestinal ischemia/reperfusion that have been previously reported (Bitterman et a1., 1991; Caty et a1., 1989; Caty et a1., 128 129 1990; Hill et a1., 1991; Schmeling et a1., 1989). The goal of this research, therefore, was to design a model that eliminated cardiovascular instability following reperfusion through aggressive fluid resuscitation. With this model, we then assessed whether this aggressive fluid resuscitation regimen had any beneficial effects on a number of parameters following 90 minutes of small bowel ischemia and reperfusion in immature rats as compared to an identical model that received conventional fluid resuscitation at the maximal rate described in the literature. The importance of adequate fluid administration following illness or injury has been understood by clinicians for decades and has been reiterated by a study in the recent pediatric literature. Following presentation of septic shock, pediatric patients demonstrated improved survival when rapid fluid resuscitation of greater than 40 ml kg“ was utilized during the first hour (Carcillo et a1., 1991). Shock was diagnosed in these children if blood pressure was less than 2 standard deviations below the mean for age, combined with three of four criteria for decreased perfusion: decreased peripheral pulses, mottled or cool extremities; tachycardia; or oliguria. In this study, aggressive fluid resuscitation decreased persistent hypovolemia but did not increase the risk of cardiogenic pulmonary edema or respiratory distress syndrome (RDS) (Carcillo et a1., 1991). 130 In other studies, resuscitation with large volumes of crystalloid solutions was tolerated well and did not lead to an increased incidence of pulmonary edema in patients following abdominal aortic surgery (Virgilio et a1., 1979). Others have suggested that expanding vascular volume to supranormal levels in high-risk post-surgical patients provides greater physiologic/hemodynamic support, yielding improved outcome (Shoemaker et a1., 1988). Moreover, crystalloid solution resuscitation of greater than 60 ml kg“ has been advocated in patients undergoing renal transplantation (Dawidson et a1., 1987). These authors state that such a fluid regimen results in a greater rates of renal allograft function and overall patient survival. The beneficial effects of aggressive fluid administration on survival of experimental subjects following intestinal ischemia/reperfusion have also been described. Improved survival with adjuvant fluid resuscitation has been demonstrated following bowel ischemia and reperfusion in dogs (Chiu et 81., 1972) and rats (Dawidson et a1., 1979; Dawidson et a1., 1981; Dawidson et a1., 1986; Dawidson et a1., 1990; Ottoson et a1., 1989). Such studies utilized variable crystalloid solution infusion rates, ranging from 42 ml kg“ hr“ (Dawidson et a1., 1986) to 1032 ml kg“ hr“ (Dawidson et a1., 1979). Improved survival has also been documented in rats following intestinal ischemia/reperfusion by giving bolus injections of autologous rat plasma to maintain mean arterial pressure 131 above 80 mmHg (van der Meer et a1., 1976). In the present study, we utilized a hypertonic (525 mOsm/L) dextrose- balanced salt solution (Appendix B) instead of 3% albumin in lactated Ringer's solution (Dawidson et al., 1979) or plasma (van der Meer et a1., 1976) because of recent concerns in the clinical arena over the administration of blood or blood’ products. In the present study, superior mesenteric artery occlusion produced a significant increase in mean arterial pressure (Figure 12, top) in both ischemia/reperfusion (IR15 and IR65) groups compared to their respective sham (SH15 and SH65) groups. This transient hypertensive period has been described previously in a similar model using adult rats subjected to 90 minutes of intestinal ischemia/reperfusion (Lefer and Ma, 1991). Superior mesenteric artery occlusion decreases blood flow to the intestine. This results in decreased intestinal baroreceptor input to the central medullary vasomotor center, reflexly increasing sympathetic output and blood pressure (Lefer and Ma, 1991). Mean arterial pressure remained elevated in both ischemia/reperfusion groups until the microvascular clamp was removed from the superior mesenteric artery and reperfusion of the ischemic intestine was initiated (Figure 12, top). At that time, the mean arterial pressure of the conventionally resuscitated IR15 group decreased Significantly to approximately 62 mmHg and remained low for the 90 minute reperfusion period. In contrast, aggressive 132 fluid resuscitation in the IR65 group attenuated the hypotension associated with reperfusion of the ischemic intestine. Hypotension has been shown to follow intestinal ischemia and reperfusion (Bitterman et a1., 1991; Brandt and Boley, 1991; Chiu et a1., 1972; Filep et a1., 1989; Glenn and Lefer, 1970; Lefer and Ma, 1991; Schmeling et a1., 1989). Preventing or attenuating