Antinociceptive properties in mice of a lectin isolated from the marine alga Amansia multifida Lamouroux

The antinociceptive effects of a lectin (LEC) isolated from the marine alga Amansia multifida were determined in Swiss mice. The LEC (1, 5, and 10 mg/kg) inhibited acetic acid-induced abdominal writhings in a dose-dependent manner after intraperitoneal or oral administration. A partial but significant inhibition of writhings was observed after the combination of LEC (10 mg/kg) with avidin (1 mg/kg), a potent inhibitor of the hemmaglutinant activity of the lectin. However, total writhing inhibition was demonstrable in the group of mice treated with LEC plus mannose (1 mg/kg), as compared to LEC alone or to control groups. Furthermore, avidin and mainly mannose also play a role in antinociception, somehow facilitating the interaction of LEC with its active cell sites. In the formalin test, although both phases of the response were significantly inhibited, the effect of LEC was predominant during phase 2, causing inhibition of licking time that ranged from 48 to 88% after oral (5 and 10 mg/kg) and intraperitoneal (1 to 5 mg/kg) administration. As is the case with morphine, the effect of LEC (2 mg/kg) was reversed by naloxone (2 mg/kg), indicating the involvement of the opioid system. LEC was also effective in the hot-plate test, producing inhibitory responses to the thermal stimulus, and its effects were blocked by naloxone. In the pentobarbital-induced sleeping time, although LEC did not alter the onset of sleep significantly, it increased the time of sleep within the same dose range compared to control. These results show that LEC presents antinociceptive effects of both central and peripheral origin, possibly involving the participation of the opioid system.

Gentle handling temporarily increases c-Fos in the substantia nigra pars compacta

Dopaminergic neurotransmission is involved in the regulation of sleep. In particular, the nigrostriatal pathway is an important center of sleep regulation. We hypothesized that dopaminergic neurons located in substantia nigra pars compacta (SNpc) could be activated by gentle handling, a method to obtain sleep deprivation (SD). Adult male C57/BL6J mice (N = 5/group) were distributed into non-SD (NSD) or SD groups. SD animals were subjected to SD once for 1 or 3 h by gentle handling. Two experiments were performed. The first determined the activation of SNpc neurons after SD, and the second examined the same parameters after pharmacologically induced dopaminergic depletion using intraperitoneal reserpine (2 mg/kg). After 1 or 3 h, SD and NSD mice were subjected to motor evaluation using the open field test. Immediately after the behavioral test, the mice were perfused intracardially to fix the brain and for immunohistochemical analysis of c-Fos protein expression within the SNpc. The open field test indicated that SD for 1 or 3 h did not modify motor behavior. However, c-Fos protein expression was increased after 1 h of SD compared with the NSD and 3-h SD groups. These immunohistochemistry data indicate that these periods of SD are not able to produce dopaminergic supersensitivity. Nevertheless, the increased expression of c-Fos within the SNpc suggests that dopaminergic nigral activation was triggered by SD earlier than motor responsiveness. Dopamine-depleted mice (experiment 2) exhibited a similar increase of c-Fos expression compared to control animals indicating that dopamine neurons are still activated in the 1-h SD group despite the exhaustion of dopamine. This finding suggests that this range (2-5-fold) of neuronal activation may serve as a marker of SD.

L-histidine enhances learning in stressed zebrafish

The aim of the present study was to determine the effect of the histaminergic precursor L-histidine and the H3 receptor antagonist thioperamide on the learning process of zebrafish submitted or not to confinement stress. On each of the 5 consecutive days of experiment (D1, D2, D3, D4, D5), animals had to associate an interruption of the aquarium air supply with food offering. Non-stressed zebrafish received an intraperitoneal injection of 100 mg/kg L-histidine, 10 mg/kg thioperamide or saline after training. Stressed animals received drug treatment and then were submitted to confinement stress for 1 h before the learning procedure. Time to approach the feeder was measured (in seconds) and was considered to be indicative of learning. A decrease in time to approach the feeder was observed in the saline-treated group (D1 = 141.92 ± 13.57; D3 = 55 ± 13.54), indicating learning. A delay in learning of stressed animals treated with saline was observed (D1 = 217.5 ± 25.66). L-histidine facilitated learning in stressed (D1 = 118.68 ± 13.9; D2 = 45.88 ± 8.2) and non-stressed (D1 = 151.11 ± 19.20; D5 = 62 ± 14.68) animals. Thioperamide inhibited learning in non-stressed (D1 = 110.38 ± 9.49; D4 = 58.79 ± 16.83) and stressed animals (D1 = 167.3 ± 26.39; D5 = 172.15 ± 27.35). L-histidine prevented the increase in blood glucose after one session of confinement (L-histidine = 65.88 ± 4.50; control = 53 ± 3.50 mg/dL). These results suggest that the histaminergic system enhances learning and modulates stress responses in zebrafish.

