Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Pro-inflammatory disequilibrium of the IL-1 beta/IL-1ra ratio in an experimental model of perinatal brain damages induced by lipopolysaccharide and hypoxia-ischemia

Bacterial infections and hypoxia/ischemia (H/I) are implicated in human neonatal brain damage leading to cerebral palsy (CP). We developed an animal model presenting similar perinatal brain damage by combining bacterial endotoxin and H/I insults. Interleukin (IL)-1beta is a mediator of brain damage and its action(s) is counteracted by its cognate anti-inflammatory IL-1 receptor antagonist (IL-1ra). We tested the hypothesis that the balance between agonist and antagonist in the IL-1 system is shifted towards inflammation in perinatal brains exposed to endotoxin and/or H/I. Lipopolysaccharide (LPS) and/or H/I enhanced both intracerebral IL-1beta mRNA and protein levels, with a maximum increase observed with combined LPS and H/I insults. Conversely, IL-1ra expression was significantly downregulated by LPS, H/I, or both combined, with a maximum magnitude of imbalance between IL-1beta and sIL-1ra noticed with the double hit. The nuclear factor (NF)kappaB component of the signaling pathway activated by IL-1beta-binding to its receptor was activated following exposure to LPS and/or H/I. We show for the first time that, perinatally, bacterial products, H/I, or both combined, induce downregulation in sIL-1ra expression concomitant with upregulation in IL-1beta. The resulting pro-inflammatory orientation in the IL-1/IL-1ra balance might play a role in the initiation of perinatal brain damages.     

Microtubule dynamics in a cytosolic extract of fetal rat brain

Brain microtubule dynamics were studied by video-enhanced differential interference contrast microscopy in a cytosolic extract from fetal rat brain, prepared under conditions designed to produce minimal alterations in microtubule stability. With urchin sperm axoneme fragments as nucleation seeds, the extract was shown to support cellular-like microtubule dynamics. Microtubules elongated from one end of the axonemes, and did not spontaneously self-assemble in the absence of axonemes. The following microtubule kinetic parameters were measured in the extract: velocity of elongation (8.1 mm/min), velocity of rapid shortening (5.8 mm/min), catastrophe frequency (0.17 min-1), and rescue frequency (1 min-1). These parameters were in close agreement with reported values for growth cones of living neurons. Microtubule properties in the fetal brain extract were shown to be affected by agents with known effects on the cytoskeleton. pp60c-src, a tyrosine kinase important in cell adhesion molecule-dependent axon growth, caused small increases in the frequency of microtubule catastrophe (0.31 min-1) and rescue (2 min-1) without changing the velocities of elongation or rapid-shortening. Although pp60c-src phosphorylated purified porcine brain tubulin in vitro, it did not elicit significant changes in its polymerization properties, suggesting that other cytoskeletal proteins in the brain extract are involved in modulating microtubule dynamics. The cytosolic extract of fetal rat brain provides a useful system for studying regulation of microtubule assembly in neuronal growth cones.     

Effect of hyperbaric oxygen on apoptosis in neonatal hypoxia-ischemia rat model.

We have previously demonstrated that a transient exposure to hyperbaric oxygen (HBO) attenuated the neuronal injury after neonatal hypoxia-ischemia. This study was undertaken to determine whether HBO offers this neuroprotection by reducing apoptosis in injured brain tissue. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% oxygen). Apoptotic cell death was examined in the injured cortex and hippocampus tissue. Caspase-3 expression and activity increased at 18 and 24 h after the hypoxia-ischemia insult. At 18-48 h, poly(ADP-ribose) polymerase (PARP) cleavage occurred, which reduced the band at 116 kDa and enhanced the band at 85 kDa. There was a time-dependent increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells. A single HBO treatment (100% oxygen, 3 ATA for 1 h) 1 h after hypoxia reduced the enhanced caspase-3 expression and activity, attenuated the PARP cleavage, and decreased the number of TUNEL-positive cells observed in the cortex and hippocampus. These results suggest that the neuroprotective effect of HBO is at least partially mediated by the reduction of apoptosis.     

