Ileal nitric oxide formation is altered in the young rat in response to endotoxin: effects of exposure to alcohol in utero.

This study was designed to test the hypothesis that the activity and expression of iNOS in the ileum will be altered in young rats exposed to alcohol in utero (FAE). The subjects, 26-29-day-old rats, were progeny of dams fed an alcohol diet during gestation and their pair-fed/control cohorts. We determined the effects of endotoxin administration on ileal iNOS enzymatic activity and on ileal iNOS immunoreactivity. The basal levels of the measured variables were not significantly different between the groups. In response to LPS, however, FAE animals responded with a greater increase in ileal iNOS activity and immunoreactivity. The findings indicate that ileal iNOS is augmented in response to immune signals, which may partly account for the altered gut functions associated with FAE.

Splenic sympathetic response to endotoxin is blunted in the fetal alcohol-exposed rat: role of nitric oxide.

This study was designed to test the hypothesis that nitric oxide (NO) mediates the blunted splenic sympathetic response to lipopolysaccharide (endotoxin) that occurs in young rats exposed to alcohol in utero (FAE). The subjects, 26-29-day-old rats, were progeny of pregnant dams fed an alcohol diet (35% of the calories were derived from ethanol) or their control and pair-fed (PFC) cohorts. We examined the effects of lipopolysaccharide (LPS) (0.5 mg/kg, i.p.) on splenic norepinephrine (NE) turnover, an index of sympathetic neural activity, splenic inducible NO synthase (iNOS) protein immunoreactivity, and NO metabolites nitrite/nitrate concentrations in plasma. In response to LPS, splenic NE turnover was increased by more than twofold in the PFC groups, but the increase did not occur in their FAE cohorts. The blockade of NOS with L-NAME (30 mg/kg, i.p.) reversed this difference. In both the PFC and FAE rats, basal levels of splenic iNOS protein immunoreactivity were equally barely detected and plasma NO metabolite levels were relatively low (25 microM in both groups). In response to LPS, however, iNOS protein displayed a marked increase in the PFC group and an even greater increase (by close to threefold) in the FAE rats. LPS also substantially increased plasma NO metabolite levels by close to eightfold in the control groups, but by 15-fold in their FAE cohorts compared to the basal levels. These findings support the hypothesis that in the FAE rat, an augmented NO formation accounts for the blunted sympathetic response to endotoxin.

Sympathetic neural response to immune signals involves nitric oxide: effects of exposure to alcohol in utero.

In response to infection, inflammation, or injury, the neural-immune-endocrine networks are activated to restore or maintain stability in the internal environment. Disruption of any one of the functional components may impair the effectiveness of the immune response to challenges, and may consequently jeopardize the wellness of the host. Studies in the author's laboratory have shown that the normal activation of splenic sympathetic neurons in response to the endotoxin lipopolysaccharide, a tool frequently used to mimic infection or inflammation, does not occur in fetal alcohol-exposed (FAE) rats. The sympathetic innervation of lymphoid organs is considered an important immune modulator. Thus, the anomalous splenic sympathetic response may partly account for the impaired immunity associated with FAE. Although the underlying mechanism is far from clear, studies described in this report suggest that nitric oxide (NO), a gaseous free radical, is involved in the altered splenic sympathetic neural response to immune signals. The suggestion is supported by the following findings: (1) blockade of NO synthesis prevented the blunted sympathetic response to lipopolysaccharide or interleukin-1 in FAE rats, and (2) there was a further increase in NO formation in response to lipopolysaccharide in the FAE rats compared to their control cohorts. This was demonstrated by an augmented increase in the inducible NO synthase immunoreactivity in the spleen as well as in circulating levels of NO metabolites. It is suggested, therefore, that the altered splenic sympathetic response to immune signals involves excessive formation of NO that may account, at least in part, for the impaired immunity associated with FAE.

Exposure to alcohol in utero blunts splenic sympathetic neural response to endotoxin and interleukin-1beta in the rat.

Systemic administration of endotoxin (LPS) or interleukin-1beta (IL-1) to prepubertal rats induced a marked increase in splenic but not cardiac norepinephrine (NE) turnover, an index of sympathetic neural activity. In contrast, the splenic neural response was blunted in their fetal alcohol-exposed (FAE) cohorts. Because the sympathetic outflow to lymphoid organs is considered an important immune modulator, the anomalous neural response to immune signals may partly account for the impaired cellular immunity and, thus, for the increased susceptibility to infections associated with FAE.