hypotension, however, should be an important consideration when studying the secondary pathophysiology of intestinal injury per so so that the additional variable of hypotension does not alter the experimental results. I Although it is well known that brief periods of hemorrhagic hypotension stimulate sympathetic nervous system activity and reduce splanchnic blood flow (Ramenofsky et a1., 1981), the effects of hypotension, in the absence of blood loss, have only recently been explored. Euvolemic, Chemically-induced hypotension produces elevated release of the prostaglandin E“ the immunosuppressive action of which leads to depression of antigen presentation capacity of peritoneal macrophages (Ertel et a1., 1992). Therefore, the secondary consequences of hypotension may influence the effects of ischemia/reperfusion and should be avoided when the objectives of the study are unrelated to hypotension. Central venous pressure was used in this study as a measure of right atrial filling pressure; declines in central venous pressure may indicate a decrease in 133 circulating blood volume. In the present study, conventional fluid resuscitation in both ischemia/reperfusion (IR15) and sham (SH15) groups yielded a decrease in central venous pressure (Figure 12, bottom). The animals in these groups were beginning to demonstrate the effects of decreased circulating blood volume. Central venous pressure tended to remain constant, however, in those groups which received aggressive fluid resuscitation (SH65 and IR65). This indicates a maintenance of circulating blood volume. Hemoconcentration occurs following intestinal ischemia/reperfusion as indicated by an increase in hematocrit (Dawidson et a1., 1981; Dawidson et a1., 1986; Dawidson et a1., 1990). Hematocrit is the percent of whole blood comprised of packed red blood cells vis-a-vis plasma. This measurement was chosen as an index of circulating blood volume (Robarts et a1., 1979; Van Beaumont, 1972) since the total red blood cell mass in this model is constant (Miki et a1., 1980). Since changes in hematocrit correlate well with changes in plasma volume in another shock model (Ottoson et a1., 1989), when hematocrit increases, plasma volume is assumed to be lost and the animal becomes hemoconcentrated. In the present study, systemic arterial hematocrit (Figure 13) increased over the course of the experiment for the conventionally resuscitated groups (SH15 and IR15). Hematocrit was significantly higher in the groups with conventional fluid resuscitation (SH15 and IR15) than in the 134 aggressively resuscitated groups (SH65 and IR65). It was higher both at the end of the ischemic period and at the end of the experiment (90 minutes post-reperfusion). Moreover, the IR15 group was significantly more hemoconcentrated than that of its respective sham (SH15) at the end ofthe experiment. Even the conventional resuscitation sham (SH15) group was significantly hemoconcentrated compared to the aggressively resuscitated sham (SH65) group. There was no hemoconcentration, however, in either aggressively resuscitated group (SH65 and IR65) at any time during the experiment (Figure 13). Just as aggressive fluid resuscitation prevented the fall in central venous pressure, it also appears to prevent the hemoconcentration, or hypovolemia, associated with intestinal ischemia/reperfusion. Blood hyperviscosity may occur with extreme cases of hemoconcentration and polycythemia (Dintenfass, 1981). Such conditions have been shown to be detrimental to survival following an insult such as mesenteric ischemia by producing a more extensive bowel injury (Dunn et 81., 1985). Conversely, experimental hemodilution (i.e., lowering hematocrit) has been shown to be beneficial during reperfusion of post-ischemic intestine by increasing mesenteric tissue oxygen consumption (Mesh and Gewertz, 1990). In the present study, the blood of animals that received aggressive fluid resuscitation was not diluted. 135 The prevention of significant hypotension and hemoconcentration by increased experimental hydration, coupled with the adequate reperfusion of post-ischemic intestine, may have contributed to the beneficial effects we observed. Moreover, the demonstration of slight reductions in central venous pressure and the significant hemoconcentration in the conventionally resuscitated sham (SH15) group signifies the occurrence of profound insensible water losses. This is most likely due to fluid evaporation from the various cannulation and laparotomy incisions, even though every means possible was employed to prevent such events (i.e., covering all incisions with plastic wrap and saline-soaked gauze). This further stresses the importance. of aggressive fluid administration during the experiment to counteract such evaporative losses. The greater rate of maintenance fluid in the IR65 group prevented hemoconcentration from occurring (Figure 13) and attenuated the