Jul 9, 2009 - To cite this article: Linda M. Ernst, Carolyn M. Salafia, Anthony M. Carter & John C. Pezzullo ..... Jones CT, Harding JE, Gu W, Lafeber HN.
Pediatric Pathology
ISSN: 0277-0938 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ipdp17
Hepatic Histology in Intrauterine Growth Retardation Following Uterine Artery Ligation in the Guinea Pig Linda M. Ernst, Carolyn M. Salafia, Anthony M. Carter & John C. Pezzullo To cite this article: Linda M. Ernst, Carolyn M. Salafia, Anthony M. Carter & John C. Pezzullo (1993) Hepatic Histology in Intrauterine Growth Retardation Following Uterine Artery Ligation in the Guinea Pig, Pediatric Pathology, 13:6, 763-772, DOI: 10.3109/15513819309048263 To link to this article: http://dx.doi.org/10.3109/15513819309048263
Published online: 09 Jul 2009.
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Date: 19 October 2016, At: 06:34
HEPATIC HISTOLOGY IN INTRAUTERINE GROWTH RETARDATION FOLLOWING UTERINE ARTERY LIGATION IN THE GUINEA PIG
Linda M. Ernst, MS, Carolyn M. Salafia, MD, Anthony M. Carter, PhD, and Department of Pathology, University of Connecticut John C. Pezzullo, PhD School of Medicine, Farmington, Connecticut 06302, Department of Physiology, University of Odense, Odense, Denmark, and Technology Resource Center, Providence, Rhode Island
Hepatic histology was examined i n guinea pig fetuses in which intrauten'ne growth retardation (IUGR) was induced unilateral uterine artery ligation and compared with that of control (well-grown) fetuses from uterine harns with intact circulations. From all animals, sections taken from the six lobes ofthe liver were prepared using hematoxylin and eosin stains. Pa'odic acid-SchiJf and Prussian blue stains (both o n j x e d samples) and Oil Red 0 stains on frozen tissues were p e r f m e d on a subset of samples. Comparisons revealed greaterf a t content in hepatocytes (P < .05) and decreased total hepatic hematopoiesis (P .01) i n IUGR animals than in controls. No diffuences in hepatocyte histology among lobes were observed in control animals. IUGR animals had increased f a t content and decreased extramedulla? hematopoiesis in the lobes receiving the portal circulation (P < .05). Thesedata indicatesignijcant abnmalities ofhepatic lipid metabolism in IUGR animals follow'ng uta'ne artery ligation. An unexpected and counterintuitivejinding was the derrease in hepatic hematopoiesis i n the right side of the liver. KEY WORDS: animal models, extramedulla? hematopoiesis, hepatic histology, IUGR.
INTRODUCTION Intrauterine growth retardation (IUGR) is a major clinical problem affecting, according to definition, from 4 to 10%of infants. IUGRcarries both immediate fetal and neonatal risks of morbidity and mortality and also long-term pediatric risks of impaired neurodevelopmental outcome ( 1). Clinical risk factors for IUGR include congenital viral infection, maternal smoking, malnutrition, substance abuse, multiple gestation, and maternal diseases (2). In the majority of cases, however, IUGR occurs in the absence of clinical risk for fetal growth impairment. The lack of basic understanding of the mechanisms involved in IUGR has resulted in failure not only to better assess risk for IUGR but also to devise effective intrauterine preventive and therapeutic strategies.
Pediatric Pathology, 13:763-772, 1993 Copyright 0 1993 laylor 0.' Francis 0277-0938/93$10.00 + .OO
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Studies of the placentas of otherwise idiopathic IUGR infants suggest that uterine vascular insufficiency contributes to fetal growth impairment. IUGR can be reproduced in the guinea pig by uterine artery ligation in midgestation. In the guinea pig, uterine blood flow progressively increases throughout gestation, in a fashion similar to that in humans. Uterine artery ligation interrupts this normal process and reduces total maternal placental blood flow (3) and total nutrient availability (4,5). The pattern of IUGR resembles that of asymmetric or “late-onset” IUGR in the human. However, the precise causal mechanisms linking reduced placental blood flow to progression of IUGR are not clear. The respective contributions to growth impairment of reduction of uterine blood flow, placental damage, and primary fetal effects have not been clarified. It is reasonable to consider the liver to play a critical role in the evolution of IUGR. First, the reduced liver size in “asymmetric” IUGR is paradoxical. The liver in the guinea pig is perfused primarily by the umbilical venous return and would therefore be the recipient of optimal nutrient supplies. Despite this, delayed maturation of hepatocyte metabolism (6) occurs. There is also delayed brain myelinization in intrauterine growth retardation (7), but liver growth in this model fails to keep pace with growth of the brain. Elevated brain/liver weight ratios are used to define this pattern of IUGR (8,9) in a manner analogous to that in humans (10). In addition, the liver is critical to midtrimester hematopoiesis and to fatty acid metabolism. Growth retardation is presumed to be produced in this model system by the reduction of nutrient and oxygen delivery to the conceptus. We expected that increased extramedullary hematopoiesis would be a feature of the hepatic histology of IUGR fetuses and hypothesized that this would be most prominent on the right side of the liver. Because the liver is recognized to be a sensitive measure of abnormal fatty acid handling, we also hypothesized that histopathologic evidence of altered fatty acid metabolism could be identified in IUGR guinea pig fetuses. This initial study was undertaken to evaluate hepatic histology and correlate histologic features between IUGR and well-grown fetuses.
