Changes in motor behavior during pregnancy in rats: the basis for a possible animal model of restless legs syndrome

PURPOSE:Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS – sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features.METHODS:We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation.RESULTS:There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05).CONCLUSION:These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder.

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca2+-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation

Malaria vaccine: roadblocks and possible solutions

Malaria remains the most prevalent and devastating parasitic disease worldwide. Vaccination is considered to be an approach that will complement other strategies for prevention and control of the disease in the future. In the last 10 years, intense studies aimed at the development of a malaria vaccine have provided important knowledge of the nature of the host immunological mechanisms of protection and their respective target antigens. It became well established that protective immune responses can be generated against the distinct stages of Plasmodium. However, in general, protective immune responses are directed at stage-specific antigens. The elucidation of the primary structure of these antigens made possible the generation of synthetic and recombinant proteins that are being extensively used in experimental immunizations against the infection. Today, several epitopes of limited polymorphism have been described and protective immunity can be generated by immunization with them. These epitopes are being tested as primary candidates for a subunit vaccine against malaria. Here we critically review the major roadblocks for the development of a malaria vaccine and provide some insight on how these problems are being solved

Possible involvement of A1 receptors in the inhibition of gonadotropin secretion induced by adenosine in rat hemipituitaries in vitro

We investigated the participation of A1 or A2 receptors in the gonadotrope and their role in the regulation of LH and FSH secretion in adult rat hemipituitary preparations, using adenosine analogues. A dose-dependent inhibition of LH and FSH secretion was observed after the administration of graded doses of the R-isomer of phenylisopropyladenosine (R-PIA; 1 nM, 10 nM, 100 nM, 1 µM and 10 µM). The effect of R-PIA (10 nM) was blocked by the addition of 8-cyclopentyltheophylline (CPT), a selective A1 adenosine receptor antagonist, at the dose of 1 µM. The addition of an A2 receptor-specific agonist, 5-N-methylcarboxamidoadenosine (MECA), at the doses of 1 nM to 1 µM had no significant effect on LH or FSH secretion, suggesting the absence of this receptor subtype in the gonadotrope. However, a sharp inhibition of the basal secretion of these gonadotropins was observed after the administration of 10 µM MECA. This effect mimicked the inhibition induced by R-PIA, supporting the hypothesis of the presence of A1 receptors in the gonadotrope. R-PIA (1 nM to 1 µM) also inhibited the secretion of LH and FSH induced by phospholipase C (0.5 IU/ml) in a dose-dependent manner. These results suggest the presence of A1 receptors and the absence of A2 receptors in the gonadotrope. It is possible that the inhibition of LH and FSH secretion resulting from the activation of A1 receptors may have occurred independently of the increase in membrane phosphoinositide synthesis.

Gap junctions in cells of the immune system: structure, regulation and possible functional roles

Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

Fighting by sleep-deprived rats as a possible manifestation of panic: effects of sodium lactate

Increased fighting is an effect of desynchronized sleep deprivation (DSD) in rats, and recently this behavior has been suggested to be spontaneous panic and equivalent to panic disorder. In the present study we tested this hypothesis by evaluating the effect of sodium lactate on this aggressiveness, because this substance is recognized to induce spontaneous panic attacks in patients. A total of 186 male albino Wistar rats, 250-350 g, 90-120 days of age, were submitted to DSD (multiple platform method) for 0, 4, or 5 days. At the end of the deprivation period the rats were divided into subgroups respectively injected intraperitoneally with 1.86, 2.98 and 3.72 g/kg of 1 M sodium lactate, or 1.86 and 3.72 g/kg of 2 M sodium lactate. The control animals were submitted to the same procedures but received equivalent injections of sodium chloride. Regardless of DSD time, sleep-deprived animals that received sodium lactate presented a significantly higher mean number of fights (0.13 ± 0.02 fights/min) and a longer mean time spent in confrontation (2.43 ± 0.66 s/min) than the controls (0.01 ± 0.006 fights/min and 0.12 ± 0.07 s/min, respectively; P<0.01, Student t-test). For the sodium lactate group, concentration of the solution and time of deprivation increased the number of fights, with the mean number of fights and mean duration of fighting episodes being greater with the 2.98 g/kg dose using 1 M lactate concentration. These results support the hypothesis that fighting induced by DSD is probably a spontaneous panic manifestation. However, additional investigations are necessary in order to accept this as a promising animal model for studies on panic disorder.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body.