Pharmacological effects and behavioral interventions on memory consolidation and reconsolidation

In this article, we will review some behavioral, pharmacological and neurochemical studies from our laboratory on mice, which might contribute to our understanding of the complex processes of memory consolidation and reconsolidation. We discuss the post-training (memory consolidation) and post-reactivation (memory reconsolidation) effects of icv infusions of hemicholinium, a central inhibitor of acetylcholine synthesis, of intraperitoneal administration of L-NAME, a non-specific inhibitor of nitric oxide synthase, of intrahippocampal injections of an inhibitor of the transcription factor NF-κB, and the exposure of mice to a new learning situation on retention performance of an inhibitory avoidance response. All treatments impair long-term memory consolidation and retrieval-induced memory processes different from extinction, probably in accordance with the "reconsolidation hypothesis".

Lactobacillus delbrueckii UFV-H2b20 induces type 1 cytokine production by mouse cells in vitro and in vivo

Lactobacillus delbrueckii UFV-H2b20 has been shown to increase clearance of bacteria injected into the blood of germ-free mice. Moreover, it induces the production of type 1 cytokines by human peripheral mononuclear cells. The objective of the present study was to investigate the production of inflammatory cytokines [interleukin-12 (IL-12 p40), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)] triggered in vitro by live, heat-killed or lysozyme-treated L. delbrueckii UFV-H2b20 and in vivo by a live preparation. Germ-free, L. delbrueckii-monoassociated and lipopolysaccharide (LPS)-resistant C3H/HeJ mice were used as experimental models. UFV-H2b20 induced the production of IL-12 p40 and TNF-α by peritoneal cells and IFN-γ by spleen cells from germ-free or monoassociated Swiss/NIH mice and LPS-hyporesponsive mice (around 40 ng/mL for IL-12 p40, 200 pg/mL for TNF-α and 10 ng/mL for IFN-γ). Heat treatment of L. delbrueckii did not affect the production of these cytokines. Lysozyme treatment decreased IL-12 p40 production by peritoneal cells from C3H/HeJ mice, but did not affect TNF-α production by these cells or IFN-γ production by spleen cells from the same mouse strain. TNF-α production by peritoneal cells from Swiss/NIH L. delbrueckii-monoassociated mice was inhibited by lysozyme treatment. When testing IL-12 p40 and IFN-γ levels in sera from germ-free or monoassociated Swiss/NIH mice systemically challenged with Escherichia coli we observed that IL-12 p40 was produced at marginally higher levels by monoassociated mice than by germ-free mice (40 vs 60 ng/mL), but IFN-γ was produced earlier and at higher levels by monoassociated mice (monoassociated 4 and 14 ng/mL 4 and 8 h after infection, germfree 0 and 7.5 ng/mL at the same times). These results show that L. delbrueckii UFV-H2b20 stimulates the production of type 1 cytokines in vitro and in vivo, therefore suggesting that L. delbrueckii might have adjuvant properties in infection in which these cytokines play a major role.

Chlorpheniramine impairs functional recovery in Carassius auratus after telencephalic ablation

We determined the effect of an H1 receptor antagonist on the functional recovery of Carassius auratus submitted to telencephalic ablation. Five days after surgery the fish underwent a spatial-choice learning paradigm test. The fish, weighing 6-12 g, were divided into four groups: telencephalic ablation (A) or sham lesion (S) and saline (SAL) or chlorpheniramine (CPA, ip, 16 mg/kg). For eight consecutive days each animal was trained individually in sessions separated by 24 h (alternate days). Training trials (T1-T8) consisted of finding the food in one of the feeders, which were randomly blocked for each subject. Animals received an intraperitoneal injection of SAL or CPA 10 min after the training trials. The time spent by the animals in each group to find the food (latency) was analyzed separately at T1 and T8 by the Kruskal-Wallis test, followed by the Student Newman-Keuls test. At T1 the latencies (mean ± SEM) of the A-SAL (586.3 ± 13.6) and A-CPA (600 ± 0) groups were significantly longer than those of the S-SAL (226.14 ± 61.15) and S-CPA (356.33 ± 68.8) groups. At T8, the latencies of the A-CPA group (510.11 ± 62.2) remained higher than those of the other groups, all of which showed significantly shorter latencies (A-SAL = 301.91 ± 78.32; S-CPA = 191.58 ± 73.03; S-SAL = 90.28 ± 41) compared with T1. These results support evidence that training can lead to functional recovery of spatial-choice learning in telencephalonless fish and also that the antagonist of the H1 receptor impairs it.

