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Obstetrics & Gynecology 2000;95:453-456
© 2000 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Preterm Delivery in Mice With Renal Abscess

GEORGE M. MUSSALLI, MD, STEVEN R. BRUNNERT, DVM and EMMET HIRSCH, MD

From the Department of Obstetrics and Gynecology, and the Institute of Comparative Medicine, Columbia University College of Physicians and Surgeons, New York, New York.

Address reprint requests to: Emmet Hirsch, MD Columbia-Presbyterian Medical Center Department of Obstetrics and Gynecology P&S Building, 16-417, 630 W. 168th St. New York, NY 10032 E-mail: eh25{at}columbia.edu


   Abstract
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Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: Our purpose was to develop a mouse model of renal abscess to study the effect of extrauterine infection on preterm delivery.

Methods: Escherichia coli or sterile medium was injected into the left kidney of 70 pregnant mice that had completed approximately 75% of gestation. Preterm delivery rates were recorded for various inocula. Kidney specimens were obtained and examined grossly and histologically for abscess formation.

Results: Thirty-one of 51 animals (60.8%) infected with 1 x 105-9 x 106 bacteria and none of 19 uninfected animals delivered prematurely (P < .001). Renal abscess was induced in 100% of mice receiving bacterial inoculation but in none receiving sterile medium.

Conclusion: Kidney injection provides a reliable method for inducing renal abscess in pregnant mice. Renal abscess induces preterm delivery at a stable rate across a wide range of bacterial inocula. This model of extrauterine infection may be particularly useful in investigations of infection-induced preterm delivery.

Preterm birth is the leading cause of perinatal mortality and neonatal morbidity in North America and Europe.1 Intrauterine2,3 and extrauterine4–6 infections have long been known to be associated with preterm delivery, however there has been no reduction in the incidence of preterm birth in the United States during the last half century.7,8 In view of the complex biochemistry of infection-induced preterm birth, as well as the difficulties in performing controlled studies in human subjects, the development of reliable and reproducible animal models of preterm labor is essential to increase our understanding of this condition. Murine models of preterm birth, in particular, offer several advantages. Mice are inexpensive, are easy to maintain, and have short gestations. Additionally, murine immunology has been well characterized, and experimental manipulation of murine gene expression is possible. There are several obvious differences between mice and humans in normal pregnancy and parturition (eg, number of gestations, dependence on progesterone withdrawal, bacterial flora). However, the biochemistry of infection-induced labor in these organisms may be similar, as reflected in responsiveness to, and production of, inflammatory cytokines and prostaglandins.9–14

Several animal models of preterm delivery following intrauterine infection or systemic inflammation have been reported.15–21 However, a MEDLINE search for the period 1966–1998 (keywords: animal, model, preterm, pregnancy, infection, inflammation, systemic, extrauterine) failed to identify models of preterm delivery following infection confined to a specific extrauterine compartment.

The purpose of this study was to develop a mouse model of localized renal abscess to study the effect of extrauterine infection on preterm delivery.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
All animal procedures were approved by the Institutional Animal Care and Use Committee of the Columbia University College of Physicians and Surgeons. Seventy virginal female CD-1 mice (Charles River Laboratories, Wilmington, MA), 6–8 weeks old, were housed in a modified barrier facility with free access to food and water. Ambient temperature was kept at approximately 22C and a light-dark cycle of 12 hours each was maintained. Females in estrus, as determined by the appearance of the vaginal epithelium,22 were placed with individually housed CD-1 males. Mating was confirmed by the presence of a vaginal plug. Surgery was performed at 14.5 days gestation (about 75% of a normal 19 to 20–day gestational period). Mice were assigned to bacterial or control injections according to a predetermined schedule by picking the first available tail from groups in cages, without any attempt to discriminate between subjects. At each session, at least four mice received bacterial injections, and at least one mouse received sterile media, serving as a control.