Possible etiologies for tropical spastic paraparesis and human T lymphotropic virus I-associated myelopathy

The epidemiology of tropical spastic paraparesis/human T lymphotropic virus I (HTLV-I)-associated myelopathy (TSP/HAM) is frequently inconsistent and suggests environmental factors in the etiology of these syndromes. The neuropathology corresponds to a toxometabolic or autoimmune process and possibly not to a viral disease. Some logical hypotheses about the etiology and physiopathology of TSP and HAM are proposed. Glutamate-mediated excitotoxicity, central distal axonopathies, cassava, lathyrism and cycad toxicity may explain most cases of TSP. The damage caused to astrocytes and to the blood-brain barrier by HTLV-I plus xenobiotics may explain most cases of HAM. Analysis of the HTLV-I/xenobiotic ratio clarifies most of the paradoxical epidemiology of TSP and HAM. Modern neurotoxicology, neuroimmunology and molecular biology may explain the neuropathology of TSP and HAM. It is quite possible that there are other xenobiotics implicated in the etiology of some TSP/HAMs. The prevention of these syndromes appears to be possible today.

Ionic radiocontrast inhibits endothelium-dependent vasodilation of the canine renal artery in vitro: possible mechanism of renal failure following contrast medium infusion

To determine if radiocontrast impairs vascular relaxation of the renal artery, segments (4-5 mm in length) of canine renal artery were suspended in vitro in organ chambers to measure isometric force (95% O2/5% CO2, at 37ºC). Arterial segments with and without endothelium were placed at the optimal point of their length-tension relation and incubated with 10 µM indomethacin to prevent synthesis of endogenous prostanoids. The presence of nonionic radiocontrast (iohexol, Omnipaque 350, 1 ml in 25 ml control solution, 4% (v/v)) did not alter endothelium-dependent relaxation to acetylcholine in rings precontracted with both norepinephrine and prostaglandin F2alpha (N = 6). When the rings were precontracted with prostaglandin F2alpha, the presence of ionic contrast did not inhibit the relaxation of the arteries. However, in canine renal arteries contracted with norepinephrine, the presence of ionic radiocontrast (diatrizoate meglumine and diatrizoate sodium, MD-76, 1 ml in 25 ml control solution, 4% (v/v)) inhibited relaxation in response to acetylcholine, sodium nitroprusside (N = 6 in each group), and isoproterenol (N = 5; P < 0.05). Rings were relaxed less than 50% of norepinephrine contraction. Following removal of the contrast, vascular relaxation in response to the agonists returned to normal. These results indicate that ionic radiocontrast nonspecifically inhibits vasodilation (both cAMP-mediated and cGMP-mediated) of canine renal arteries contracted with norepinephrine. This reversible impairment of vasodilation could inhibit normal renal perfusion and act as a mechanism of renal failure following radiocontrast infusion. In the adopted experimental protocol the isoproterenol-induced relaxation of renal arteries precontracted with norepinephrine was more affected, suggesting a pivotal role of the cAMP system.

Possible in vivo mechanisms involved in photodynamic therapy using tetrapyrrolic macrocycles

Photodynamic therapy (PDT) mediated by oxidative stress causes direct tumor cell damage as well as microvascular injury. To improve this treatment new photosensitizers are being synthesized and tested. We evaluated the effects of PDT with 5,10,15,20-tetrakis(4-methoxyphenyl)-porphyrin (TMPP) and its zinc complex (ZnTMPP) on tumor levels of malondialdehyde (MDA), reduced glutathione (GSH) and cytokines, and on the activity of caspase-3 and metalloproteases (MMP-2 and -9) and attempted to correlate them with the histological alterations of tumors in 3-month-old male Wistar rats, 180 ± 20 g, bearing Walker 256 carcinosarcoma. Rats were randomly divided into five groups: group 1, ZnTMPP+irradiation (IR) 10 mg/kg body weight; group 2, TMPP+IR 10 mg/kg body weight; group 3, 5-aminolevulinic acid (5-ALA+IR) 250 mg/kg body weight; group 4, control, no treatment; group 5, only IR. The tumors were irradiated for 15 min with red light (100 J/cm², 10 kHz, 685 nm) 24 h after drug administration. Tumor tissue levels of MDA (1.1 ± 0.7 in ZnTMPP vs 0.1 ± 0.04 nmol/mg protein in control) and TNF-α (43.5 ± 31.2 in ZnTMPP vs 17.3 ± 1.2 pg/mg protein in control) were significantly higher in treated tumors than in controls. Higher caspase-3 activity (1.9 ± 0.9 in TMPP vs 1.1 ± 0.6 OD/mg protein in control) as well as the activation of MMP-2 (P < 0.05) were also observed in tumors. These parameters were correlated (Spearman correlation, P < 0.05) with the histological alterations. These results suggest that PDT activates the innate immune system and that the effects of PDT with TMPP and ZnTMPP are mediated by reactive oxygen species, which induce cell membrane damage and apoptosis.

