Patterns of growth deficiency in rats exposed in utero to undernutrition, ethanol, or the neuroteratogen methylazoxymethanol (MAM).

Children and experimental animals exposed to ethanol (EtOH) in utero commonly have low birthweights, and many remain small at maturity. Low body weight or small stature in adulthood may reflect an inability to recover from in utero growth retardation, or it may reflect a separate, postnatal growth deficiency. In this study, daily body weights (postnatal days 1 to 60) were compared among the offspring of the following groups of Long Evans rats: dams fed liquid diet containing 35% EtOH-derived calories; their pair-fed and chow-fed controls; and dams exposed to methylazoxymethanol (MAM) in two previous studies, in which offspring exhibited reduced numbers of growth hormone releasing factor (GRF) neurons. All treatments produced a number of offspring with weight deficits beginning after birth and persisting into maturity. Three distinct patterns of growth deficiency were observed: (1) weight loss relative to controls in the first weeks of life, seen in offspring exposed to EtOH, pair feeding, or MAM on gestation day 13 (G13); (2) a delay in the onset of the prepubertal growth spurt, seen in all EtOH-exposed offspring and in G13 MAM-exposed dwarfs; and (3) failure to sustain the prepubertal growth spurt, seen only after exposure to MAM on G14. The results of this study support the view that prenatal EtOH exposure is capable of affecting postnatal growth specifically; moreover, the pattern of growth deficiency seen in EtOH-exposed offspring was distinct from that of the undernourished offspring of pair-fed dams.

Changes in the reproductive system following acute prenatal exposure to ethanol or methylazoxymethanol in the rat: I. Effects on immunoreactive LHRH cell number.

It has long been recognized that ethanol (EtOH) interferes with the hypothalamo-pituitary-gonad axis in adults of many species, and recent studies have provided evidence for similar effects after prenatal EtOH exposure. Since EtOH is capable of injuring dividing cells, we investigated the possibility that a single acute in utero EtOH exposure during the period of LHRH neuron formation might change the number of immunoreactive LHRH cells in the hypothalamus. Final LHRH cell division in Long-Evans rats was determined by [3H]thymidine autoradiography to take place over a short period between gestation days 12 and 13. Subsequently, pregnant rats were treated acutely with either EtOH or methylazoxymethanol (MAM), a known neuroteratogen, and the numbers of immunoreactive LHRH cells were counted. On gestation day 22, LHRH-positive cell numbers were significantly fewer than control numbers in both EtOH- and MAM-exposed offspring. On postnatal day 60, cell numbers in EtOH-exposed offspring did not differ from control numbers, whereas cells in MAM-exposed offspring remained significantly reduced. In controls, there were 40% fewer LHRH-positive cells on postnatal day 4 than in late gestation or at maturity. We conclude that 1) acute exposure to a high dose of EtOH at a critical time in early gestation can alter the expression of LHRH in late gestation; 2) exposure to MAM in the same period alters LHRH expression before birth and in the adult; and 3) in the early postnatal period, LHRH expression decreases profoundly.

Selective vulnerability and early progression of hippocampal CA1 pyramidal cell degeneration and GFAP-positive astrocyte reactivity in the rat four-vessel occlusion model of transient global ischemia.