significant drop in mean arterial pressure following reperfusion as seen in the IR15 group (Figure 12, top). The large volume of fluid did not, however, produce fluid overload in these animals since central venous pressure (Figure 12, bottom) and systemic arterial hematocrit (Figure 13) remained essentially unchanged throughout the experiment. ,The conventionally resuscitated ischemia/reperfusion (IR15) group displayed a significant metabolic acidosis at the end of the experiment (Table 2). This fact was 136 indicated by a significantly lower arterial blood pH and bicarbonate levels versus its respective initial values and that of the SH15 group. As a result, significant respiratory compensation occurred (signified by a decreased arterial Pcoz); however, this was not sufficient to overcome the acidosis (Table 2). In contrast, animals that received aggressive fluid resuscitation displayed a significantly decreased metabolic acidosis (Table 2). Although respiratory compensation was evident in the IR65 group, this method of acid-base balance plus bicarbonate buffering was sufficient to maintain arterial blood pH at near-normal levels. This suggests that systemic hypoperfusion and tissue hypoxia were attenuated in the ischemia/reperfusion animals that received aggressive fluid resuscitation. As a result, attenuation of anaerobic metabolism at the tissue level may have lead to less lactic acid formation and thus, decreased metabolic acidosis. The attenuation of systemic hypoperfusion with aggressive fluid resuscitation was further supported by the maintenance of peripheral organ blood flow following reperfusion as measured by laser Doppler flowmetry (Tables 4 and 5). Laser Doppler flowmetry has been described as a continuous and non-invasive method of measuring tissue blood flow (Oberg, 1990). This methodology has been used successfully to measure tissue blood flow in the liver (Almond and Wheatley, 1992), skeletal muscle (Lombard and Roman, 1990), renal papilla (Roman and Smits, 1986), and the 137 small intestine of both animals (Johansson, 1988; Wolfman, 1989) and humans (Ahn et a1., 1986; Johansson, 1988; Johansson et a1., 1989; Perbeck et al., 1990). Data from the present study indicate that in the aggressively resuscitated ischemia/reperfusion (IR65) group, there was a full reestablishment of splanchnic and renal blood flow following reperfusion. This was in sharp contrast to the blood flow deficits demonstrated in the intestine and kidney of the IR15 group at the end of the experiment (Tables 4 and 5). This failure of the IR15 group to fully reperfuse the ischemic intestine demonstrates well the phenomenon of ”no reflow", also known as persistent tissue ischemia (Menger et a1., 1991). Poor survival time and the absence of 72 hour survivors in the IR15 group (Table 3) again indicates that systemic perfusion deficits can aggravate the already severe effects of post-ischemic bowel injury. Previous studies have demonstrated that intestinal blood flow during periods of hypovolemia in neonatal piglets was impaired due to the preferential shunting of blood to perfuse the brain and heart (Ramenofsky et a1., 1981). If this is true in rats, the sustained hypovolemic hypotension in the conventionally resuscitated IR15 group could have exacerbated the degree of gross bowel necrosis observed (Table 8) and accounted for the decreased tissue blood flow demonstrated in the post- reperfusion kidney and small intestine (Tables 4 and 5). 138 The observation (Figure 14) that animals in the IR65 group that survived the insult tended to gain weight at a slower rate than their respective sham (SH65) group raises the question as to a potential mechanism. One such possibility would be a decreased intestinal absorptive capacity inherent to ischemic mucosal damage (Fujimoto et a1., 1991b). Secondly, the release of some factor(s) into the circulation as a result of the intestinal damage may have yielded an animal was uninterested in eating. The exact mechanism for the decreased post—experimental weight gain, however, remains unknown. I Previous studies utilizing a model of adult feline intestinal ischemia and reperfusion have shown that post- ischemic bowel becomes extremely permeable to water due to significantly increased vascular permeability (Granger et a1., 1982; Parks and Granger, 1983). This results in' significant fluid filtration and sequestration in the intestine, leading to intestinal tissue edema (Dawidson et a1., 1979; Granger et a1., 1980). In the present study, the increase in tissue water content in all segments of bowel was consistent with this observation (Table 7). The relatively greater amount of intestinal tissue water content in animals with ischemia/reperfusion but with a high intravenous fluid resuscitation rate (i.e., IR65 group) further suggests that this fluid was derived from the intravascular compartment. 