Pyrogen-free Saline Research Articles

Detoxification of LTA by intracanal medication: analysis by macrophages proinflammatory cytokines production.

The aim of this study was to evaluate in vitro the effect of calcium hydroxide [Ca(OH)2], 2% chlorhexidine gel (CHX) on macrophages (RAW 264.7) to produce pro-inflammatory cytokines and nitric oxide after pretreatment with lipoteichoic acid (LTA) of Enterococcus faecalis. Forty-eight human single-rooted teeth were instrumented with R25.08 (RECIPROC) and sterilized by gamma irradiation. LTA was inoculated in the root canal of each specimen for 96 hours. Specimens were instrumented with 40.06 and 50.05 (RECIPROC) and medicated with: I) Pyrogen-free saline solution (SS); II) 2% CHX gel; III) Ca(OH)2 + SS; or IV) Ca(OH)2 + CHX for 14 days. Three samples (S) were performed of the root canal of each specimen at: S1) immediately after instrumentation; S2) after Ethylenediaminetetraacetic acid (EDTA); S3) after intracanal medication removal. Subsequent quantification of cytokines (IL-1β, TNF-α, MIP-1α, IP-10, G-CSF and IL-6) by immunosorbent assay (ELISA) and nitric oxide by the Griess method was carried-out. Data were submitted to a normality test and then analyzed with one-way ANOVA and Tukey test with a significance level of 5% using GraphPad Prism 6. Ca(OH)2 + SS and Ca(OH)2 + CHX presented lower levels of TNF-α, TNF-α, IL-6, G-CSF and nitric oxide. Ca(OH)2 + SS was the most effective in reducing MIP-1α. CHX was effective in reducing IL-6 and G-CSF. Therefore, the combined intracanal medication of calcium hydroxide and chlorhexidine is effective in reducing the cytokines TNF-α, IL-1β, IL-6, G-CSF and nitric oxide.

Differential immune response to two Staphylococcus aureus strains with distinct adaptation genotypes after experimental intramammary infection of dairy cows

The aim of this study was to evaluate and compare the ability of two S. aureus strains with different adaptation genotypes (low and high) to the bovine mammary gland (MG) to establish an intramammary infection (IMI) and induce an immune response after an experimental challenge in lactating cows. Two isolates (designated 806 and 5011) from bovine IMI with different genotypic profiles, harboring genes involved in adherence and biofilm production, belonging to different capsular polysaccharide (CP) type, accessory gene regulator (agr) group, pulsotype (PT) and sequence type/clonal complex (ST/CC) were selected. Strains 806 and 5011 were associated with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 806 (NP) was characterized as agr group II, cap5 positive and ST350; strain 5011 (P) agr group I, cap8 positive and CC188. Three groups of clinically healthy cows, 4 cows/treatment group, were inoculated by the intramammary route with strain 806 (NP), strain 5011 (P) and pyrogen-free saline solution. All mammary quarters challenged with strain 806 (NP) developed mild clinical mastitis between 1 and 7 d post inoculation (pi). Quarters challenged with strain 5011 (P) developed a persistent IMI; bacteria were recovered from milk from d 7 pi and up to d 56 pi. In quarters inoculated with strain 806 (NP) the inflammatory response induced was greater and earlier than the one induced by strain 5011 (P), since a somatic cell count (SCC) peak was observed at d 2 pi, while in quarters inoculated with strain 5011 (P) no variations in SCC were observed until d 4 pi reaching the maximum values at d 14 pi; indicating a lower and delayed initial inflammatory response. The highest levels of nitric oxide (NO) and lactoferrin (Lf) detected in milk from quarters inoculated with both S. aureus strains coincided with the highest SCC at the same time periods, indicating an association with the magnitude of inflammation. The high levels of IL-1β induced by strain 806 (NP) were associated with the highest SCC detected (d 2 pi); while quarters inoculated with strain 5011 (P) showed similar IL-1β levels to those found in control quarters. In quarters inoculated with strain 806 (NP) two peaks of IL-6 levels on d 2 and 14 pi were observed; while in quarters inoculated with strain 5011 (P) IL-6 levels were similar to those found in control quarters. The strain 806 (NP) induced a higher total IgG and IgG1 response; while strain 5011 (P) generated a higher IgG2 response (even against the heterologous strain). The present study demonstrated that S. aureus strains with different genotype and adaptability to bovine MG influence the local host immune response and the course and severity of the infectious process.