Selective, delayed-onset vulnerability of hippocampal CA1 pyramidal cells has been reported as a unique phenomenon in man and the rat four-vessel occlusion (4-VO) model of global ischemia. This has become of great interest for clarification of CA1 pathophysiology and pharmacological intervention after global ischemia. Studies of pathophysiology and pharmacotherapy appear to be impeded by variability in specific criteria and duration of 4-VO ischemia for producing selective CA1 and differential CA1-CA3 damage. The goals of this study were to: (1) develop specific criteria for 4-VO ischemia to ensure selective, bilaterally symmetrical CA1 pyramidal cell damage, (2) examine the effects of 15 min of ischemia on concomitant CA1 cell necrosis and presence of remaining and/or "viable" neurons postischemia, (3) compare 15 and 30 min of ischemia on differential vulnerability of CA1-CA3 subfields, and (4) evaluate the effects of 15 min of ischemia on CA1 pyramidal cell necrosis and glial fibrillary acidic protein (GFAP)-positive astrocyte reactivity in CA1. After 15 min of ischemia, hippocampal pyramidal cell damage was well delineated, with CA1 severely damaged, but leaving CA3 virtually intact. In contrast, 30 min of ischemia produced severe CA1 and less severe CA3 necrosis. Histological evaluations across Days 1, 3, 6, and 14 indicated a significant delayed onset of CA1-CA3 cell necrosis by Day 3. Counting of remaining cells indicated a detectable loss of some large pyramidal neurons even 1 day after ischemia. Compared to controls, there was a differential increase in GFAP-positive astrocytes in CA1-CA3 after ischemia. The results provided quantitative data on the effects of specific 4-VO criteria and durations on: (1) selective CA1 cell necrosis, (2) differential CA1-CA3 cell vulnerability, (3) presence of postischemic remaining and/or viable neurons, and (4) prospect of a "therapeutic window" for pharmacological treatment of CA1 neuronal injury.

The relationship of rat brain weight and pituitary weight to postnatal growth after prenatal exposure to methylazoxymethanol.

Teratogens can affect body weight in various ways, but the association of brain damage with postnatal growth abnormalities suggests a role for neuroendocrine growth-controlling systems. Growth deficiencies follow methylazoxymethanol (MAM) exposure during the period when the growth hormone releasing factor (GRF) cells of the hypothalamus form, and the pattern of growth of the animals is like that of animals deficient in growth hormone. The present studies were designed to examine the growth, body proportions, brain weight, and pituitary weight of animals treated with 20 mg/kg MAM on the 13th day of gestation, a peak period for production of GRF neurons. Among the offspring, this treatment produced about 25% dwarfs (animals smaller than the smallest control of the same sex). Significantly more females than males were categorized as dwarfs. The weight effect occurred long after birth, as is characteristic of animals and humans with growth hormone deficiency. Analyses of weights over the course of development indicated that prenatal factors, rather than factors operating between birth and weaning, predicted the adult body weight of dwarfs, while both sets of factors were significant in other animals. The growth reduction was symmetrical, as would be expected if the animals were growth hormone deficient, with an 18% reduction in weight reflecting a 6% reduction in bone length. The remaining treated animals were similar to controls in absolute weight, body proportions, and rate of growth. Neither pituitary weight nor brain weight appears to play the key role in determining which animals will exhibit growth deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of hypothalamic cell numbers in dwarf and normal weight rats exposed prenatally to methylazoxymethanol (MAM).

Growth deficiencies follow MAM exposure during the period when the growth hormone releasing factor (GRF) cells of the hypothalamus form, while animals exposed slightly later in gestation when the inhibitors of growth hormone release are forming, exhibit giantism. Counts of sample regions of the hypothalamus have shown that rats treated in utero on the 14th day of gestation have reductions in the number of GRF cells, increases in the number of SRIF (somatotropin release inhibiting factor) cells, and alterations of pituitary structure. These effects occurred in all treated subjects, even though obvious effects on body size were present in a small fraction of the treated animals. The present study was designed to examine the effect of 20 mg/kg MAM on the 13th day of gestation (a peak period for production of GRF cells) on GRF and SRIF cell numbers, in a large sample of dwarf-treated rats, normal weight-treated rats, and controls. The results of total counts of hypothalamic cells identified by immunocytochemistry demonstrated significant reductions in GRF cells in both dwarf and normal weight rats exposed to MAM, compared to controls, with no difference between the two treated groups. Like pituitary weights, the neuron counts were significantly correlated with body weight only in dwarf animals. SRIF cell numbers were equivalent to those in controls, suggesting that the increase reported earlier may have been due to a rebound effect in proliferation rather than some response of SRIF cells to GRF cell reduction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prenatal exposure to methylazoxymethanol (MAM) on brain weight, hypothalamic cell number, pituitary structure, and postnatal growth in the rat.