139 Although histologic injury was similar in both ischemia/reperfusion groups (IR15 and IR65), the highly resuscitated IR65 group had 23% less necrotic bowel upon gross observation (Table 8). Further evidence that systemic factors exacerbate intestinal injury in the conventionally resuscitated ischemia/reperfusion group (IR15) was the presence of a greater degree of histologic injury to the duodenum compared to its sham (SH15) group (Table 8). Since the duodenum is supplied by the celiac axis in addition to the superior mesenteric artery (Kornblith et a1., 1992; Marston and Pegington, 1989), midgut ischemia alone should not affect duodenal blood flow as much as jejunal and ileal blood flow. Yet, persistent hypoperfusion of the small intestine in the IR15 group existed even after reperfusion had occurred (Table 5). It appears that hypovolemia and hypotension could account for the observed reduction in intestinal blood flow, potentially leading to duodenal damage. Moreover, the persistent hypoperfusion of the intestine may also explain the greater overall amount of gross bowel necrosis in animals with less exogenous fluid resuscitation (Table 8). There appears to be minor polymorphonuclear granulocyte (neutrophil) margination in the lung following intestinal ischemia/reperfusion. This consisted of adherence to the vascular endothelium without migration into perivascular spaces or alveOli. In this model, polymorphonuclear leukocyte infiltration was not evident. This finding was 140 consistent with the observations of others in models of lung ischemia/reperfusion (Steimle et a1., 1992) and intestinal ischemia/reperfusion in immature (Crissinger and Tso, 1992; Hill, 1987; Strauss and Snyder, 1983) and mature (Leung et a1., 1992) animals. This is contrary, however, to what is reported in the literature using an adult model of intestinal ischemia/reperfusion (Karasawa et a1., 1991b; Kurtel et a1., 1991; Otamiri, 1989; Otamiri and Tagesson, 1989). Other than the minor neutrophil margination, there appears to be no histologic change produced in the lung following intestinal ischemia/reperfusion. Indeed, the lungs of the ischemic groups tended to be drier than their respective sham groups (Table 7). These results could be explained when one considers the hemoconcentration that occurs in the IR15 group. The increased oncotic pressure of the concentrated blood could cause water to flow down its osmotic gradient and out of the extravascular lung tissue. Another explanation for the decreased lung tissue water content in the ischemia/reperfusion groups may be a result of the respiratory compensation of the metabolic acidosis (Table 2). The rat uses the lungs and respiratory tract as a major means of thermoregulation. Since these animals have a stable body temperature during these carefully controlled experiments, and there was increased respiratory drive stimulated by the metabolic acidosis, the rats could have evaporated and expired more water from the extravascular 141 lung space than their respective sham groups. The greater oncotic pressure of the blood (due to hemoconcentration) would not allow water to equilibrate in the lung tissue, and a drier lung may result. These explanations, however, are speculative at best; the precise mechanism remains unknown. Although severe lung injury was not shown within the confines of this study, functional lung damage is not conclusively excluded. The severe lung injury reported in other studies (Caty et a1., 1990; Gerkin et a1., 1992; Hill et a1., 1991; Hill et al., 1992a; Koike et a1., 1992a; Koike et a1., 1992b; Schmeling et a1., 1989; Terada et a1., 1992), however, may be due, in part, to uncompensated cardiovascular instability following intestinal ischemia/reperfusion. Since one of four IR15 animals (25%) yielded a positive Clostridium peritoneal fluid culture, bacterial translocation and transmural necrosis may occur to a greater extent in the conventionally resuscitated IR15 group. Bacterial translocation across the bowel wall following intestinal ischemia/reperfusion has been well studied (Deitch, 1990; Patel et a1., 1992; Wells et a1., 1989); however, the data in this study are not sufficient to evaluate the role of translocation in this model. Intestinal ischemia/reperfusion produces a devastating cascade of intravascular events. The systemic release of various ”shock factors" such as lysosomal hydrolases (Abe et a1., 1972), cardiac depressant factors (Williams et a1., 142 1969), histamine (Kusche et a1., 1981), prostanoids (Lefer, 1985), leukotrienes (Feuerstein and Hallenbeck, 1987; Lefer, 1985), and platelet-activating factor (Feuerstein and Hallenbeck, 1987), as well as inflammatory cytokines (Bitterman et a1., 1991; Caty et a1., 1990), have all been described as playing a role in intestinal ischemia/reperfusion "shock". Although fluid administration, regardless of the rate, statistically affected some steady state sera measurements (Tables 9 and 11), most