Lactate as an oxidizable substrate for rat brain in vitro during the perinatal period.

Foetal brain slices showed a high capacity for lactate oxidation in vitro during late gestation. This capacity remained high during the very early postnatal period, suggesting that lactate may play an important role as an energy substrate in the brain during the early neonatal period. The capacity for lactate oxidation decreased markedly during the first 2 days of extra-uterine life and thereafter remained low.     

Oxytocin receptor in human fetal membranes at term and during labor

Human fetal membranes, taken from 30 patients submitted to caesarean section during the final stages of gestation and labor, were examined in order to evaluate the presence and characteristics of the oxytocin receptor. The presence of oxytocin receptors in human fetal membranes, both in the amnion and in the chorion-decidua, was demonstrated in this study. The receptor binding to oxytocin showed a significant increase during early and advanced labor compared with before the onset of labor. When the pre-labor level was taken as the normalized form (control = 100) the increase with respect to the control (10 cases) for the amnion in early labor (2.27 times +/- 0.11, mean +/- SEM, P less than 0.001, 10 cases) and in advanced labor (2.53 times +/- 0.15, 10 cases, P less than 0.001) was highly significant. In the chorion-decidua the increase was 1.61 times +/- 0.09, P less than 0.001 in early labor and 1.66 times +/- 0.19, P less than 0.001 in advanced labor. Scatchard analysis showed a single receptor site for oxytocin in amnion and chorion decidua. The dissociation constant (Kd) did not change during the various stages of labor; the mean values found were 0.228 +/- 0.02 (mean +/- SEM) nM in the amnion and 0.193 +/- 0.03 nM in the chorion-decidua respectively. These findings suggest that human fetal membranes are target organs for oxytocin and that they might play a role in the onset of labor through an increase of receptor binding.     

Insulin-like growth factors and binding proteins in the fetal rat: Alterations during maternal starvation and effects in fetal brain cell culture

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction.Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [¹²⁵I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity. Conversely, addition of physiological amounts of rat IGFBP-1 inhibited the effects of fed fetal plasma on brain cell growth, and bioactivity was reduced even further with higher concentrations of IGFBP-1. Based on these results, we conclude that reciprocal changes in circulating levels of IGFBP-1 (increased) and IGF-I (decreased) may combine to reduce the availability of IGF-I to this tissue and limit fetal brain cell growth when maternal nutrition is impaired.     

Morphine formation from codeine in rat brain: a possible mechanism of codeine analgesia

The O-demethylation of codeine to morphine was demonstrated in rat brain homogenate. Maximal formation occurred at 10 minutes, with a Vmax of 5.93 +/- 0.16 nmol/g brain/h and Km of 37.82 +/- 4.99 microM. The formation was significantly (P less than 0.05) greater in the microvessel-rich brain fraction. Intraperitoneal injection of codeine in the rat resulted in brain concentrations of morphine which could not be solely attributed to transfer of morphine from the blood stream across the blood-brain barrier. Morphine formed in the brain after codeine administration may be an important mechanism for codeine-induced analgesia.     

Immunochemical comparison of estradiol-binding molecules in perinatal rat brain cytosol and serum.

Estradiol-binding macromolecules in fetoneonatal rat brain cytosol and serum were compared by immunochemical techniques. When treated by a double diffusion procedure, both cytosol and serum formed precipitin lines with rabbit antiserum specific for perinatal rat serum proteins. These lines fused completely, indicating, within the limits of detection of this particular antiserum, the presence of identical antigenic determinants in the brain and serum. Prior removal of immunoprecipitable material from cytosol or serum, after incubation with the specific antiserum, prevented formation of such precipitin lines. The procedure similarly presented specific estradiol-binding to macromolecules. It was therefore concluded that the specifically perinatal, antigenically similar, components in rat brain cytosol and serum (possibly representing alphafetoprotein) are responsible for the estradiol-binding activity in these two tissue compartments. Measurements of heme concentrations indicated that the alphafetoprotein-like material in the cytosol does not reflect blood contamination, but rather a separate population of similar or identical molecules.     