Congenital brain damage syndromes typically are described in terms of behavioral symptoms. Many brain functions are not reflected in behavior, however, and prenatal injury to the developing nervous system could alter these functions, as well. To test the hypothesis that prenatal brain injury can result in postnatal endocrine malfunction, rats were exposed in utero to 20 mg/kg of methylazoxymethanol acetate, a potent neuroteratogen, at two stages of gestation when different sets of growth-controlling neurons of the hypothalamus are forming. The growth hormone releasing factor (GRF) neurons stimulate release of growth hormone from the somatotropes of the anterior pituitary, contributing to rapid growth in the period between weaning and puberty. The somatotropin release inhibiting factor (SRIF) neurons have the opposite effect on the pituitary and can inhibit the GRF cells directly. Growth of treated animals was monitored daily from birth to 40 days and compared to that of controls. Treatment on the 14th day of gestation produced a small number of dwarf animals characterized by normal weight at birth and a sudden decrease in growth rate at the beginning of the fourth postnatal week that led to a body weight about 50% of normal. Treatment on day 16 yielded an acceleration of postnatal growth (significant in males). In each group, most treated animals were like controls in adult size and pattern of growth. As adults, both treatment groups demonstrated massive reductions in brain weight which characterized all the subjects, whether or not they exhibited growth anomalies. The animals treated on day 14 were confirmed to have a significant, selective reduction in growth hormone releasing factor neurons. Reductions were greatest in the middle and posterior levels of the GRF cell distribution, the regions forming most actively at the time of exposure. Unexpectedly, the same group also had increased numbers of periventricular SRIF neurons. Neither type of neurons was significantly altered in the later treatment group. Examination of pituitary structure indicated that dwarfs had very small pituitaries, with an immature pattern of somatotrope distribution, and giants had very large pituitaries, with some hypertrophy of somatotropes. The results suggest that endocrine anomalies which manifest themselves long after birth may originate as birth defects of the nervous system.

Birthdates of the growth hormone releasing factor cells of the rat hypothalamus: an autoradiographic study of immunocytochemically identified neurons.

Growth hormone releasing factor (GRF) neurons in the arcuate nucleus of the hypothalamus and somatostatin (SRIF) neurons in the anterior periventricular region of the hypothalamus act to control the release of growth hormone from the anterior pituitary. To investigate the possibility that the growth-controlling functions of these cells might be compromised by injuries to the developing brain, it is important to know the details of the production and differentiation of these small, specialized cell groups. The overall pattern of cell production in the hypothalamus is known from autoradiographic studies with general nuclear stains, but no data are available on the birthdates (times of final mitoses) of GRF-producing cells. The present study was undertaken to determine when the GRF cells form. Counts of immunocytochemically identified GRF cells labeled on given days were taken from serial coronal sections through the hypothalamus of adult rats labeled on the 10th-17th days of gestation (day of finding a vaginal plug = day 1). As has been shown for the hypothalamus in general, the GRF cells showed a gradient of production from anterior to posterior. The peak of anterior cell proliferation was on day 13, middle cells on day 14, and posterior cells on day 15. These dates are 1 or 2 days earlier than those of GRF-negative cells in the same regions. No lateral to medial gradient of formation was seen in GRF cells. Rather, the laterally placed cells along the base of the brain and those surrounding the ventromedial nucleus formed simultaneously with the GRF cells of the arcuate nucleus. The birthdating results presented here are in agreement with the results of studies of teratogens which suggest that rat postnatal growth is reduced most severely by exposure to neurotoxic agents on days 12 or 13 of gestation. On the basis of data for the whole hypothalamus, such treatments would appear to be too early to interfere with cell production for the arcuate nucleus, but the timing fits the period of vulnerability as defined by the birthdates determined in the present study for the subpopulation of cells destined to produce GRF.