values still remained within normal Limits (Harkness and Wagner, 1983; Loeb and Quimby, 1989). Idoreover, all sham animals survived the protocol (Table 3), therefore, the physiological significance of these alterations appears to be minor. The exception to this was the increased serum glucose levels. This can most obviously be explained by the use of a 5% dextrose-containing lactated Ringer’s solution (Appendix B). Nevertheless, it appears that fluid administration did not seriously alter the baseline circulatory status of the rat. By the end of the experiment (90 minutes post- reperfusion), both ischemia/reperfusion groups (IR15 and IR65) displayed significant hyponatremia as compared to their respective sham animals (Table 10). This may be related to fluid retention in the extracellular space (Table 7) and/or secondary to hyperglycemia (Bakerman, 1984); _however, the values were still close to normal ranges. Hyponatremia has also been reported during pediatric 143 bacterial infections (Gonzalez et a1., 1964; Shann and Germer, 1985). Enhanced absorption of bacteria from the bowel has been reported in other models of intestinal ischemia/reperfusion (Schoenberg and Berger, 1990) and in models of canine colonic ischemia/reperfusion (Bennion et al., 1984a; Bennion et a1., 1984b). Unfortunately, cultures of portal blood were not performed within the confines of these studies. In a similar model of intestinal ischemia/reperfusion using 5-week-old rats, neither portal nor systemic bacteremia was apparent when blood cultures were obtained at 90 minutes post-reperfusion (O’Neill et a1., Unpublished Observations). Therefore, the role of bacteremia-induced hyponatremia in this model remains unknown. Significant hypocalcemia occurred in the IR65 group at the end of the experiment (Table 10). A similar finding was noted following reperfusion in a canine model of intestinal ischemia/reperfusion (Mozes et a1., 1989). More importantly, however, the IR15 group displayed significant hyperkalemia at the end of the experiment (Table 10). Hyperkalemia has been reported to be the cause of death in other models of intestinal ischemia/reperfusion (Mozes et a1., 1989; van der Meer et a1., 1986). Excessive serum potassium levels (12.5 mmol/L) in vitro produced changes in rat electrocardiograms, such as P-wave elongation, P-Q interval doubling, a split QRS complex, and a decreased T- wave (van der Meer et a1., 1986). The authors state that 144 such effects in vitro occur at in vivo plasma potassium concentrations of 10-12 mmol/L. In the present study, serum potassium concentrations in the IR15 group (11.2:O.4 mEq/L) fit that range (Table 10). The cause of death in the IR15 group, therefore, may be related to cardiac abnormalities secondary to serum hyperkalemia. The precise origin of the hyperkalemia remains obscure. Some of the possible sources of the potassium have been ruled out (van der Meer et a1., 1986). In that study (van der Meer et a1., 1986), mild metabolic acidosis was not sufficient to elicit significant hyperkalemia. There was some evidence that hypotension (60 mmHg for 12 hours) induced a slight elevation in plasma potassium. Moreover, reduced renal function in nephrectomized rats also produced a slight elevation in plasma potassium levels (van der Meer et a1., 1986). Since both hypotension (Figure 12, top) and reduced renal blood flow (Table 4) occur in the IR15 group at the end of the experiment, this may be sufficient to produce the elevated serum potassium levels demonstrated in this study (Table 4). Aggressive fluid resuscitation in the IR65 group, however, prevented the hyperkalemia from occurring. This may have contributed to the improved survival observed in the IR65 group (Table 3). Possible explanations include the attenuation of hypotension (Figure 12, top) and the restoration of renal blood flow at the end of the experiment (Table 10). 145 Hyperphosphatemia was present in the IR15 group (Table 10). This has been previously reported in patients with extensive bowel necrosis (May and Berenson, 1983). Aggressive fluid resuscitation again prevented hyperphosphatemia from occurring in the IR65 group. This may be explained by the fact that the IR65 group had 23% less grossly necrotic bowel than the IR15 group at the end of the experiment (Table 8). Profound hypotension, hypovolemia, and the concomitant decrease in collateral perfusion (Ramenofsky et a1., 1981) of the intestine from the unmanipulated celiac axis and inferior mesenteric arteries could be producing a more extensive bowel injury. On the other hand, hyperphosphatemia may also indicate acute renal insufficiency (Bakerman, 1984). The hyperglycemia displayed in all groups can be at least partially due to the glucose load that was given as 5% dextrose in lactated Ringer's solution (Appendix B). Placed in perspective, these samples were obtained following 4 hours of fluid resuscitation with a 