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The Role of Experimental Maternal Liver Pathology in the Development of Physiological Immaturity in the OffspringG. V. Bryukhin and M. L. Sizonenko

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 154, No. 11, pp. 544-546, November, 2012Original article submitted August 26, 2011

We compared the parameters of physiological maturity in the offspring of albino Wistar rats with experimental chronic liver disease of various genesis (toxic, autoimmune, and alcoholic). It was found that chronic liver disease in female rats can be a cause of physiological imma-turity of the offspring (increased stillbirth rate, reduced viability, and delayed disappearance of immaturity).

Key Words: chronic liver disease; offspring; physiological immaturity

Department of Histology, Embryology and Cytology, Chelyabinsk

State Medical Academy, Russia. Address for correspondence: maximus_74.79@mail.ru. M. L. Sizonenko

Reproduction of healthy offspring is one of the ur-gent problems of modern medicine. In light of this, the attention of scientists is focused on woman health protection as a prerequisite for reproduction of new healthy generation [4]. At the same time, extragenital diseases in mothers, especially hepatobiliary system pathology, are among the causes of perinatal pathology including birth of physiologically immature offspring [3]. Despite widespread increase in the prevalence of chronic liver disease (CLD), among them in wom-en of childbearing age, impact of this disease on the growth and development of the offspring is not fully understood. We have previously shown [1,2,5] that the offspring of female rats with experimental CLD were characterized by delayed morphological and functional development of the life-support systems including the macrophage, hemopoietic, reproductive, endocrine, and nervous systems.

Here we analyzed the parameters of physiological maturity in the offspring of female rats with experi-mental CLD at different postnatal terms.

MATERIALS AND METHODS

The experiments were performed on Wistar albino rats and their offspring at various postpartum terms (on

postnatal days 1, 15, 30, and 45). Toxic and autoim-mune CLD was modeled in adult mature females. In-tact animals (320 rat pups from 112 litters) comprised the control group (group 1).

For modeling toxic CLD (group 2; 346 rat pups from 84 litters), 0.1 ml 40% CCl4 in vegetable oil was subcutaneously injected to rats twice a week over 2 months. The development of toxic CLD was evalu-ated by morphological changes (fatty degeneration of hepatocytes, dilatation of sinusoidal capillaries and space of Disse, focal intralobular necrotic changes in hepatocytes, moderate lymphocytic and histiocytic infi ltration of the liver stroma).

Autoimmune CLD (group 3; 374 rat pups from 90 litters) was induced via long-term sensitization by homologous liver antigen with Freund’s adjuvant [7]. Complete cycle of immunization lasted for 4 months, the animals received ~200 mg liver antigen. Autoim-mune CLD was diagnosed by high titer of anti-liver autoantibodies (1:640, 1:1280), increased γ-globulin fractions in the blood, and morphologic changes (mar-ked perivascular lymphocytic and histiocytic infi ltra-tion of portal tracts, focal necrotic changes in hepa-tocytes, their discomplexation, hyperplasia of liver macrophages, and dilatation of sinusoidal capillaries).

For modeling chronic alcohol intoxication with predominant damage to the liver (group 4; 177 rat pups from 41 litters), the rats were allowed free ac-cess to 15% ethyl alcohol rats instead of water for

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3 months. The development of alcoholic CLD was verifi ed by morphological (focal necrotic alterations of hepatocytes, moderate lymphocytic and histiocytic infi ltration of the liver stroma, hepatocytes discom-plexation, and dilatation of sinusoidal capillaries), bio-chemical (increased transaminase activity, changes in protein fractions, increased alkaline phosphatase activity etc.), and immunological changes (high titer of anti-liver autoantibodies; 1:640).

According to the Regulations of the UN Environ-ment Programme, International Labour Organization and the World Health Organization on the principles of valuation of offspring in experimental animals, the fol-lowing parameters were used to estimate physio logical maturity in control and experimental groups: number of live born fetuses, fetal deaths, litter size, sex, and weight of newborns. In addition, we evalua ted the state of the life-supporting systems of the offspring in animal models. To this end, the survival rate of ani-mals during the early postnatal ontogeny (by the end of the 2nd and 15th postnatal days), dynamics of body weight gain (BW), and dynamics of thymus, spleen, and popliteal lymph node weight were evaluated. The degree of physiological maturity of the offspring was assessed by timing of certain stages of physical devel-opment in each litter (eye and acoustic meatus open-ing, appearance of primary and secondary hairs, and eruption of incisors).

RESULTS

Female rats with CLD of different genesis had im-paired reproductive function. Thus, impaired estrous cycle characterized by the development of persistent diestrus or estrus was detected in the majority of ani-mals (58% animals in group 2, 49% in group 3, and 53% in group 4). No cases of estrous cycle distur-bances were reported in group 1 (controls).

When female rats were caged with males on day 1 of estrus, mating occurred in all the animals in group 1 and in 33.35 and 42.5% females in groups 2 and 3, respectively. During pregnancy, prenatal mortality in experimental animals signifi cantly exceeded the control level (Table 1). The number of newborns per litter was signifi cantly reduced in groups with CLD. However, comparative analysis of litters by gender showed more marked predominance of females in the experimental animals then in controls.