Histological localization of methylmercury in mouse brain and kidney by emulsion autoradiography of 203Hg.

Some investigators have abandoned the use of 203Hg emulsion autoradiography in favor of chemical methods of microscopic localization of mercury. However, recent studies indicate that the latter methods identify only inorganic mercury, or some product of inorganic mercury, making them of little or no value for studies of methylmercury toxicity. Doubts about the use of 203Hg for microscopic localization arose because of the high maximum energy of its emissions and the concern that its latent images might be confounded with silver grains produced by chemical reactions between tissue Hg and the silver supplied by photographic emulsions. Examination of the spectrum of emissions from 203Hg demonstrates that its maximum energy emissions are rare. The mean energy of 203Hg emissions is in the 50-ke V range and the modal emissions are close to 0 ke V, indicating sufficient low energy emissions for autoradiography. In preliminary experiments, methylmercury content of mouse brain was shown to be stable through the steps of tissue processing for plastic sections. A direct comparison of autoradiographic grain counts from tissue treated with "cold" or "hot" methylmercury demonstrated that no grains above background were produced in the absence of nuclear emissions--only "hot" samples affected emulsion. In the kidneys of mice killed 24 hr after dosing, grains were most numerous over cortical tubules and significantly less numerous over glomeruli. In the cerebellum, the molecular layer was significantly more heavily labeled than the granular layer. The number of grains was greatly increased in every region by increasing the specific activity of the methylmercury dosing solution while holding the dose of methylmercury constant. Like the differential effect of "hot" vs "cold" tissue, the differential effect of low vs high specific activity confirms that the grain counts reflect nuclear emissions from the sample tissues, rather than a chemical effect dependent only on mercury content. Grain counts provided a measure of methylmercury content that matched the content measured by atomic absorption (AA). For example, the ratio of kidney/brain content was 32 by AA and 31 by grain counts in one experiment. Thus, 203Hg emulsion autoradiography appears to be a useful approach to localization of methylmercury in tissue sections processed for light microscopy.

Mercury localization in mouse kidney over time: autoradiography versus silver staining.

Several methods of silver staining have been employed to localize mercury in tissue, under the assumption that the techniques represent total Hg, but recent reports have suggested that these stains are specific for a limited fraction of the Hg present in some samples. Magos et al. (1985, Arch. Toxicol. 57, 260-267) hypothesized that the stains actually vary with inorganic mercury content. The purpose of the present study was to compare localization by radiolabeling to localization by one silver stain, the photoemulsion histochemical technique, in tissues prepared to contain a range of levels of total Hg and a range of levels of inorganic Hg. Mice dosed with 8 mg Hg/kg as MeHg were killed 24 hr, 1 week, or 2 weeks after exposure, to allow a decrease in total Hg and an increase in the proportion of demethylated Hg over time. Mice dosed with 4 mg Hg/kg as HgCl2 provided samples in which all the Hg present was in the inorganic form. Atomic absorption of kidneys of mice dosed with MeHg showed that total Hg fell from 55 micrograms/g to 39 to 25 over 2 weeks, while the inorganic fraction climbed from about 2 to 27 to 35%. Grain counts from autoradiographs of 203Hg-labeled sections correlated with total Hg content at +0.88, but silver staining was correlated with inorganic Hg content, appearing only at late termination times in MeHg-exposed animals, but soon after dosing in mice exposed to inorganic Hg. The photoemulsion histochemical technique revealed a substance strictly localized in the proximal tubules, while autoradiographs and grain counts showed total Hg to be present throughout the kidney tissue. These results support the contention that silver stains are selective for inorganic Hg and suggest that the distribution of inorganic Hg, whether introduced experimentally or by gradual demethylation, is different from the distribution of MeHg. If subsequent studies support the association of silver stains with inorganic Hg, it should be possible to localize Hg in histologic sections, distinguishing between organic and inorganic forms, which differ in toxicity.