5% dextrose-containing solution. More than likely, serum glucose levels would be substantially lower if these measurements were repeated at some time after the conclusion of the experiment. The serum hyperglycemia was not, however, necessarily a detrimental effect. Previous studies have demonstrated that glucose administration improved survival following intestinal ischemia/reperfusion produced by portal vein occlusion (van der Meer et a1., 1981). Interestingly, 146 despite the equality of exogenous glucose administration within fluid resuscitation groups in the present study, both ischemia/reperfusion groups displayed significantly greater serum glucose concentrations than their respective sham groups by the end of the experiment. This suggests a coexistent endogenous release of glucose, possibly due to an elevation in catecholamine levels following this type of insult (van der Meer et a1., 1981). The exact mechanism for this ischemia/reperfusion-induced glucose elevation, however, remains unknown. The metabolic derangements at the end of the experiment (Table 12) in the IR15 group versus its respective sham (SH15) suggests liver damage, renal compromise, and impending circulatory collapse. Elevations in alkaline phosphatase, serum glutamic oxalacetic transaminase, serum glutamic pyruvate transaminase, lactic dehydrogenase, and bilirubin may imply liver damage has occurred as a result of the intestinal ischemia/reperfusion as well as hypovolemic shock. Such observations are consistent with that of others (Turnage et a1., 1991). Moreover, elevations in creatinine and blood urea nitrogen (Table 12) suggest acute renal insufficiency (Bakerman, 1984) and are consistent with the. hyperkalemia/hyperphosphatemia data (Table 10) and the _reduced renal blood flow (Table 4). Furthermore, increased levels of alkaline phosphatase and creatine phosphokinase indicate lysosomal enzyme release (Aktan et a1., 1990) and are potential indicators of impending circulatory shock (Abe 147 et a1., 1972). Most of these same parameters (Table 12) were significantly elevated in the SH15 group versus the SH65 group, suggesting a systemic (non-splanchnic) mechanism possibly related to relative fluid deprivation. Aggressive fluid therapy attenuated the increases in alkaline phosphatase, creatinine, and bilirubin displayed in the IR15 group. Most indicators of acute hepatic injury (serum glutamic pyruvate transaminase, serum glutamic oxalacetic transaminase, lactic dehydrogenase) were still elevated in the IR65 group, suggesting liver damage still occurred secondarily to bowel ischemia. Aggressive fluid resuscitation, however, moderated these increases compared to the IR15 group (Table 12). Aggressive fluid resuscitation in the IR65 group also prevented the dramatic increases in serum levels of blood urea nitrogen, creatinine, and potassium evident in the IR15 group at the end of the experiment (Table 12). In a similar model of intestinal ischemia/reperfusion, reduced renal function was demonstrated by decreased urine production and potassium excretion (van der Meer et a1., 1986). Our indicators of reduced kidney function (increased serum levels of blood urea nitrogen, creatinine, and potassium) in the conventionally resuscitated animals following intestinal ischemia/reperfusion are consistent with their findings (van der Meer et a1., 1986). Moreover, renal blood flow was reduced at the end of the experiment (Table 4), further 148 suggesting acute renal insufficiency in the IR15 group. It appears that aggressive fluid resuscitation attenuated the acute renal damage (reflected by decreased levels of serum blood urea nitrogen, creatinine, and potassium). These results suggest that gut-related factors may be less important than the degree of resuscitation in the manifestation of renal compromise following intestinal- ischemia/reperfusion. Portal serum levels of lipopolysaccharide were not significantly elevated in any group at any time (Figure 15). Although some studies have identified elevated portal levels of lipopolysaccharide in models of intestinal ischemia/reperfusion (Caty et a1., 1989; Caty et a1., 1990; Cuevas and Fine, 1971), others have not (Squadrito et a1., 1992). In a model similar to ours (Caty et a1., 1990), the peak lipopolysaccharide levels were reported toward the end of the ischemic period and during early (less than 30 minutes) reperfusion. Disappearance of portal lipopolysaccharide levels was complete by 90 minutes post- reperfusion (Caty et a1., 1990). In the present study, lipopolysaccharide was not significantly elevated in any group at 90 minutes post-reperfusion; however, this does not rule out a significant endotoxemia during the earlier stages of reperfusion. Tumor necrosis factor was not detected by bioassay in the serum of any group at any measured time. Using a similar model, previous studies demonstrated transient 149 elevations in serum tumor necrosis