The effect of exposure to diazepam through the placenta or through the mother's milk

Neuropharmacology Vol. 23, No. 9, pp. 1099-l104, Printed in Great Britain.All rightsreserved

1984

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THE EFFECT OF EXPOSURE TO DIAZEPAM THROUGH THE PLACENTA OR THROUGH THE MOTHER’S MILK HISTOLOGICAL

FINDINGS

IN SLICES OF RAT BRAIN

B. FRIEDER,A. MESHORERand VERONIKAE. GRIMM Isotope Department, Weizmann Institute of Science, Rehovot, Israel (Accepted 30 November 1983)

Summary-Exposure to diazepam during the prenatal or early postnatal developmental period has been reported to result in later behavioural deficits. In the present study morphological changes in the brains of rats that were exposed to diazepam (DZP) prenatally or through the mother’s milk postnatally were investigated. The results showed that prolonged prenatal exposure (16 days) to diazepam (10 mg/kg) resulted in characteristic and extensive pathological changes, i.e. gliosis and perivascular cuffing in the brains of the rats. These changes could be observed under the light microscope a long time after exposure to the drug had been terminated. Limiting the prenatal exposure to a single trimester of 7 days reduced somewhat the number of lesions but did not prevent their occurrence. Rats exposed to diazepam postnatally through the mothers’ milk showed very few lesions. Key words: diazepam, prenatal treatment, development, gliosis, perivascular cuffing.

An increasing number of studies draw attention to the effects of drugs taken by pregnant women on foetal development (Cohen, 1964; Karnovsky, 1965; Forfar and Nelson, 1973). For example, exposure to the tranquillizer diazepam during gestation was reported to result in the so called “floppy infant syndrome” characterized by hypotonia, hypothermia, respiratory complications, hyperbilirubinemia and poor sucking response (Joyce and Kenyon, 1972; McCarthy, O’Connell and Robinson, 1973; Flowers, Rudloph and Desmond, 1969; Rowlatt, 1978; Woods and Malan, 1978; McAllister, 1980). Recently, a number of animal studies have shown that prenatal exposure to various neuroleptic and anxiolytic agents resulted in significant alterations of development and behaviour detected at some later time. Orally administered diazepam (DZP) throughout pregnancy resulted in delayed sexual maturation and impaired acquisition of a conditioned avoidance response in rat offspring (Ljubimov, Smolmikova and Stirekalova, 1974). Exposure of pregnant rats to diazepam during the final week of gestation interfered with the development of arousal processes, studied later in the pups (Kellogg, Tervo, Ison, Parisi and Miller, 1980). of brain cells, In rats, rapid proliferation differentiation and growth in neural tissue take place from about the second week of gestation until the third postnatal week (Winnick, 1974). Diazepam, a lipophilic drug with a small molecular weight, crosses the placenta rapidly, since the capacity of the foetus or newborn to dispose of the drug is very small

(Marcucci, Musini, Airopodi, Guaitani and Garatini, 1973; Morselli, Principi, Tognoni, Reali, Belvedere, Standen and Sereni, 1973; Mandelli, Morselli, Nordio, Pardi, Principi, Sereni and Tognoni, 1975) the drug and its pharmacologically active metabolites accumulate in the foetus. Studies with diazepam have shown concentrations in foetal plasma to be greater than those in maternal plasma (Cavanagh and Condo, 1964; Idanpaan-Heikkila, Jouppila, Puolakka and Vorne, 1971; Cree, Meyer and Hailey, 1973; Erkkola, Kangas, and Pekkarinen, 1973). The resultant large concentration of diazepam in foetal tissue has been noted (Erkkola, Kanto and Sellman, 1974) to be comparable to those which have deleterious effects on human cells in tissue culture (Breen and Stenchever, 1970). Some of the long range implications of this phenomenon are being investigated in this laboratory. It has been shown that exposure to diazepam (5 and 10 mg/kg) of rat dams throughout most of the gestation period (16 days) resulted in the pups showing reduced exploratory behaviour in an open field and a dosedependent learning deficit that manifested itself in a complex choice-discrimination task, but not in simple learning at two months of age (Gai and Grimm, 1982). When the exposure to diazepam (10 mg/kg) was restricted to only the second trimester of pregnancy (days 8-14) no learning deficits were found. However such exposure during the final week of gestation (days 14-20) caused significantly poorer performance both in acquisition and on the recall test of the complex discrimination task compared to the