Treatment of hemorrhagic shock with hypertonic saline solution modulates the inflammatory response to live bacteria in lungs

Shock and resuscitation render patients more susceptible to acute lung injury due to an exacerbated immune response to subsequent inflammatory stimuli. To study the role of innate immunity in this situation, we investigated acute lung injury in an experimental model of ischemia-reperfusion (I-R) followed by an early challenge with live bacteria. Conscious rats (N = 8 in each group) were submitted to controlled hemorrhage and resuscitated with isotonic saline (SS, 0.9% NaCl) or hypertonic saline (HS, 7.5% NaCl) solution, followed by intratracheal or intraperitoneal inoculation of Escherichia coli. After infection, toll-like receptor (TLR) 2 and 4 mRNA expression was monitored by RT-PCR in infected tissues. Plasma levels of tumor necrosis factor α and interleukins 6 and 10 were determined by ELISA. All animals showed similar hemodynamic variables, with mean arterial pressure decreasing to nearly 40 mmHg after bleeding. HS or SS used as resuscitation fluid yielded equal hemodynamic results. Intratracheal E. coli inoculation per se induced a marked neutrophil infiltration in septa and inside the alveoli, while intraperitoneal inoculation-associated neutrophils and edema were restricted to the interseptal space. Previous I-R enhanced lung neutrophil infiltration upon bacterial challenge when SS was used as reperfusion fluid, whereas neutrophil influx was unchanged in HS-treated animals. No difference in TLR expression or cytokine secretion was detected between groups receiving HS or SS. We conclude that HS is effective in reducing the early inflammatory response to infection after I-R, and that this phenomenon is achieved by modulation of factors other than expression of innate immunity components.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL·kg-1·day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-α1 (-53%), TIMP-1 (-31.7%), TGF-β1 (-57.7%), and MMP-2 (-41.6%), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1%), ALT (-17.6%), and AST (-12.2%) serum levels; c) increased liver weight (11.3%), and reduced liver collagen (-37.1%), regenerative nodules size (-22.1%), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and “re-couples” NOS function.

L-histidine provokes a state-dependent memory retrieval deficit in mice re-exposed to the elevated plus-maze

The effects of L-histidine (LH) on anxiety and memory retrieval were investigated in adult male Swiss Albino mice (weight 30-35 g) using the elevated plus-maze. The test was performed on two consecutive days: trial 1 (T1) and trial 2 (T2). In T1, mice received an intraperitoneal injection of saline (SAL) or LH before the test and were then injected again and retested 24 h later. LH had no effect on anxiety at the dose of 200 mg/kg since there was no difference between the SAL-SAL and LH-LH groups at T1 regarding open-arm entries (OAE) and open-arm time (OAT) (mean ± SEM; OAE: 4.0 ± 0.71, 4.80 ± 1.05; OAT: 40.55 ± 9.90, 51.55 ± 12.10, respectively; P > 0.05, Kruskal-Wallis test), or at the dose of 500 mg/kg (OAE: 5.27 ± 0.73, 4.87 ± 0.66; OAT: 63.93 ± 11.72, 63.58 ± 10.22; P > 0.05, Fisher LSD test). At T2, LH-LH animals did not reduce open-arm activity (OAE and OAT) at the dose of 200 mg/kg (T1: 4.87 ± 0.66, T2: 5.47 ± 1.05; T1: 63.58 ± 10.22; T2: 49.01 ± 8.43 for OAE and OAT, respectively; P > 0.05, Wilcoxon test) or at the dose of 500 mg/kg (T1: 4.80 ± 1.60, T2: 4.70 ± 1.04; T1: 51.55 ± 12.10, T2: 43.88 ± 10.64 for OAE and OAT, respectively; P > 0.05, Fisher LSD test), showing an inability to evoke memory 24 h later. These data suggest that LH does not act on anxiety but does induce a state-dependent memory retrieval deficit in mice.

Effect of gallium-arsenide laser, gallium-aluminum-arsenide laser and healing ointment on cutaneous wound healing in Wistar rats