MATERIALS AND METHODS Guinea pigs of outbred stock (Ssc:AL) were housed as previously described (11). The female guinea pig usually has a regular estrous cycle with a mean interval between ovulations of 16 days at 10-12 weeks of age, at which time body weight is about 500 g. Pregnancy was determined by daily inspection of the vaginal membrane as described by Elvidge (12) with day zero of pregnancy defined as the first day on which the vaginal membrane was fully open. The surgical technique was as previously described (8). Briefly, at day 29-33 of pregnancy, guinea pigs were anesthetized and access to the lower
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abdomen was gained through a 2-cm midline incision caudal to the umbilicus. The uterine artery and vein embedded in the mesometrial fat pad of one uterine horn were ligated from the dorsal side with surgical silk at the level of the cervix. The fetuses were then examined at 60-64 days. Only cases with at least one normally grown fetus in the control horn and/or a growth-retarded fetus on the ligated side, defined as such by a braidliver weight ratio of >0.85, were included in this study (8).The liver was weighed with the gallbladder intact. The brain was cut loose from the spinal cord to include the medulla oblongata. Natural runts were excluded. On the date mothers were sacrificed, maternal weight, fetal and placental weights, fetal location in the uterus, and brain and liver weights of all fetuses on the ligated side were recorded. A total of 15 IUGR fetuses and 6 controls from nonligated uterine horns formed the population of the present study. The livers of the control and growth-retarded fetuses were dissected and divided into six segments: the caudate, the right lobe (right and left sublobes), the quadrate lobe (right and left sublobes), and the left lobe (Fig. 1). Each of these segments was further divided into two or three subsegments. All subsegments of the liver were embedded in paraffin and 5-mm sections were cut, stained with hematoxylin and eosin, and mounted on glass slides. The slides were then reviewed by a single obeserver (L.M.E.),who was “blinded” as to the experimental details. To evaluate hepatic hematopoiesis, erythroid colonies were counted in three different fields at 4 0 ~To . evaluate hepatocyte vacuolation, a subjective scale was developed, with tissues graded from 0 to 7 considering the proportion of cells in which vacuoles were identified and the extent of replacement of the
FIGURE 1. The guinea pig liver showing division into lobes and sublobes. Adapted from Cooper and Schiller (24).
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cytoplasm by vacuoles in involved cells. In addition, slides from a subset of tissue samples were stained for subjective evaluation of the presence and extent of iron/hemosiderin (Prussian blue), glycogen (PAS with and without diastase), and fat (Oil Red 0) content in hepatic tissues. The means and standard deviations of both the extent of vacuolization and extramedullary hematopoiesis for each lobe of the livers and for the whole liver were calculated, and comparisons were performed using unpaired Student’s &tests with P< .05 considered significant.
RESULTS Pertinent morphometric data are presented in Table 1. As previously described, midgestational uterine artery ligation produced profound IUGR (8,13).By definition, the IUGR fetuses had a markedly increased brain/liver weight ratio. The patterns of organ growth in the IUGR fetuses in the present study resemble those of late-onset IUGR in the human. Reduced placental weights were also observed in IUGR as compared to control fetuses (P< .05), and fetoplacental weight ratios were similar in the two groups. Histologic assessment of the livers of IUGR and control fetuses showed striking differences in the extent of hematopoiesis and hepatocyte vacuolation and in the distribution of these processes among the liver lobes. Figure 2A shows the raw scores of extent of extramedullary hematopoiesis in the liver and in each hepatic lobe. Representative photomicrographs of hematopoiesis in the right and left lobes of the liver of IUGR and control fetuses are presented in Figs. 3 to 6. Two features are noteworthy. First, there is an overall decrease in total hepatic hematopoiesis in IUGR fetuses compared with controls ( P
