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JOURNAL OF SURGICAL RESEARCH 50,170-174 991)

Outcome Following Femur Fracture and S~bsequent Cecal Ligation-and Puncture in Endotoxin-Sensitive (C3H/HeN)

and Endotoxin-Resistant (C3H/HeJ) Mice 1

Dep¡;

CHRISTOPHER C. BAKER, M.D.,. TRAcy NlVEN-FAIRCHILD, B.S.,.CHERYL CARAGNANO, B.S.,. AND THOMAS S. KUPPER, M.D.,.,t

*Surgery and tDermatalagy, Yate University Schoal al Medicine, New Haven, Cannecticut 06510

Submitted for publication January 2, 1990

This study examined the effect of gengjg folIowin2~~in a reproducible model of sepsis-cecalliga-tlon and puncture (CLP)-in endotoxin-sensitive(C3H/HeN) and endotoxin-resistant (CeH/HeJ) mice.Studies used CLP with a 25-gauge needle at differenttime intervals following injury, as induced by femurfracture (FF), to determine the effects of sublethal sep-gis on survival alter trauma. There was a 3% mortalityfor FF alone in both groups. Mortality in C3H/HeJ micewas not significantly increased over FF alone exceptwhen CLP followed FF by 3 days (45%, P < 0.02, Chi-square). In contrast, C3H/HeN mice had significantlyincreased mortality rates (75 to 90%, P < 0.001) versusFF alone at all intervals between FF and CLP. Mortal-ity for FF plus CLP was significantly greater for C3H/HeN compared to C3H/HeJ (P < 0.001) for all time in-tervals between FF and CLP. In conclusion, animalsexposed to a septic episode following FF had signifi-cantly greater mortality than FF animals without aseptic challenge. Endotoxin-sensitive mice had signifi-cantly higher mortality alter CLP and significantly in-creased mortality when CLP followed FF (regardless oftiming) compared to endótoxin-resistant mice. @ 1991Academic Presa, Inc.

several years in the role of macrophage dysfunction inimmunosuppression following trauma. Since the macro-phage is activated by endotoxin [4], it is possible thatabnormal responses to endotoxin may lead to early mac-rophage dysfunction after trauma.

This study was undertaken to evaluate t,he interactionoftrauma and sepsis. Initial studies used a valid model ofintra-abdominal sepsis (CLP) in endotoxin-sensitive(C3HfHeN) and resistant mice (C3HfHeJ) and devel-oped a model system for studying the interaction oftrauma and sepsis. These mice were chosen because ofthe differential response of their macrophage to endo-toxin [8], in anticipation of subsequent studies on themechanisms of immunologic dysfunction after trauma.The cecalligation and puncture model in mice [5] waschosen for the sepsis model for the reasons previouslyoutlined by Chaudry [6]. Open isolated femur fracturewas used as the model for trauma a~ reported by Stone[7]. Using this model system ofFF followed by CLP, theeffects of sepsis at various times after injury on survivalwere investigated. The experimental protocol is outlinedin Fig. 1.

MA TERIALS AND METHODS

INTRODUCTIONAnimals

C3HjHeJ mice were obtained from J ackson Laborato-ries (Bar Harbor, ME) and C3HjHeN mice were pur-chased from NIH (Bethesda, MD). AII mice used werepathogen-free 4- to 6-week-old males (25-30 g) housedin laminar flow cabinets. Animals were cared for in ac-cordance with NIH and Yale Department of AnimalCare Protocols.

Systemic sepsis continues to plague critically ill andinjured surgical patients, carrying a mortality of 50 to60% [1]. Although deaths from surgical sepsis have beenthought to be due to the failure of host defense mecha-nisms, positive blood cultures have only been found in athird of trauma patients with sepsis [2]. Recently thedemonstration of translocation of bacteria and endo-toxin across the gut has been advanced as a solution tothis paradox [3]. Our laboratory hasbeen interested for

Surgical ProceduresThe cecalligation and puncture (CLP) procedure was

adapted to micefrom the model described in rats byChaudry [6]. Animals were anesthetized intramuscu-larlywith a combination of xylazine (16 mg/kg) and ke-

1 Supported by NIH Grant GM35909 and AI25082. (No reprints

available.)