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offspring of dams exposed to vehicle. Shorter prenatal treatment did not affect activity in open field. A third group of rat dams were treated with diazepam (10 mg/kg) during lactation (16 days) so that their pups were exposed to diazepam through the milk. This postnatal group showed consistent and lasting hyperactivity in the open field (tested at 20 and 35 days of age) and significant deficits in acquisition and recall deficits on the complex discrimination task at two months of age (Frieder, Epstein and Grimm, unpublished). The purpose of the present study was to test the hypothesis that there are morphological changes in the brain following early exposure to diazepam, and these changes may be the basis for the observed behavioural impairments. METHODS

Nulliparous female Wistar rats, two months of age, were selected as dams. Pregnancy was detected by daily vaginal smears. The day after the first positive smear was defined as day one of gestation. At this time the rats were transferred into individual housing cages in the colony room of the Brain and Behaviour group at the Weizmann institute. Constant temperature, humidity and 12-hr light-dark cycle (lights on 7 a.m.) were maintained in this room. Food and water were available ad lib&m. Treatment of dams: 10 mg/kg diazepam (DZP) (“Assival 10” injectable ampules, TEVA, Israel) or equal volumes of the vehicle of diazepam (40% propylene glycol, 8% alcohol in distilled water) were injected subcutaneously. The injections to the dams were carried out during different periods of pregnancy or during lactation in the following four treatment conditions: (a) Diazepam (or vehicle) for 16 consecutive days from the 4th to the 20th day of pregnancy. (b) Diazepam (or vehicle) for 7 consecutive days, during the second trimester of gestation (days 8-14). (c) Diazepam (or vehicle) for 7 consecutive days, during the third trimester of gestation (days 14-20). (d) Diazepam (or vehicle) for 16 consecutive days during the lactation period, days 3-19 postnatally. Within 24 hr of birth the pups were counted, weighed and redistributed to foster dams (10 pups per dam). All foster dams were vehicle-treated during pregnancy. For the histological tests offspring of mothers from the four treatment groups together with their controls were selected randomly and were killed by decapitation between 45 and 65 days of age.

The entire brain was removed and was fixed for 72 hr in 3.7% formalin. Following this the brain was cut into four cross sections and these were placed again in formalin for 24 hr. The sections were then dehydrated in a graded series of alcohols, cleared in xylene and embedded in paraffin. Sections of 7~ thickness were cut and these were stained with luxol-fast-blue (stains myelin and red blood cells) and nuclear fast red (stains nuclei). The sections were scanned under the light microscope and the number of foci of gliosis and perivascular cuffing per visual field (750 p in diameter) were counted. For each animal 10-20 brain sections were scanned. Sections were taken every 250~. Twenty to one hundred visual fields were scanned for each region depending on the size of the region. Table 1 summarizes the experimental conditions and shows the number of animals whose brains were used for the histological examination.