During physiological pregnancy, the load to the liver is greatly increased due to necessity to inactivate waste products of the fetus and sex hormones. It is logical to assume that toxic and autoimmune CLD in female rats impairs many vital functions of the liver, e.g. detoxifi cation, which causes accumulation of met-abolic products, including nitrogenous wastes (creati-nine, residual nitrogen, and others) with pronounced toxic effect. Metabolites cross the placental barrier and enter the fetus body, which impairs the conditions of intrauterine growth, complicate pregnancy in females with CLD, and lead to birth of physiologically imma-ture offspring. Thus, rat pups in all the experimental groups showed delayed physical development, which manifested in more protracted signs of immaturity (Table 2). In addition, rat pups were weak suckers, sluggish and inactive.

According to modern views [1], reduced BW gain in the early postpartum period is a sign of physiologi-cal immaturity, because most of the stages of physical development are related to this parameter. Decelera-tion of BW gain after birth was revealed in animals of all experimental groups in comparison with the control group (Fig. 1).

In control pups, BW gradually increased after birth reaching the highest value by at the end of pu-berty (day 45). The same dynamics was found in rats of all experimental groups, but BW in these pups was

TABLE 1. Characteristics of Litters of Female Rats with CLD of Different Etiologies (M±m)

Group

Number of animals per litter

Stillbirths per litter,

%

Alive, %

total

females males

abs. % abs. % after 2 days after 15 days

1 8.00±0.09 4.20±0.05 52.5±0.6 3.8±0.6 47.5±0.6 1.25±0.27 96.69±2.50 94.6±1.7

2 5.55±0.13* 3.30±0.15* 59.5±1.1* 2.25±0.15* 40.5±1.5* 5.35±0.63* 83.89±2.00* 77.01±3.50*

3 6.72±0.50* 4.52±0.30 67.2±1.5* 2.20±0.30 32.8±1.5* 2.75±0.43* 80.77±2.10* 77.49±3.80*

4 6.41±0.31* 3.84±0.25 59.91±1.00* 2.57±0.25 40.09±1.00* 3.14±0.33* 74.35±3.00* 71.40±2.85*

Note. *Here and in Table 2: p≤0.05 in comparison with group 1.

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lower than in controls at most time points. These data are consistent with the data on daily BW gain. Thus, daily BW gain during the fi rst 2 weeks after birth was 0.91±0.05 g in group 1 (controls), and 0.86±0.09, 0.80±0.09, and 0.61±0.10 g in groups 2, 3, and 4, re-spectively. On postpartum days 15-30, BW gain was 1.69±0.21 g in group 1, whereas in group 2, 3, and 4 it was 1.05±0.14, 1.07±0.10, and 1.01±0.08 g, re-spectively. Finally, daily BW gain on days 30-45 was 1.59±0.11 g in group 1 and 1.35±0.14, 0.99±0.15,

TABLE 2. Physical Signs in Offspring of Female Rats with CLD (M±m)

Group Primary fur Secondary furEye

openingAcoustic

meatus openingPinna

detachmentEruption of

incisors

1 (n=30) 5.067±0.095 9.267±0.095 14.033±0.339 12.233±0.104 2.30±0.85 8.100±0.100

2 (n=40) 6.550±0.113* 10.250±0.099* 16.035±0.127* 13.925±0.109* 3.075±0.084* 9.450±0.133*

3 (n=31) 5.323±0.085* 9.711±0.115* 15.033±0.122* 12.774±0.137* 2.968±0.109* 8.516±0.102*

4 (n=35) 5.755±0.123* 9.824±0.105* 15.609±0.121* 13.472±0.113* 2.897±0.104* 8.914±0.109*

and 0.86±0.11 g in groups 2, 3, and 4, respectively. Thus, daily BW gain in animals of groups 2 and 3 during the early postpartum period was signifi cantly lower, which determined lower BW during the inves-tigated period.

Viability of the animals is one of the most impor-tant indicators of physiological maturity. The survival rate in offspring of female rats with CLD of different genesis was signifi cantly below the control (Table 1). Thus, female rats with experimental CLD of toxic, autoimmune, and alcoholic genesis bore physiologi-cally immature offspring.

REFERENCES

1. G. V. Bryukhin, Morfologiya, 108, No. 1, 35-38 (1995).2. G. V. Bryukhin and E. V. Vtorushina, Probl. Reprod., 11, No.

3, 54-57 (2004).3. E. T. Mikhaylenko, A. A. Zakrevskiy, N. G. Bogdashkin, et al.,

Pregnancy & Childbirth in Chronic Diseases of the Hepatobi-liary System [in Russian], Kiev (1990).

4. L. B. Nikolaeva and G. A. Ushakova, First Pregnancy and First Childbirth [in Russian], Мoscow (2010).

5. M. L. Sizonenko and G. V. Bryukhin, Probl. Reprod., 10, No. 2, 45-47 (2008).

6. A. B. Kuznetsova and G. V. Bryukhin, Izv. Chelyabinskogo Nauch. tsentra, No. 2, 110-113 (2006).

7. A. W. Lohse and M. Manns, Hepatology, 11, No. 1, 24-30 (1990).

Fig. 1. BW of the offspring of experimental animals in different per-

iods of postnatal ontogeny. 1-4: groups 1-4, respectively.

G. V. Bryukhin and M. L. Sizonenko