1700022.4804/91 $1.50Copyright @ 1991 by Academic Press, Inc.All rights of reproduction in any form reserved.

BAKER ET AL.: SEPSIS, ENDOTOXIN, AND TRAUMA171

Femur Fracture (FF) Alone > 96 hoursMortality Assessed

Cecal Ligation and Puncture (CLP) -> 96 hoursMortality Assessed

2 DaysJ,

4 DaysJ,

6 DaysJ.-FF

FIG. l. The experimental protocol is outlined here, showing the intervals between FF and CLP.

tamine (80 mg/kg). After shaving the anterior abdomi-nal wall, a 15-mm midline incision was made and thececum exposed and ligated at its base with 2-0 silk. Thececum was punctured with the appropriate gauge needle,and approximately 0.1 mI of reces were squeezed out inorder to be certain that the puncture holes were open.The abdominal incision was closed with 4-0 Nylon, andthe mice rehydrated with 1 mI of normal saline subcuta-neously.

Our murine model of femur fracture (FF) was adaptedfrom Stone et al. [7). Mice were anesthetized and shavedin the left thigh region. A 10-mm incision was made lat-eral to the femur, which was dissected from thesurrounding muscle. The femur was then sharply cut atits midpoint with scissors. The incision was closed with4-0 Nylon, and the mice were resuscitated as above.

Data Analysis

AII animals were assessed for survival twice daily forup to 10 days after the experimental procedure; in-creases in mortality, however, were not seen after 96 hrin mice undergoing CLP. When mice were subjected toCLP after FF, mortality was determined twice daily for 7days and did not increase thereafter. Difference~_9.~,:- -tween groups were assessed using Chi-square analysiswith the Yates cóntinuity correction [9].

I'1

IRESULTS ever, the results were different. Mortality for

CLP for C3H/HeN was significantly

I CLP (P < 0.001). Mortality for FF plusHeN was algo increased (P < 0.001)alone in C3HjHeN (3%).

The Effect 01 Endotoxin Sensitivity on Outcomealter CLP

The effect of endotoxin sensitivity on mo[tality at 96hr after CLP for the two groups of mice is depicted inTable 1. In several hundred previous experiments, wehave rarely seen deaths occurring at times greater than96 hr following CLP, which was algo the case in thisstudy. As can be seen, CLP was lethal for C3H/HeN forall needle sizes except 25 gauge (45% mortality). The

alone (3%) and CLP alone (47%)