RESULTS

Table 2 summarizes the findings of the histological examination of brain sections from control and animals exposed to diazepam. None of the nine control animals showed any abnormalities. Animals that were exposed to diazepam during early development showed two types of pathological patterns: gliosis and perivascular cuffing (mononuclear infiltration surrounding blood vessels) (see Figs 1 and 2). Lesions were not seen in all the animals in any given treatment group. Four of the six rats chosen randomly for histological study, from the group that received exposure to diazepam throughout gestation (days 4-20) showed pathological patterns. Two rats out of five exposed in the second trimester (days 8-13) and, four out of four of those treated in the third trimester of pregnancy showed some pathological signs. In the postnatally exposed group few lesions were found (see Table 2). Generally the lesions were not limited to specific brain regions. In the 16 days prenatal exposure group, signs of gliosis tended to be more numerous in the cortex, while perivascular cuffing appeared to be more frequent in subcortical areas. The cerebellum was relatively free of either type of lesions. In the second trimester exposed group, two rats out of five showed pathological patterns, Gliosis was found with high frequency in the cortex and hippo-

Table 1. Showing the developmental periods in which the dams were exposed to diazepam or vehicle, the length of the exposure, the number of dams treated and the number of offspring of these, that were randomlv selected for the histoloaical studv Developmental period in which diazepam was given Long term prenatal Second trimester Third trimester Postnatal, lactation Prenatal vehicle

Days of exposure to diazepam/vehicle 16 I 7 16 16

Nux’nber of dams

Number of offspring used for the histology

5 4 3 2 6

6 5 4 5 9

Fig. 1. Example of gliosis found in the cortex of rats treated prenatally with diazeparn. N = neuronal cell bodies; G = glia cells; L = lumen of blood vessel; GL = region of gliosis. Stained with luxol-fast-blue and counterstained with nuclear fast red. Magnification x 260.

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Fig. 2. Gliosis and perivascular cuffing in the brain of rats exposed prenatally to diazepam. N = neuronal cell body; L = lumen of blood vessel; PC = perivascular cuffing :mononuclear infiltration surrounding the blood vessel; GL = region of gliosis. Stained with luxol-fast-blue and counterstained with nuclear fast red. Magnification x 200.

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Effect of prenatal DZP on brain histology

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Table 2. Gliosis (gl.) and perivascular cuffing (p.c.) in brain sections of adult rats exposed to diazepam prenatally and postnatally* Prenatal Period of exposure: Total number of rats: Region Cortex gl. P.C. Caudate gl. P.C. HPC gl. P.C. Talanus gl. p.c. RF gl. P.C. Septum gl. D.C. Cerebellim gl. p/Z. Frontal gl. Cortex p.c.

Postnatal 3rd trimester 16 days Control (4) (5) (9) tMean number of lesions per microscopic field (750 p diameter) and number of rats showing lesions (n) 16 days (6)

0.73f0.15 0.25 f 0.03 0.44 + 0.06 0.47 f 0.08 0.35 & 0.07 0.53 f0.15 0.53 * 0.1 1.04 f 0.5 0.4 + 0.1 0.57 f 0.25 0.5io.13 0.71 f 0.15 0.2 + 0.03 0.3 +_0.2 1.13*0.05 0.35 ? 0.08

2nd trimester (5)

n=4 n=4 n=2 n=4 n=4 n =4 n=4 n=4 n=4 n=4 n=4 n=3 n= 3 n=2 n =4 n=4

0.6+0.1 0.45 * 0.05 0.08 0.6 f 0.2 0.56 k 0.22 1.44 f 0.5 0.07 0.5 + 0.16 0.4 1.2 0.44 0.55

n=2 n= 2 il=l n=2 n=2 n=2 n=l n= 2 n=l n=l n=l n=l

0.325 + 0.2 0.064 + 0.02 0.34 k 0.08 0.12 0.96 0.12 0.24 i 0.06 0.18+0.08 0.45 f 0.05 0.35 + 0.1 0.36 + 0.2

not examined 0.43 f 0.2 0.6 k 0.03

n=2 n=2

0.45 + 0.32 0.13_+0.01

n=4 n= 2 n=2 n=l n=l fl=l n=4 n =4 n= 2 I?=2 n=l n=O n=O n=O n= 3 n =2

0.1+0.04 0.05 0.3 0.1

0.13f0.01

0.1 0.1

n=2 il=l n=l fl=l n=O n=O n=O n=2 n=O n=O n=O n=O n=O n=O n=l II=1

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

*Three prenatal groups are animals receiving diazepam during gestation for 16 days, for 7 days during the second trimester, and for 7 days during the third trimester. One postnatal group received diazepam through mother’s milk for 16 days. tMean f SEM. The sections were taken from the followine; brain areas: cortex. caudate, hippocampus (HPC), talamus, reticular formation (RF), septum, cerebellum and frontal cortex.