factor levels that peaked at 30 minutes post-reperfusion and disappeared 30 minutes later (Caty et a1., 1990). In the present study, our 90 minute post—reperfusion sample may have missed a transient tumor necrosis factor elevation. In contrast, serum interleukin-6 was markedly elevated in both ischemia/reperfusion groups (IR15 and IR65) versus their respective sham groups (SH15 and SH65) at the end of the experiment (Figure 16). Aggressive fluid resuscitation again attenuated the systemic release of interleukin-6 in the IR65 group. Reduced serum interleukin-6 levels may have been related to the prevention of secondary cardiovascular instability (hypotension and hemoconcentration) following reperfusion. Since high serum levels of interleukin-6 have been associated with fatal outcome in man during septic shock (Hack et a1., 1989; Waage et a1., 1989a), the greater levels of interleukin-6 in the conventionally resuscitated animals may have been related to the 100% mortality in the IR15 group (Table 3). Conversely, the aggressively resuscitated IR65 group displayed a significantly greater survival of 27% (Table 3) with significantly lower serum levels of interleukin-6 (Figure 16). A cause-effect relationship between serum interleukin-6 levels and 'mortality in this model, however, remains unknown. Although the beneficial effects of aggressive fluid resuscitation may be due to the expansion of vascular volume and the dilution of the toxic waste products and cellular 150 enzymes, this appears not to be entirely the case. An approximate 39% decrease in plasma volume was reflected by the changes in hematocrit (Van Beaumont, 1972). This cannot explain the electrolyte imbalances, nor can it fully explain the greater than two-fold increases in creatine phosphokinase, lactic dehydrogenase, bilirubin, blood urea nitrogen, and interleukin-6, in the IR15 group as compared to the IR65 group. Nonetheless, the significantly increased levels of. toxic waste products or the increased immunological alterations in the conventionally resuscitated animals, regardless of the etiology, may indicate the development of circulatory shock following intestinal ischemia and reperfusion. These additional variables, due to shock, may not allow a truly selective study of the secondary consequences of intestinal ischemia/reperfusion per se. Despite the fact that aggressive fluid resuscitation had only a modest effect on long-term survival, the fact remains that such a regimen did significantly increase the survival time of the non-surviving animals. The increased survival time of approximately 8 hours (Table 3) nonetheless provides an opportunity to administer pharmacologic agents in order to further enhance the survival rates. An interesting caveat to this study is the question of whether continued infusion of crystalloid solutions alone over the 72 hour observation period could further improve survival, provided this regimen maintains critical 151 cardiovascular parameters throughout the study period. Unfortunately, we did not assess this due to the technical difficulty of chronic instrumentation and fluid management in rodents of this size. Nevertheless, use of this model sets the stage for future studies to examine possible pharmacologic therapies to further improve outcome following intestinal ischemia/reperfusion in immature rats. SUMMARY AND CONCLUSIONS In the present study, significant hypotension and hemoconcentration occurred in the IR15 group as compared to its sham group (SH15) and the aggressively resuscitated ischemia/reperfusion (IR65) group after reperfusion and at the end of the experiment. This was associated with lower overall group survival and survival time, as well as a greater proportion of grossly necrotic intestine. In addition, the conventionally resuscitated IR15 group displayed a significantly greater metabolic acidosis and respiratory compensation. Moreover, there appears to be persistent tissue hypoperfusion as indicated by reduced blood flow in the kidney, duodenum, and ileum following reperfusion. Even the conventionally resuscitated sham (SH15) group was hemoconcentrated when compared to the high resuscitation sham group (SH65); this demonstrates significant insensible fluid loss and volume contraction independent of third space loss into damaged tissue. Aggressive fluid resuscitation following bowel ischemia and reperfusion is a necessity; hyperkalemia, hyperphosphatemia, acute renal insufficiency, and the enhanced release of the inflammatory cytokine IL-6 in the conventionally resuscitated animals may act in concert to 152 153 produce systemic cardiovascular instability and increase mortality. By providing adequate hemodynamic stability through aggressive fluid resuscitation throughout the study period, many of these metabolic derangements were attenuated. In conclusion, maintenance of cardiovascular stability by aggressive fluid therapy avoids the