Effects of nerve growth factor on splenic norepinephrine and pineal N-acetyl-transferase in neonate rats exposed to alcohol in utero: neuroimmune correlates.

Prenatal alcohol exposure (FAE) has been associated with multiple anomalies, including a selective developmental delay of sympathetic innervation in lymphoid organs. Sympathetic neurons require nerve growth factor (NGF) for their development and maintenance, and recent evidence has suggested that alcohol impairs the synthesis and/or biological activity of NGF in selected central and peripheral neurons. Thus, the present study examined the hypothesis that NGF administration to FAE rats during early postnatal development would reverse some of the peripheral sympathetic deficits. Neonate rats, FAE and the corresponding control cohorts, received daily treatments of NGF or cytochrome C (0.3 mg/kg; s.c.) for various time intervals, and were killed 24 hr or 10 days after the last treatment. The measured parameters included norepinephrine (NE) concentrations in the spleen and heart, which receive nor-adrenergic innervation from the coeliac ganglion and the superior cervical ganglion (SCG), respectively. In addition, we measured the activity of pineal N-acetyltransferase (NAT), the rate-limiting enzyme of melatonin biosynthesis, which depends on sympathetic innervation from the SCG. The data show that chronic, but not acute, NGF treatments reversed the FAE-related deficits in splenic NE concentrations as well as in pineal NAT activity in a time- and age-dependent manner. Sympathetic neurons play an important role in immune modulation. Thus, the altered splenic NE levels and pineal NAT activity may play a role in immune deficits associated with exposure to alcohol in utero.

Ferritin effect on the transverse relaxation of water: NMR microscopy at 9.4 T.

Accumulation of ferritin, the iron storage protein, has been linked recently to aging and a number of pathologies. Noninvasive detection of iron storage by MRI relies on its extremely strong effect on water relaxation. The aim of this article is to characterize the effect of ferritin on transverse water relaxation in a high magnetic field, using an imaging Carr-Purcell Meiboom-Gill (CPMG) preparation sequence. Ferritin-induced water relaxation showed quadratic dependence on the iron loading factor, implying a paramagnetic mechanism. However, an additional zero order term was found, that could be due to the initial stages of the iron core loading. Significant enhancement of ferritin contrast was obtained at very short Tau CPMG durations. This approach for enhancing ferritin contrast was demonstrated by NMR microscopy of ferritin-injected Xenopus oocytes, thus showing the feasibility of ferritin detection in a high magnetic field, even in systems with short transverse relaxation.

Prenatal alcohol exposure selectively suppresses cell-mediated but not humoral immune responsiveness.

The present study examined effects of fetal alcohol exposure (FAE) on the ability of peripubertal male and female rats to mount a humoral immune response against T-cell-dependent as well as independent antigens. The appropriate pair-fed (PF) and control (C) cohort rats were included. Serum immunoglobulins (Ig) levels were determined following a primary or secondary immune response. In addition, plasma corticosterone levels were measured in conscious, freely moving FAE, PF and C rats following sensitization with the T-cell-dependent antigen sheep red blood cells (SRBC). The study demonstrates that, in response to primary or secondary immunization, serum Ig levels in FAE rats were not significantly different from those in the PF or C cohorts. On the other hand, a marked reduction in mitogen-induced T-cell proliferative response was observed in FAE male rats in the same age group. Plasma corticosterone concentrations were increased almost four-fold 7 days after the primary immunization with SRBC, but there were no significant differences among the FAE, PF or C groups. Taken together, evidence from in vivo and in vitro studies indicates that FAE is associated with a selective impairment of cell-mediated immune function, including mitogen-induced T-cell proliferation, graft versus host as well as contact sensitization responses, but not of humoral immune function.

Systemic interleukin-1 induces early and late patterns of c-fos mRNA expression in brain.