This study determined the effects of gallium-aluminum-arsenide laser (GaAlAs), gallium-arsenide laser (GaAs) and Dersani® healing ointment on skin wounds in Wistar rats. The parameters analyzed were: type I and III collagen fiber concentrations as well as the rate of wound closure. Five wounds, 12 mm in diameter, were made on the animals’ backs. The depth of the surgical incision was controlled by removing the epithelial tissue until the dorsal muscular fascia was exposed. The animals were anesthetized with ketamine and xylazine via intraperitoneal injection. The rats were randomly divided into five groups of 6 animals each, according to the treatment received. Group 1 (L4): GaAs laser (4 J/cm²); group 2 (L30): GaAlAs laser (30 J/cm²); group 3 (L60): GaAlAs laser (60 J/cm²); group 4 (D): Dersani® ointment; group 5 (control): 0.9% saline. The applications were made daily over a period of 20 days. Tissue fragments were stained with picrosirius to distinguish type I collagen from type III collagen. The collagen fibers were photo-documented and analyzed using the Quantum software based on the primary color spectrum (red, yellow and blue). Significant results for wound closing rate were obtained for group 1 (L4), 7.37 mm/day. The highest concentration of type III collagen fibers was observed in group 2 (L30; 37.80 ± 7.10%), which differed from control (29.86 ± 5.15%) on the 20th day of treatment. The type I collagen fibers of group 1 (L4; 2.67 ± 2.23%) and group 2 (L30; 2.87 ± 2.40%) differed significantly from control (1.77 ± 2.97%) on the 20th day of the experiment.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

Increased levels of glutamate in the central nervous system are associated with behavioral symptoms in experimental malaria

Cerebral malaria (CM) is a severe complication resulting from Plasmodium falciparum infection. This condition has been associated with cognitive, behavioral and motor dysfunctions, seizures and coma. The underlying mechanisms of CM are incompletely understood. Glutamate and other metabolites such as lactate have been implicated in its pathogenesis. In the present study, we investigated the involvement of glutamate in the behavioral symptoms of CM. Seventeen female C57BL/6 mice (20-25 g) aged 6-8 weeks were infected with P. berghei ANKA by the intraperitoneal route using a standardized inoculation of 10(6) parasitized red blood cells suspended in 0.2 mL PBS. Control animals (N = 17) received the same volume of PBS. Behavioral and neurological symptoms were analyzed by the SmithKline/Harwell/Imperial College/Royal Hospital/Phenotype Assessment (SHIRPA) battery. Glutamate release was measured in the cerebral cortex and cerebrospinal fluid of infected and control mice by fluorimetric assay. All functional categories of the SHIRPA battery were significantly altered in the infected mice at 6 days post-infection (dpi) (P ≤ 0.05). In parallel to CM symptoms, we found a significant increase in glutamate levels in the cerebral cortex (mean ± SEM; control: 11.62 ± 0.90 nmol/mg protein; infected at 3 dpi: 10.36 ± 1.17 nmol/mg protein; infected at 6 dpi: 26.65 ± 0.73 nmol/mg protein; with EGTA, control: 5.60 ± 1.92 nmol/mg protein; infected at 3 dpi: 6.24 ± 1.87 nmol/mg protein; infected at 6 dpi: 14.14 ± 0.84 nmol/mg protein) and in the cerebrospinal fluid (control: 128 ± 51.23 pmol/mg protein; infected: 301.4 ± 22.52 pmol/mg protein) of infected mice (P ≤ 0.05). These findings suggest a role of glutamate in the central nervous system dysfunction found in CM.

GM1 improves neurofascin155 association with lipid rafts and prevents rat brain myelin injury after hypoxia-ischemia

White matter injury characterized by damage to myelin is an important process in hypoxic-ischemic brain damage (HIBD). Because the oligodendrocyte-specific isoform of neurofascin, neurofascin 155 (NF155), and its association with lipid rafts are essential for the establishment and stabilization of the paranodal junction, which is required for tight interaction between myelin and axons, we analyzed the effect of monosialotetrahexosyl ganglioside (GM1) on NF155 expression and its association with lipid rafts after HIBD in Sprague-Dawley rats, weighing 12-15 g, on day 7 post-partum (P7; N = 20 per group). HIBD was induced on P7 and the rats were divided into two groups: one group received an intraperitoneal injection of 50 mg/kg GM1 three times and the other group an injection of saline. There was also a group of 20 sham-operated rats. After sacrifice, the brains of the rats were removed on P30 and studied by immunochemistry, SDS-PAGE, Western blot analysis, and electron microscopy. Staining showed that the saline group had definite rarefaction and fragmentation of brain myelin sheaths, whereas the GM1 group had no obvious structural changes. The GM1 group had 1.9-2.9-fold more GM1 in lipid rafts than the saline group (fraction 3-6; all P < 0.05) and 0.5-2.4-fold higher expression of NF155 in lipid rafts (fraction 3-5; all P < 0.05). Injection of GM1 increased the content of GM1 in lipid rafts as well as NF155 expression and its lipid raft association in HIBD rat brains. GM1 may repair the structure of lipid rafts, promote the association of NF155 (or other important proteins) with lipid rafts, stabilize the structure of paranodes, and eventually prevent myelin sheath damage, suggesting a novel mechanism for its neuroprotective properties.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.