confounding variable of severe hypovolemia, resulting in hypotension and hemoconcentration. First, the significant cardiovascular instability profoundly alters the physiologic response to bowel ischemia and adversely affects both the amount of gross bowel injury and survival. Second, due to the excessive increases in post-ischemic bowel permeability, massive amounts of fluid are required to provide a hemodynamically stable model. The aggressive fluid administration we advocate, however, does not adversely affect peripheral organ tissue water content or induce volume overload. Thus, data on distant organ effects of such ischemic bowel lesions should be viewed with caution if the model does not demonstrate adequate hemodynamic stability. In closing, we strongly advocate the use of this model of intestinal ischemia/reperfusion in immature rats utilizing aggressive fluid resuscitation. This model allows for identifying the systemic effects of intestinal ischemia and reperfusion per se without the additional influences of cardiovascular instability and secondary hemodynamic shock. APPENDICES APPENDIX A: Diet Composition Rodent Laboratory Chow® (Purina # 5001) Tgtnl Diggntlnlg Nutrients 76.00 3 - 0 tr t 9.00 §I2££_331£§! .11Z§_£Q§1L§m Phygiglggic :nel vnlue 30 c 21112.3 £91 ..1152.& Arginine 1.38 Cholesterol 270.0 ppm Cystine 0.32 Glycine 1.20 112;; (cnnde) Histidine 0.55 Neutral Detergent Fiber 16.00 Isoleucine 1.18 Acid Detergent Fiber 8.20 Leucine 1.70 Lysine 1.42 Ann Z,§Q 3 Methionine 0.43 Phenylalanine 1.03 V tamin _ppn_ Tyrosine 0.68 Carotene 4.5 Threonine 0.91 Biotin 0.1 Tryptophan 0.29 Menadione --- Valine 1.21 Thiamine 15.0 Riboflavin 8.0 t Niacin 95.0 Calcium 1.00 Pantothenic Acid 24.0 Phosphorus 0.61 Choline (x 100) 22.5 Potassium 1.10 Folic Acid 5.9 Magnesium 0.21 Pyridoxine 6.0 Sodium 0.40 Chlorine 0.56 nchkg Vitamin B12 22.0 .221. Fluorine 35.0 _lQLgn Iron 198.0 Vitamin A 15.0 Zinc 70.0 Vitamin D 4.5 Manganese 64.3 Copper 18.0 _lngg Cobalt 0.6 Vitamin E 40.0 Iodine 0.7 Chromium 1.8 .Jnngn Selenium 0.2 Ascorbic Acid --- 154 APPENDIX B: 5% Dextrose Lactated Ringer’s Solution Composition Calculated Osmolality = 525 mOsm/L Approximate pH = 4.9 9282911119 29.2.1112: Dextrose (anhydrous) 50,000 mg Sodium Lactate (anhydrous) 310 mg Sodium Chloride (dihydrate) 600 mg Potassium Chloride 30 mg Calcium Chloride (dihydrate) 20 mg We Sodium 130 mEq/L Potassium 4 mEq/L Calcium ' 3 mEq/L Chloride 109 mEq/L Lactate 28 mEq/L 155 APPENDIX C: Intestinal Histologic Grading Score m QESCRIPIION 0 Normal villi 1 Apical villus edema 2 Mild fusion of the villi 3 Prominent fusion of the villi 4 Apical villus slough (ulceration); involving less than one third of the villous height 5 Apical villus slough; involving one third to two thirds of the villus height; basal crypts intact 6 Apical villus slough; involving greater than two thirds of the villus height 7 Focal necrosis of muscularis propria 8 Transmural necrosis 156 APPENDIX D: ‘ 5' _ESLL Arginine 200.0 Asparagine 50.0 Aspartic acid 20.0 Cystine 50.0 Glutamic acid 20.0 Glutamine 300.0 Glycine 10.0 Histidine 15.0 Hydroxyproline 20.0 Isoleucine 50.0 Leucine 50.0 Lysine HCl 40.0 Methionine 15.0 Phenylalanine 15.0 Proline 20.0 Serine 30.0 Threonine 20.0 Tryptophan 5.0 Tyrosine 20.0 Valine 20.0 WM Glucose 2000.0 Phenol red 5.0 Vltamlns Biotin Vitamin B12 Calcium pantothenate Choline chloride Folic acid i-Inositol Nicotinamide p-Aminobenzoic acid Pyridoxine HCl Riboflavin Thiamine HCl lnorganic salts NaCl KCl NaZHPO, - 7 H20 Mgso4 - 7 H20 Ncho3 Ca(NO3)2 - 4 H20 Glutathione 157 RPMI 1640 (Cell Culture Media) Composition .2911 0.2 0.005 3.0 1.0 35.0 .9911: 6000.0 400.0 1512.0 100.0 2000.0 100.0 APPENDIX E: Solution Composition Hank's Buffered Saline Solution W111 SE11: Calcium Chloride (CaClfi 0.14 Potassium Chloride (KCl) 0.40 Potassium Phosphate (KH2P04) 0.06 Magnesium Chloride (MgC12~ 6 H5» 0.10. Magnesium Sulfate (MgSO4"7IfiO) 0.10 Sodium Chloride (NaCl) 8.00 Sodium Bicarbonate (NaHCOQ 0.35 Sodium Phosphate (dibasic; NaZHPO4 ' 7 H20) 0.09 gtng; Qomponentg D-glucose 1.00 Phenol red ' 0.01 Sodium Dodecyl Sulfate (10%) in Phosphate Buffered Saline 1n2121812_93118 2111 Potassium Chloride (KCl) 0.20 Potassium Phosphate (KH2P04) 0.20 Sodium Chloride (NaCl) - 8.00 Sodium Phosphate, dibasic (NaZHPO4 ' 7 H20) 2.16 Sodium Dodecyl Sulfate (C(2H25048Na) 100.00. 158 10. LITERATURE CITED Qgtlgnd’s Illustrated Medical Dictionany. W.B. Saunders Co., Philadelphia, 1965. e a e nd se 0 ab t 'm . 0.8. Government Printing Office, Washington, D.C., 1985. ' e e . Medical Economics Company Inc., Oradell, N.J., 1990. a 's e ' . Williams & Wilkins, Baltimore, 1990. Abe, H., J. Carballo, H.E. Appert, and J.M. Howard. The release and fate of the intestinal lysosomal enzymes after acute ischemic injury of the intestine. §BI91_§¥n§§Ql1_QQ§£§£1 135: 531-535: 1972- Aderka, D., J. Le, and J. Vilcek. 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