campus but not in other subcortical regions, while perivascular cuffing was present in most regions. In the third trimester exposed group all four randomly selected rats showed some lesions. Perivascular cuffing was much less frequent compared to the two former groups, whereas gliosis was substantial and especially evident in the cortex. No significant relationship was found between the degree of pathological changes and the time of killing the animals (45-65 days). Postnatally-exposed rats showed very few lesions. DISCUSSION

The results show that prolonged prenatal exposure to diazepam resulted in characteristic and fairly extensive pathological changes in the brains of mature animals. These changes could be observed under the light microscope and long after exposure to the drug had been termina.ted. Limiting the prenatal exposure to a single trimester of seven days reduced somewhat the number of lesions but did not prevent their occurrence. It would appear that this is the first report of a phenomenon of this kind concerning the effects of diazepam, a lipid-soluble drug which crosses the placenta rapidly and can accumulate in lipidcontaining tissues. The enzymes of degradation and mechanisms of elimination of drugs start developing only postnatally (Marcucci et al., 1973; Mandelli et al., 1975; Morselli et al., 1973). This may lead to relatively large concentrations of unmetabolized diazepam in the foetus. The observed lesions, gliosis and perivascular cuffing, may provide some clues as to the underlying mechanism. Perivascular cuffing is an inflammatory response. It N.P.23,-

appears in infections, allergies and autoimmune disease (Paterson, 1966). In the present study, perivascular cuffing was shown in two-month-old animals, a long time after diazepam was completely eliminated from their body; A number of possibilities may be considered. The extensive gliosis found in brain slices of the rats exposed to diazepam may be a sign of neuronal death (Jacobson, 1972). Diazepam has been shown to cause some degenerative changes in cells (Breen and Stenchever, 1970). Particles from such damaged or destroyed cells may stimulate reactions aimed at the digestion and removal of this cellular debris. Another possibility is that rats treated prenatally with diazepam developed some autoimmune disease. Tolerance, or lack of response to “self” antigens is developed according to one theory, in early life by exposure to massive amounts of the antigen (self tissues) (Patterson, 1966). If diazepam can suppress immunological responses as was shown in mice (Destotes, Tedone and Evreux, 1982) then this early development of tolerance may be disturbed, potentially leading to an autoimmune condition. A third possibility may lie in the effect of diazepam on membrane stucture (Breen and Stenchever, 1970). Prenatal exposure may lead to changes in membrane structure in brain which may make the blood-brain barrier more permeable to inflammatory agents. Finally, the possibility exists that the perivascular cuffing observed is a result of exogenous agents. Prenatally exposed animals may be more susceptible than controls to inflammation caused by exogenous agent (virus, bacteria etc.). This hypersensitivity to infections may result from the possible suppressive effect of diazepam on the development of defensive systems, or the pups may have been infected in utero. While normal foetuses are protected through the

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immune system of their mother, pups treated with diazepam would lack this protection if the immune mechanisms of their mother was depressed by the drug. All these possibilites will require further research. Although the mechanisms resnonsible for the inflammatory reaction in the brains of adult rats after prenatal exposure to diazepam is not known, demonstration of the existence of these lesions may have important implications concerning the therapeutic use of diazepam during pregnancy and early development. Acknowledgements-This project was generously supported by the J. Ira and Nicki Harris fund for Research on Learning Disabilities and the Israel Center for Psychobiology-the Charles E. Smith family foundation No. 2183.

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