Alzheimer's disease: is a vaccine possible?

The cause of Alzheimer's disease is still unknown, but the disease is distinctively characterized by the accumulation of β-amyloid plaques and neurofibrillary tangles in the brain. These features have become the primary focus of much of the research looking for new treatments for the disease, including immunotherapy and vaccines targeting β-amyloid in the brain. Adverse effects observed in a clinical trial based on the β-amyloid protein were attributed to the presence of the target antigen and emphasized the relevance of finding safer antigen candidates for active immunization. For this kind of approach, different vaccine formulations using DNA, peptide, and heterologous prime-boost immunization regimens have been proposed. Promising results are expected from different vaccine candidates encompassing B-cell epitopes of the β-amyloid protein. In addition, recent results indicate that targeting another protein involved in the etiology of the disease has opened new perspectives for the effective prevention of the illness. Collectively, the evidence indicates that the idea of finding an effective vaccine for the control of Alzheimer's disease, although not without challenges, is a possibility.

Expression of β-catenin and c-myc during human common bile duct development: a possible role in the morphogenesis of the common bile duct

β-catenin and c-myc play important roles in the development of tissues and organs. However, little is known about their expression patterns during the development of the human common bile duct. Immunohistochemistry was used to detect β-catenin and c-myc expression in common bile duct samples from postmortem tissues of 14 premature infants and 6 spontaneously aborted fetuses. The expression of β-catenin and c-myc was also analyzed by Western blot. The samples were divided into four groups based on the stage of human fetal development: 12, 13-27, 28-37, and >37 weeks. The Image-Pro Plus v. 6.0 image analysis software was used to calculate the mean qualifying score (MQS). At fetal stages 12, 13-27, 28-37, and >37 weeks, MQS of β-catenin were 612.52±262.13, 818.38±311.73, 706.33±157.19, and 350.69±110.19, respectively. There was a significant difference in MQS among the four groups (ANOVA, P=0.0155) and between the scores at >37 and 13-27 weeks (Student-Newman-Keuls, P<0.05). At fetal stages 12, 13-27, 28-37, and >37 weeks, the MQS of c-myc were 1376.64±330.04, 1224.18±171.66, 1270.24±320.75, and 741.04±219.19, respectively. There was a significant difference in MQS among the four groups (ANOVA, P=0.0087) and between the scores at >37 and 12 weeks, >37 and 13-27 weeks, and >37 and 28-37 weeks (all P<0.05, Student-Newman-Keuls). Western blots showed that β-catenin and c-myc expression were significantly higher in fetal than in postnatal control duct tissue (P<0.05). c-myc and β-catenin are involved in the normal development of the human common bile duct.

Possible roles of COX-1 in learning and memory impairment induced by traumatic brain injury in mice

People who suffer from traumatic brain injury (TBI) often experience cognitive deficits in spatial reference and working memory. The possible roles of cyclooxygenase-1 (COX-1) in learning and memory impairment in mice with TBI are far from well known. Adult mice subjected to TBI were treated with the COX-1 selective inhibitor SC560. Performance in the open field and on the beam walk was then used to assess motor and behavioral function 1, 3, 7, 14, and 21 days following injury. Acquisition of spatial learning and memory retention was assessed using the Morris water maze on day 15 post-TBI. The expressions of COX-1, prostaglandin E2 (PGE2), interleukin (IL)-6, brain-derived neurotrophic factor (BDNF), platelet-derived growth factor BB (PDGF-BB), synapsin-I, and synaptophysin were detected in TBI mice. Administration of SC560 improved performance of beam walk tasks as well as spatial learning and memory after TBI. SC560 also reduced expressions of inflammatory markers IL-6 and PGE2, and reversed the expressions of COX-1, BDNF, PDGF-BB, synapsin-I, and synaptophysin in TBI mice. The present findings demonstrated that COX-1 might play an important role in cognitive deficits after TBI and that selective COX-1 inhibition should be further investigated as a potential therapeutic approach for TBI.