This study was designed to examine the mechanisms by which systemic interleukin-1 affects neuroendocrine systems in the brain. Intraperitoneal injections of interleukin-1 beta (1.25 micrograms/rat) were administered to rats. One or three hours after injection, the expression levels of the immediate-early gene c-fos and of genes for several neuropeptides, receptors, and enzymes were examined by in situ hybridization histochemistry. In the brainstem at 1 hr, c-fos mRNA was elevated in the area postrema and nucleus of the solitary tract, but not in the locus coeruleus. At 3 hr, the c-fos mRNA levels had increased further in the nucleus of the solitary tract. Rostrally, elevations in c-fos mRNA levels were found in the hypothalamic and thalamic paraventricular nuclei, central nucleus of amygdala, bed nucleus of the stria terminalis, and medial preoptic area, peaking at 1 hr and diminishing at 3 hr. In addition, at 3 hr a new pattern of c-fos activity emerged--the arcuate nucleus and cells at the external margins throughout the brain now expressed c-fos mRNA. Corticotropin-releasing hormone mRNA levels were doubled in the paraventricular nucleus at 1 and 3 hr, concomitant with elevations in plasma adrenocorticotrophic hormone (ACTH) and corticosterone. Tyrosine hydroxylase mRNA levels in the brainstem did not change. The c-fos mRNA induction patterns reveal a temporally dynamic response to interleukin-1 administration. We propose that the early set of structures responding to interleukin-1 initiates the neuroendocrine response to cytokines. Coactivation of the area postrema and nucleus of the solitary tract may reflect entry into the brain and neural transduction of the peripheral signal. The late set--including the nucleus of the solitary tract, arcuate nucleus, and the brain's edge--may reflect cellular activation along the diffusion routes traveled by interleukin-1 or a bioactive transduction product, because the pattern of edge labeling is similar to the autoradiographic pattern of flow lf radiolabeled tracer substances in the cerebrospinal fluid. The late c-fos mRNA response to interleukin-1, therefore, may represent a demonstration of information transfer in the parasynaptic mode, also known as volume transmission.

Fetal alcohol exposure and adult tumorigenesis.

Adult rats exposed to prenatal alcohol were evaluated for their susceptibility to either hormone- or chemical-inducing tumors. In the first study, rats exposed to prenatal alcohol displayed an increased propensity to beta-estradiol (E2)-induced adenohypophyseal prolactinoma. The susceptibility was manifest as a potentiated increase in anterior pituitary weight as well as in serum prolactin levels after 1 and 3 weeks but not 5 weeks of hormone treatment. Two weeks after withdrawing the E2-implant, the prolactinoma underwent involution and serum prolactin reversed to baseline levels. The high concentrations of serum corticosterone were also reduced but did not return to baseline levels after E2 removal. In the second study, nitrosomethylbenzylamine (NMBA) was utilized to induce esophageal cancer in adult rats. There were no significant differences in tumor incidence or size between the prenatal alcohol-exposed and the pair-fed cohorts. However, the NMBA-treated prenatal alcohol-exposed rats displayed a marked decrease in thymus: body wt ratio as well as adrenal gland hyperplasia. The results suggest that no single mechanism can account for the variable susceptibility displayed by the prenatal alcohol-exposed rats to chemical carcinogens. Some of the observed changes, however, may be attributable to the long-lasting adverse effects of prenatal alcohol exposure on the well-being of the adult host.

Maternal and paternal alcohol use: effects on the immune system of the offspring.

There is no single mechanism which can account for such a complex biological phenomenon as immune regulation, nor is it clear how alcohol teratogenicity exerts its multiple adversive effects, including lasting immune deficits. Much of the research aimed at unravelling effects of pre- or early postnatal alcohol exposure on the organism's defense mechanisms and long-term health risks has been phenomenological. A better understanding of mechanisms which underlie alcohol effects on immune competency will require integrated studies of the neuro-immune-endocrine networks.

Prenatal alcohol exposure alters the development of sympathetic synaptic components and of nerve growth factor receptor expression selectivity in lymphoid organs.

Exposure to alcohol in utero has been associated with long-term immune deficits. In addition, adult mice exposed to alcohol prenatally display altered noradrenergic synaptic transmission selectively in lymphoid organs. This is consistent with the hypothesis that sympathetic neurons play an important role in immunomodulation. The development and maintenance of sympathetic neurons are critically dependent on nerve growth factor (NGF). Furthermore, NGF has been shown to modulate immune responses and NGF receptor expression has been localized to lymphoid organs. The present work examined the effects of prenatal alcohol exposure on the development and maturation of pre- and postsynaptic sympathetic components, including norepinephrine and beta-adrenoceptors, respectively, as well as the early expression of NGF receptors in lymphoid and other organs of the C57BL/6 mouse. Infant mice that were exposed to alcohol in utero displayed reduced levels of norepinephrine and beta-adrenoceptor density, as well as increased NGF receptor expression in the thymus and spleen, but not the heart. These selective changes may account, in part, for the persistent immune incompetence characteristic of fetal alcohol syndrome.

Developmental delays associated with prenatal alcohol exposure are reversed by thyroid hormone treatment.