Escherichia coli bacteria (American Type Culture Collection No. 12014), received freeze-dried, were suspended in liquid medium, cultured overnight at 37C, and frozen in aliquots. All liquid culturing and dilution of bacteria were performed in Luria-Bertani medium.23 Before each experiment, a 100 µL E coli aliquot was thawed, suspended in 6 mL of Luria-Bertani medium, and grown at 37C for 16 hours. A 100 µL sample was then removed from this suspension, diluted by a factor of 104, and reincubated at 37C for 4 hours to achieve log-phase growth.

Mice were inoculated using a method we previously have described in the nonpregnant animal.24 Briefly, mice were anesthetized with 0.015 mL/g body weight of 2.5% tribromoethyl alcohol and 2.5% tert-amyl alcohol mixed in phosphate-buffered saline. The mouse was immobilized in the prone position, and a flank incision was made to expose the left kidney. A volume of 0.02 mL of either E coli suspension or Luria-Bertani medium was injected into the lower pole of the kidney to the depth of the bevel of a 27 guage needle (Becton-Dickinson & Co., Franklin Lakes, NJ). Because live cultures were used, inoculum sizes were estimated based on preliminary experiments and determined definitively by post hoc plating. Escherichia coli inocula were divided for purposes of analysis into four ranges: 1–2 x 105, 6–12 x 105, 2–3 x 106, and 3–9 x 106 organisms. The muscle was closed with interrupted synthetic absorbable 4-0 glycolide/lactide copolymer sutures (Polysorb; United States Surgical Corp., Norwalk, CT), and the skin was closed with 9-mm stainless steel wound clips (Clay Adams, Parsippany NJ). Animals then were placed individually in clean cages and returned to the animal facility. The duration of the entire procedure was 5–10 minutes per animal.

Mice were monitored each morning and evening by a single observer throughout the postoperative period for nonspecific signs of illness such as decreased mobility and piloerection. Illness was scored as mild, moderate, or severe based on the degree of fur ruffling and immobility, according to a predetermined scale. The approximate time from surgery to delivery was recorded. Preterm delivery was defined as delivery of one or more fetuses within 72 hours.

Mice were killed by carbon dioxide inhalation after delivery. Kidney and uterine specimens were harvested at random from a subset of mice from each inoculum range. These tissues were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. Histologic examination was performed in a masked fashion by a veterinary pathologist.

Categoric variables were analyzed by Fisher exact and {chi}2 tests. P <= .05 was considered significant. An a priori power analysis revealed that 11 mice per group would be required for each comparison of control to infected animals, assuming an increase in the rate of preterm delivery with infection from 5% to 50%, with {alpha} = .05 and ß = .2.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
 References
 
In total, 70 mice underwent kidney injection. Thirty-one of 51 animals (60.8%) injected with E coli delivered prematurely (Figure 1AGo). All control mice delivered at term (P < .001, {chi}2 test). When stratified by inoculum (Figure 1BGo), the rates of preterm delivery were not significantly different across the inoculum range of 6 x 105 - 9 x 106 organisms, with an average preterm delivery rate of 71% (P > .5, Fisher exact tests). When the preterm delivery rate at the lowest inoculum range (1–2 x 105 organisms) is compared with the average preterm delivery rate at the higher inocula (6 x 105 - 9 x 106 organisms), a dose-response relationship between inoculum and preterm delivery is observed (P = .025, Yates corrected {chi}2). All infected mice, but no control mice, experienced some degree of infectious morbidity gauged by piloerection and decreased mobility prior to delivery. There was no difference in morbidity between infected mice delivering at term and those delivering preterm. Three infected mice died (bacterial inocula: 2 x 105, 5 x 106, and 9 x 106), whereas all remaining mice recovered completely after preterm delivery.



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  		Figure 1. Preterm delivery rates following kidney inoculation. A) Incidence of preterm delivery, infected mice compared with controls; B) Incidence of preterm delivery in infected mice by inoculum.