Expression of peroxiredoxin 1, 2, and 6 in the rat brain during perinatal development and in response to dexamethasone

Peroxiredoxins (Prdx), a family of antioxidant proteins, have important defensive roles in the degenerative brain diseases and neuronal cell death in adult subjects. However, little is known in the neonatal brain. Here, we studied the developmental expression of Prdxs and their response to dexamethasone in the perinatal rat brain. Prdx 1 expression increased during late gestations and peaked at postnatal-day 1, when its expression gradually decreased. Prdx 2 expression remained largely unchanged. Prdx 6 expression continually increased as growing. Using immunohistochemistry, each Prdx showed a strong expression in the cerebral cortex and hippocampus. Prdx 1 was strongly expressed in the corpus callosum. The dexamethasone injection increased the expression of Prdx 6. In conclusion, we reveal for the first time that Prdx 1, 2 and 6 are found in abundance in the perinatal rat brain and are differentially expressed during development. The expression of Prdx 6 was affected by dexamethasone treatment.     

Alterations in liver chromatin during perinatal development of the rat

Chromatins were isolated from liver nuclei of 19-day fetuses, 2-, 5-, 21-day old and adult rats. Very little variation was observed in the mass ratio of total histones to DNA or in the spectrum of histones as determined by polyacrylamide gel electrophoresis. On the other hand, the amount and banding pattern of acidic proteins indicated pronounced changes during liver development.The composition of acidic proteins may be specific for the stage of development as evidenced immunochemically. Antibody against acidic protein-DNA complexes from adult rat liver were produced in rabbits. Whereas adult liver acidic protein-DNA complexes interacted strongly with the antibody, fetal liver preparations showed very little affinity. Complexes from 2-day-old animals reacted more strongly than fetal complexes while preparations from 5-day-old and 21-day-old displayed further increases in affinity. The results support the idea that chromatin acidic proteins play an important role in genetic expression during the ontogeny.     

The catecholamine system in rat fetal brain with a disrupted corticosteroid balance]

The disturbance of corticosteroids balance of female rats on the 16 and 18 days of pregnancy by injections of exogenous corticosterone or methopyrone--blocker of endogenous hormone formation--decreased both body weight and activity of the rate-limiting catecholamine synthesising enzyme--tyrosine hydroxylase (TH) in the stem half of the 21 day fetal brain. Concomitantly with inhibitory action, which may be caused by general retardation of the organism development, corticosteroids stimulated TH activity during prenatal ontogenesis. Fetuses developed under elevated corticosteroid level had lower body weight but higher TH activity in comparison with fetuses endured the deficit of these hormones. Besides, corticosterone injection to the females on the 20th day of gestation increased in 6 hours TH activity in stem half of their fetus brain. The data obtained suggested the prominent role of corticosteroids in the prenatal development of brain catecholaminergic system.     

Changes in blood flow distribution during the perinatal period in fetal sheep and lambs.

We have measured total blood flows and blood flows per 100 g tissue to major tissues at 120 and 140 days gestation in fetal sheep and at 3 and 21 days of age in lambs (gestation period = 144 +/- 2 days). Between 120 and 140 days gestation, flow per 100 g tissue increased by 74, 150, and 317% in the renal, intestinal, and hepatic arterial beds, but no further significant change in flow was observed at 3 or 21 days postpartum. Blood flows per 100 g to cerebral hemispheres and cerebellar tissues also increased dramatically during late gestation (142 and 121%, respectively), but declined sharply by 3 days postpartum (73 and 75%, respectively). Brain blood flows at 21 days postpartum remained substantially below late gestational levels. Adrenal blood flows per 100 g more than doubled during late gestation, fell by more than half at birth, and only partially recovered by 21 days of age. Blood flows to carcass tissues did not change in late gestation, fell at birth, then partially recovered. Pre- and post-natal increases in brain blood flows were almost entirely attributable to increased perfusion rather than tissue growth, whereas large perinatal increases in flow to the diaphragm paralleled tissue growth. Tissue growth and increased perfusion per 100 g contributed almost equally to increased blood flows to kidneys postnatally, and to adrenal glands and the gastrointestinal tract prenatally.