of 1, 3, 5, and 7 days.

~~~~~~~

/ Hortality Assessedt 1 t t 96 hours after CLPL~ 3 Days ~ 5 DayS__~~:::~

Intervals at which CLP performed

mortality for C3H/HeJ, however, was greatest for the18-gauge needle (50%) and dropped to zero for the 25-gauge needle. Mortality for CLP in the endotoxin-sensi-tive C3H/HeN mice was significantly increased (P< 0.001, x2, Yates correction) for all needle sizes com-pared to the endotoxin-resistant C3H/HeJ mice. In ad-dition, mortality for CLP in the C3H/HeJ mice tendedto parallel needle size.

Studies on the Combined Elfect o{ FF and CLP

Studies on FF alone as a model of injury demonstrateda low mortality of 3% (N = 30 animals/group) in bothstrains of mice. In the experimental studies, FF was fol-lowed by sublethal sepsis (CLP with a 25-gauge needle)in arder to eyaluate the effect of sepsis following traumain the two strains of mice. It should be recalled thatmortality following CLP alone with a 25-gauge needlewas 45% for C3H/HeN and 0% for C3H/HeJ.

The data for this set of experiments are summarizedin Table 2 andFig. 2. In general, the C3H/HeJ mice weremore resistant to the combined ~ffects of FF and CLPthan the C3H/HeN mice-mortality in C3H/HeJ micewas increased ayer FF alone (expected 3%) and CLPalone (expected 0%) when CLP followed FF by 1 dayand,3d~Y8- (35% and 45%, P < 0.02). There were no signifi-cant increases ayer expected mortality in C3H/HeJmice, however, when the interval between FF and CLP..

172 JOURNAL OF SURGICAL RESEARCH: VOL. 50, NO. 2, FEBRUARY 1991

TABLElPercent Mortality at 96 br alter CLP

Needle size (gauge)

Strain (N = 30/group) 18 19 20 21 23 25

50%blOO%a.b

40%blOO%a.b

30%lOO%o,b

20%90%a,b

16.6%90%o.b

0%46.6%4

C3H/HeJ (resistant)C3H/HeN (sensitive)

.Significantly greater than mortality for C3H/HeJ (P < 0.001).b Within strains, mortality greater than mortality for 25-gauge needle (P < 0.001).

DISCUSSION

As surgical and anesthetic management has improvedíor patients with multiple injuries and those undergoingmajor elective surgery, the clinical problems preventingsurvival in this setting have become systemic sepsis andmultiple organ íailure (MOF), which cause 75% oí thelate deaths aíter major trauma [10]. Uníortunately,many oí the human studies have evaluated heteroge-neous groups oípatients, and most previous animal mod-els oí sepsis-e.g., injection oí livebacteria [11] or endo-toxin [12]-were inadequate analogs oí human sepsis.The animal model oí CLP used in these studies is a goodanalog oí surgical sepsis, as pointed out by Chaudry et al.[6], and closely parallels the cardiovascular patterns oísepsis in man described by MacLean et al. [13]. Asshown in Table 1 oí this study, mortality in C3HjHeJmice closely parallels needle size, suggesting that thismodel represents a graded íorm oí sepsis in these ani-mals. In addition, it appears that CLP with a 25-gaugeneedle can be used to study the effect oí sublethal sepsisíollowing an immunosuppressive challenge (e.g.,trauma, thermal injury), paralleling the clinical eventsoften seen in patients. This principIe was actually uti-lized in a previous study by"Kupper, in which the sub-

.: Íethal model oí CLP with' á 25-gauge needle was con-verted to a lethal model in animals that were immuno-

.~

~

TABLE2

Percent Mortality When FF Is Followed by CLP

~

Strainof mice

C3H/HeJ (resistant)(N)C3H/Hen (sensitive) 68.4%6 75%0.6 750.6 68.2%0.6(N) (19) (20) (20)

~~~~~

suppressed by injection with T cells from burned mice 20hr prior to CLP [14]. In the current study, we evaluatedthe immunosuppression of trauma using FF as a traumamodel, followed by CLP.

The results of the current study have demonstratedseveral findings. While endotoxin sensitivity does notseem to affect mortality after FF, clearly late mortalityafter CLP is significantly increased in C3H/HeN mice.In the experiments on the combined effect of FF andCLP, the timing of CLP appeared to playa role. espe-cially in the C3H/HeJ mice, which only had increasedmortality compared to FF alone when CLP followed FFby 1 or 3 days but not at longer intervals. These datasuggest that there may be a window of time early afterFF during which the endotoxin-resistant animals aremore susceptible to a septic challenge. Conversely, theC3H/HeJ mice had a significantly decreased mortalitycompared to C3H/HeN (P < 0.001) when CLP followedFF at all intervals tested except 1 day. This parallels thedifferences between C3H/HeJ and C3H/HeN seen forCLP alone (Table 1), but the increased susceptibility ofthe endotoxin-sensitive mice to seps,is is more strikingfor FF plus CLP (Table 2). It should be pointed out thata positive control or sham was not performed in thisstudy, namely FF followed by anesthesia and laparot-omy alone. Part of the reason for this was a desire on thepart of our Animal Care Protocol Committee to limit thenumber of animals in control groups. In previous experi-

BAKER ET AL.: SEPSIS, ENDOTOXIN, AND TRAUMA 173

o C3H/HeJ

8C3H/HeN .t

>-f-:J«f-a:o~~o

DA Y 1 DAY 3 DA Y 5 DA Y 7

INTERVAL BETWEEN FF ANO CLP

FIG. 2. This bar graph depicts mortality 7 days after CLP whenCLP followed FF by 1, 3, 5, or 7 days. The open bars represent resultsfor C3H/HeJ mice and closed bars are for C3H/HeN mice. A cross (+)over the bar represents increased mortality compared to FF alone (P< 0.0001), and an asterisk (*) signifies increased mortality of C3H/HeN versus C3H/HeJ (P < 0.0001) for the same time intervalo Thenumber of animals in each group is shown in Table 2.

sion that activates the alternate complement pathway,producing a "septic" response with MOF and a 35% mor-tality [16]. AIso Schirmer and Fry have studied the sys-temic and hemodynamic effects of FF and soft-tissueinjury in Sprague-Dawley rats, suggesting that soft-tis-sue injury may be the pathophysiologic stimulus leadingto MOF [17].