Exposure to alcohol in utero has been associated with hypothyroidism and a variety of developmental defects characteristic of thyroid dysfunction. The present work examined whether these abnormalities could be reversed in infant rats treated with thyroid hormones. Subjects were offspring of dams which were on the following diet regimen during gestation: (1) free access to liquid diet containing ethanol (alcohol pups); (2) an equal volume of isocaloric liquid diet (pair-fed pups); or (3) ad libitum control diet (control pups). Neonates from each group were foster-nursed by control dams, and received triiodothyronine (T3; 0.1 mg/kg/day; s.c.) or saline treatments on postnatal days 1 to 10. The alcohol neonates displayed reduced serum thyroxine which was restored to normal by postnatal day 14. In addition, these pups showed a delayed appearance of developmental landmarks, including righting reflex, dental eruption, auditory startle response and eye opening. The retarded incisor eruption and eye opening were reversed in alcohol pups by T3 treatments. The present data suggest that at least some of the developmental abnormalities associated with prenatal alcohol exposure are attributable to perinatal hypothyroidism and can be restored by early hormone replacement therapy.

Lactational alcohol exposure elicits long-term immune deficits and increased noradrenergic synaptic transmission in lymphoid organs.

Increasing evidence suggests that the sympathetic nervous system plays an important role in immunomodulation. While chronic alcohol consumption has been associated with immune deficits, the effects of exposure to alcohol during early postnatal life on subsequent immunocompetence and activity of sympathetic neurons in lymphoid organs are not known. This study examined the long-term effects of lactational alcohol consumption on cellular immune responses and noradrenergic synaptic transmission in lymphoid and other organs of the young adult C57BL/6 mouse. The data show that exposure to alcohol via the mother's milk was associated with long-term deficits in cellular immunity, including suppression of the local graft vs host and contact hypersensitivity responses. The animals also displayed enhanced noradrenergic synaptic transmission and decreased beta-adrenoceptor density selectively in lymphoid organs. These neuroimmune changes are particularly striking since body weight-gain of the suckling pups was normal and their blood alcohol concentration was considerably lower than that of the alcohol-consuming dam. This suggests an increased sensitivity of the nascent immune and nervous systems during the critical period of early postnatal development.

Prenatal ethanol exposure alters immune capacity and noradrenergic synaptic transmission in lymphoid organs of the adult mouse.

Clinical and experimental evidence indicates that exposure to alcohol in utero is associated with altered immune capacity. The mechanisms underlying such abnormalities are not clear. However, the suggestion that reciprocal interactions between the immune and the nervous systems are necessary for a competent immune response may be relevant. This work examined the consequences of prenatal ethanol exposure on cellular immune responses and noradrenergic synaptic transmission in lymphoid organs of the adult C57B1/6 mouse. Pregnant mice were fed a liquid diet containing 25% of the calories as ethanol (4.8% w/v) or pair-fed an isocaloric equivalent of this diet throughout gestation, followed by foster-nursing the neonates on normal dams. As young adults, mice exposed to ethanol prenatally displayed immunologic and selective neurochemical changes: (1) depressed ability to produce cellular immune responses, including contact hypersensitivity and a local graft-vs-host response, and (2) altered noradrenergic synaptic transmission, including enhanced norepinephrine turnover, and a reduction in norepinephrine levels and beta-adrenoceptor density in the thymus and spleen, but not the heart. However, both the integrity and compartmentation of noradrenergic nerve fibres in the spleen were intact. It is suggested that altered noradrenergic synaptic transmission selectively in lymphoid organs may contribute to the impaired immune capacity associated with fetal alcohol exposure.

Prenatal ethanol exposure impairs lesion-induced plasticity in a dopaminergic synapse after maturity.

This study examined the consequences of alcohol (ethanol) exposure during fetal life on lesion-induced dopaminergic synapse responsiveness (plasticity) in the olfactory tubercle of the adult rat. Normally, in the olfactory tubercle, olfactory bulbectomy elicits alterations in pre- and postsynaptic dopaminergic markers, including, respectively, (1) increased tyrosine hydroxylase activity and immunoreactivity, which is associated with dopaminergic axon sprouting, and (2) increased dopaminergic receptor density and potentiated dopamine activation of adenylate cyclase. We have utilized biochemical and quantitative immunocytochemical methodology to examine these synaptic markers in olfactory bulbectomized or sham-operated adult rats. These animals were offspring of dams which were administered one of the following diets during pregnancy: (1) liquid diet containing 35% ethanol-derived calories ad libitum; (2) liquid diet containing an isocaloric amount of maltose-dextrin instead of ethanol, pair-fed; or (3) unaltered liquid diet ad libitum. The results show that prenatal alcohol exposure leads to suppression of the lesion-elicited dopaminergic synapse responsiveness in the olfactory tubercle. There were no significant differences between offspring born to control and pair-fed animals, indicating that the observed abnormalities were not due to alterations in their nutritional status. In conclusion, the present data are a biochemical and quantitative immunocytochemical demonstration of impaired lesion-induced synaptic responsiveness. This renders a new dimension in support of previous evidence indicating that prenatal alcohol exposure leads to altered neuroanatomical, neuroendocrinological and behavioral responsiveness to various challenges. Such impaired synaptic responsiveness may underlie brain functional abnormalities characteristic of fetal alcohol syndrome.