 
Histologic study revealed renal abscess formation (Figure 2Go) in all examined kidneys injected with bacteria (n = 29), and no abscesses in any of the 12 control specimens examined (P < .001, {chi}2 test). Further histologic analyses of kidneys was halted when the complete correlation between treatment group and abscess formation became apparent. The severity of infection was histologically similar across all E coli inoculum ranges and was unrelated to timing of delivery. Renal abscess was present in the inoculated kidney only and was localized to the injection site within the lower kidney pole. The contralateral kidney and the upper pole of the injected kidney revealed normal histology.



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  		Figure 2. Kidney histology. A) Abscess formation following bacterial injection showing massive leukocyte infiltration and various stages of tubular necrosis; B) normal kidney in a control mouse following sterile Luria-Bertani injection (original magnification x40).

 
Examination of right and left uteri 0–2 days after delivery from eight infected mice that delivered preterm revealed a minimal to moderate degree of endomyometritis and pyometra, whereas normal histology was found in two postpartum control mice (P = .22, Fisher exact test).


   Discussion
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Abstract
 Materials and Methods
 Results
 Discussion
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Clinical studies have demonstrated the association between preterm delivery and extrauterine infections such as pyelonephritis, appendicitis, and lobar pneumonia.25,26 The pathophysiologic mechanisms of preterm delivery associated with a localized abscess are largely unknown.

In this study we have shown that unilateral renal injection of bacteria results in localized abscess formation. The severity of infection as judged histologically was the same over a wide range of inocula. The opposite pole of the injected kidney as well as the contralateral kidney remained unaffected, reflecting the localization of abscess formation.

A dose-dependent relationship between bacterial inoculum and preterm delivery is observed at the lower inoculum range (1–6 x 105 organisms), however a plateau in the rate of preterm delivery occurs at inocula above 6 x 105 organisms. In previous work, intrauterine inoculation of relatively small inocula (2–10 x 103 organisms) led to preterm delivery in 91% of mice.19 In contrast, renal infection requires greater inocula to induce a rate of 60.8% preterm delivery. Despite this discrepancy in delivery rates, minimal to moderate metritis was induced in uterine- and kidney-infected mice. It is unclear why delivery rates after renal inoculation plateau at 60.8%; one explanation may be that a portion of the bacterial inoculum is excreted in the urine, thus limiting the extent of infection. Current theory regarding intrauterine infection-induced preterm delivery, however, implicates proinflammatory cytokines as mediators of signals for preterm delivery.12,27,28 It is possible that in the setting of localized extrauterine infection, anti-inflammatory host defense mechanisms may be sufficient to limit bacterial dissemination and the production of inflammatory cytokines in some individuals.

The results from histologic study of uterine tissue await confirmation with larger sample sizes. However, the finding of uterine inflammation in renally infected mice suggests that secondary bacterial seeding of uteri may occur. Similarly, in a small sampling, two of three sets of uterine horns and corresponding fetuses cultured from infected mice grew E coli bacteria. Whether extrauterine infection causes labor by secondary seeding of the uterus or through the expression of systemic mediators awaits further investigation.


   Footnotes
 
The authors thank Karla Damus, PhD, for her assistance with the statistical analyses.

Supported in part by grant No. 5-FY96-1140 from the March of Dimes Birth Defects Foundation.

PII S0029-7844(99)00571-2

Received May 10, 1999. Received in revised form August 11, 1999. Accepted August 19, 1999.