A better understanding of the basic mechanisms ofimmune dysfunction in the early period postinjury willberequired before targeted therapy (e.g., monoclonal an-tibodies, immunomodulators) can be translated intoclinical benefits for trauma patients. Future studies willbe directed at evaluating whether the immunosuppres-sion oftrauma is mediated directly by endotoxin or by itsliberation of other inflammatory mediators (e.g., tumornecrosis factor, gamma interferon, prostaglandins, in-terleukins, and leukotrienes).

Clearly, a number of future studies must be performedin order to achieve a better understanding of the basicpathophysiology of sepsis following trauma. Nonethe-less, two things can be concluded from this study. First,endotoxin-resistant mice have a better survival follow-ing a septic challenge (CLP), compared to endotoxin-sensitive mice, which is accentuated when sepsis followstrauma (FF). Second, the interaction of trauma and sep-Bis can be appropriately studied by combining the mod-els of femur fracture and cecalligation and puncture.

ACKNOWLEDGMENTS

The authors thank Dr. Anthony Meyer for editorial assistance andMs. Sandra Oronzo and Ms. Linda Deese for preparation ofthe manu-script.

REFERENCES

1. Baker, C. C., Degutis, L. C., DeSantis, J. G., and Baue, A. E. Theimpact of a trauma service in a u~ersity hospital. Amer. J.Surg. 114: 453, 1985.

2. Goris, R. J. A., Boekhorst, P. A., Noytinck, K. S., and Gimbrere,J. S. F. Multiple organ failure: Generalized autodestructive in-

::::-7~~~mation? Arch. Surg. 120: 1109, 1985.3: -Dei.tch, E. A., Berg, R., and Specion, R. Endotoxin promotes the

trans!ocation of bacteria from the gut. Arch. Surg. 122: 185,1987.

4.Adere~, -'\. A., Caben, ~. S., \'(right, S. D.,and Cohn,~.Z.Bactenal lIpopolysacchandes prime macrophages for enhancedrelease of arachidonic acid metabolites. J. Exp. Med. 164: 165,1986. c

...'5. Baker, C. C., Chaudry, l. H., Garnes, H. O., and Baue. A.E.Evaluationmurine cecal ligati~n and puncture modelo1983.

6. Wichterman, K. A., Baue, A. E., andChaudry, l. H.septic shock-A review of laboratory models and a proposal. J.Surg. Res. 29: 189, 1980. -' -

C ' I7. Stone, J. M., Kudsk, K. A.,Carpenter, G., and Sheldon:G.'F.,

I

ments [15], an~sthesia alone and laparotomy alone weregenerally associated with 0% mortality. We felt there-fore that it was acceptable to utilize the comparison ofFF /CLP to FF alone and CLP alone.

It should be reiterated that the only major differencebetween these two strains of mice is that the macro-phage of C3H/HeJ mice do not respond to endotoxin.Basedonpreviouswork [14] and thisstudy, thecombina-tion of open femur fracture followed by CLP appears tobe an appropriate model for further study ofthe interac-tion of the immunosuppression of trauma and the subse-quent development of sepsis.

It is not possible to qopclude from these studies thatendotoxin is the causal factor that leads to the increasedmortality in the C3H/HeN animals beca use endotoxinlevels were not measured. Experiments to evaluate en-dotoxin levels, antigen recognition, macrophage- T cellinteractions, and macrophage production of infla:tnma~-tory mediators such as interleukin -1 and tumor necrosisfactor are planned for the future. Nonetheless it appearslikely that early steps of macrophage activation mayplaya role here given the apparent immunosuppressionseen in the C3H/HeJ mice from 1 to 3 days after FF. If itis possible to understand why endotoxin resistanceconfers protection from sepsis (e.g., CLP), then it maybe possible to abrogate the mortality from sepsis afterinjury. .

Two other relevant models have recently beetÍ de-scribed in this area. Goris et al. in the Netherlands havereportedon a model ofMOF inWistar rats usingintra~peritoneal implantation of azymosan-paraffin suspen-

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