Chronic exposure to random restraint stress retards the development of estrogen-induced pituitary prolactinoma in rats.

The effects of stress on carcinogenesis have been equivocal. The present work examined the influence of an unpredictable pattern of chronic restraint stress on estradiol-induced pituitary hyperplasia in male Fischer 344 rats. The animals were grouped as follows: (1) control, (2) stressed, (3) estradiol (31 mg), subcutaneous implant for 40 days and (4) estradiol with stress, 1 h daily, randomly, for 40 days. The pituitaries increased markedly in mass in groups 3 and 4 compared to the first two groups. However, the gland weight in rats exposed to estradiol plus stress was significantly (P less than 0.04) reduced as compared to estradiol per se. Prolactin levels increased due to estradiol treatment and decreased following exposure to stress. Weights of adrenal glands increased as a result of estradiol treatment when compared to those in stressed or untreated animals. This adrenal hypertrophy correlated well with plasma corticosterone levels. The data demonstrate that chronic immobilization stress retarded estradiol-induced pituitary prolactinoma. Modulation of the immune response by stress-related factors may play a role in the inhibition of tumorigenesis.

Deafferentation elicits a transient decrease in Na+, K+-ATPase activity and ouabain binding in the olfactory tubercle.

This work examines the effects of olfactory bulbectomy on Na+, K+-ATPase activity and ouabain binding in the olfactory tubercle. The activity and number of enzyme sites were reduced significantly in olfactory tubercle, but not in corpus striatum or hippocampus, 14 and 21 days after bulbectomy. Enzyme activity and ouabain binding returned to normal by 42 days after the lesions. The time of the reduction in Na+, K+-ATPase coincides with that observed earlier for dopaminergic sprouting and increased dopamine-sensitive adenylate cyclase activity.

Perinatal, not adult, hypothyroidism suppresses dopaminergic axon sprouting in the deafferented olfactory tubercle of adult rat.

We reported recently that chronic thyroid deficiency in rat, beginning in utero and terminating after maturity, suppresses lesion-induced central catecholaminergic axon sprouting in the adult brain [Gottesfeld et al, 1985]. The present work was undertaken to define the critical period of hypothyroidism on subsequent neuronal sprouting. Thyroid hormones deficiency was induced in rats by methimazole during (a) gestational days 8-21 (20 mg/kg/day in the drinking water); (b) postnatal days 1-15 (0.2 or 0.4 mg/pup/day; i.p.), or (c) in the mature animal for 4 weeks (20 mg/kg/day in the drinking water). The olfactory tubercles (OTs) were used as a model to study sprouting of dopaminergic (DA) nerve terminals, elicited by olfactory bulbectomy. Animals in each group received lesions or sham operations as adults, and sacrificed 3 weeks after the operation. Thus, for each of the above treatments four subgroups were formed: (a) euthyroid/sham-operation, (b) euthyroid/lesion, (c) hypothyroid/sham-operation, and (d) hypothyroid/lesion. Sprouting of DA axon terminals in the OTs was identified by biochemical assays and quantitative immunofluorescent microscopy, using tyrosine hydroxylase (TH) as a marker. Serum thyroxine levels served as an index of the thyroid status. The results demonstrate that lesion-induced sprouting of DA axon terminals in OTs of adult rats is suppressed by hypothyroidism induced prenatally or during the early postnatal period, but not after maturity. Thus, there is a perinatal critical period during which altered thyroid function exerts long-term effects on neuronal plasticity.

Restraint stress during late pregnancy in rats elicits early hypermyelination in the offspring.

Female rats were subjected to a regular, daily schedule of 2 hr of restraint stress during the final 6 days of pregnancy. During the first 2 postnatal weeks, adrenal weights were greater than normal in the offspring of the stressed dams. The concentration of brain myelin was higher than control at 14 and 21 days of age but similar to normal by day 40. Early hypermyelination may be partly responsible for early motor development, as previously observed in prenatally stressed rats.