   References
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 Materials and Methods
 Results
 Discussion
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1. Berkowitz GS, Papiernik E. Epidemiology of preterm birth. Epidemiol Rev 1993;15:414–43.[Free Full Text]

2. Guzick DS, Winn K. The association of chorioamnionitis with preterm delivery. Obstet Gynecol 1985;65:11–6.[Abstract/Free Full Text]

3. Romero R, Sirtori M, Oyarzun E, Avila C, Mazor M, Callahan R, et al. Infection and labor. V. Prevalence, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes. Am J Obstet Gynecol 1989;161:817–24.[Medline]

4. McLane CM. Pyelitis of pregnancy: A five-year study. Am J Obstet Gynecol 1939;38:117–23.

5. Kass EH. Pyelonephritis and bacteriuria: A major problem in preventive medicine. Ann Intern Med 1962;56:46–53.

6. Fan YD, Pastorek JG, Miller JM Jr, Mulvey J. Acute pyelonephritis in pregnancy. Am J Perinatol 1987;4:324–6.[Medline]

7. Committee to Study the Prevention of Low Birthweight, Division of Health Promotion and Disease Prevention, Institute of Medicine. Preventing low birthweight. Washington, DC: National Academy Press, 1985:1–41.

8. Creasy RK. Preterm birth prevention: Where are we? Am J Obstet Gynecol 1993;168:1223–30.[Medline]

9. Hirsch E, Blanchard R, Mehta S. Differential fetal and maternal contributions to the cytokine milieu in a murine model of infection-induced preterm birth. Am J Obstet Gynecol 1999;180:429–34.[Medline]

10. Hillier SL, Witkin SS, Krohn MA, Watts DH, Kiviat NB, Eschenbach DA. The relationship of amniotic fluid cytokines and preterm delivery, amniotic fluid infection, histologic chorioamnionitis, and chorioamnion infection. Obstet Gynecol 1993;81:941–8.[Abstract/Free Full Text]

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12. Romero R, Brody DT, Oyarzun E, Mazor M, Wu YK, Hobbins JC, et al. Infection and labor III. Interleukin-1: A signal for the onset of parturition. Am J Obstet Gynecol 1989;160:1117–23.[Medline]

13. Romero R, Manogue KR, Mitchell MD, Wu YK, Oyarzun E, Hobbins JC, et al. Infection and labor IV. Cachectin-tumor necrosis factor in the amniotic fluid of women with intraamniotic infection and preterm labor. Am J Obstet Gynecol 1989;161:336–41.[Medline]

14. Dudley DJ, Chen CL, Branch DW, Hammond E, Mitchell MD. A murine model of preterm labor: Inflammatory mediators regulate the production of prostaglandin E2 and interleukin-6 by murine decidua. Biol Reprod 1993;48:33–9.[Abstract]

15. Dombroski RA, Woodard DS, Harper MJ, Gibbs RS. A rabbit model for bacteria-induced preterm pregnancy loss. Am J Obstet Gynecol 1990;163:1938–43.[Medline]

16. Bry K, Hallman M. Transforming growth factor-beta 2 prevents preterm delivery induced by interleukin-1 alpha and tumor necrosis factor-alpha in the rabbit. Am J Obstet Gynecol 1993;168:1318–22.[Medline]

17. Fidel PL Jr, Romero R, Wolf N, Cutright J, Ramirez M, Araneda H, et al. Systemic and local cytokine profiles in endotoxin-induced preterm parturition in mice. Am J Obstet Gynecol 1994;170:1467–75.[Medline]

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23. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual. Vol. 3. New York: Cold Spring Harbor Laboratory Press, 1989.

24. Mussalli GM, Brunnert SR, Hirsch E. A murine model of renal abscess formation. Clin Diagn Lab Immunol 1999;6:273–5.[Abstract/Free Full Text]

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27. Hillier SL, Witkin SS, Krohn MA, Watts DH, Kiviat NB, Eschenbach DA. The relationship of amniotic fluid cytokines and preterm delivery, amniotic fluid infection, histologic chorioamnionitis, and chorioamnion infection. Obstet Gynecol 1993;81:941–8.

28. Baggia S, Gravett MG, Witkin SS, Haluska GJ, Novy MJ. Interleukin-1 beta intra-amniotic infusion induces tumor necrosis factor-alpha, prostaglandin production, and preterm contractions in pregnant rhesus monkeys. J Soc Gynecol Investig 1996;3:121–6.[Medline]




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