The effects of apelin-13 against cisplatin-induced nephrotoxicity in rats

Acute kidney injury (AKI) is observed in nearly 60% of patients undergoing cisplatin (CP) therapy. The aim of this study was to reveal the potential effects of apelin-13 (AP-13) in the prevention of CP-induced renal toxicity, together with its antioxidant and anti-inflammatory effect mechanisms. Four experimental groups were established. Group 1, the control group, received 0.9% saline solution alone intraperitoneally (IP). Group 2, the CP group, received CP IP at 5 mg/kg once weekly for four weeks for induction of nephrotoxicity. In Group 3, the CP + Apelin-13 (AP-13) group, AP-13 was prepared at 20 nmol kg/d in sterile pyrogen-free saline before injection every day for four weeks and administered IP. CP was administered IP at 5 mg/kg once weekly for four weeks for induction of nephrotoxicity. In Group 4, the AP-13 group, AP-13 was prepared at 20 nmol kg/d in sterile pyrogen-free 0.9% saline before injection every day for four weeks and administered IP. Thiobarbituric acid reactive substances (TBARS), thiol (-SH), interleukin-1 beta, cleaved caspase-3, 8-hydroxy 2-deoxyguanosine (8-OHdG), and nuclear factor kappa B (NF-κβ/p65) levels were then measured. Increased oxidative stress, inflammation, and apoptosis as a result of CP application activated the cascade. However, AP-13 administration reduced the oxidative stress increased by CIS with the determined antioxidant effect and reduced the damage by increasing total -SH levels. 8-OHdG and NF-κβ/p65, which were up-regulated by triggering oxidative stress and inflammation, were down-regulated through the antioxidant and anti-inflammatory effects of AP-13.

Abstract # 3118 Acylated ghrelin’s anti-inflammatory actions on lipopolysaccharide (LPS)-induced cytokine release

Ghrelin regulates hypothalamic-pituitary-adrenal (HPA) axis responses to psychological stress at the brain and pituitary. Ghrelin is also a potent anti-inflammatory agent. However, its role in the HPA axis response to immune challenge is still imprecisely described. We hypothesized ghrelin’s anti-inflammatory actions on lipopolysaccharide (LPS)-induced cytokine release are mediated by activation of the HPA axis. We gave Wistar rats acylated ghrelin (AG; 1 mg/kg sc), concomitantly with lipopolysaccharide (LPS; E. coli, 100 μ g/kg i.p.) or pyrogen-free saline. We then assessed circulating cytokines and paraventricular nucleus of the hypothalamus (PVN) neuronal activation (c-Fos) as well as glucocorticoid and adrenocorticotropic hormone (ACTH) profiles. Our data show that AG markedly suppresses the circulating cytokine response to LPS, particularly that of tumour necrosis factor alpha, interleukin 1 and interleukin 10. In vitro, high AG can stimulate release of ACTH from the anterior pituitary, but in vivo the ACTH and glucocorticoid responses to LPS are not influenced by AG. AG alone activates the medial parvocellular region of the PVN, but it does not alter the LPS-induced activation of this region. These data suggest that AG markedly suppresses the circulating cytokine response to LPS but probably does not do this by modulating the HPA axis response in vivo.

Impact of prenatal immune challenge on the demyelination injury during adulthood

Brain inflammation is associated with several brain diseases such as multiple sclerosis (MS), a disease characterized by demyelination. Whether prenatal immune challenge affects demyelination-induced inflammation in the white matter during adulthood is unclear. In the present study, we used a well-established experimental model of focal demyelination to assess whether prenatal immune challenge affects demyelination-induced inflammation. Pregnant rats were injected with either lipopolysaccharide (100μg/kg, ip) or pyrogen-free saline. A 2μL solution of the gliotoxin ethidium bromide (0.04%) was stereotaxically infused into the corpus callosum of adult male offspring. The extent of demyelination lesion was assessed using Luxol fast blue (LFB) staining. Oligodendrocyte precursor cells, mature oligodendrocytes, markers of cellular gliosis, and inflammation were monitored in the vicinity of the demyelination lesion area. Prenatal lipopolysaccharide reduced the size of the demyelination lesion during adulthood. This reduced lesion was associated with enhanced density of mature oligodendrocytes and reduced density of microglial cells in the vicinity of the demyelination lesion. Such reduction in microglial cell density was accompanied by a reduced activation of the nuclear factor κB signaling pathway. These data strongly suggest that prenatal immune challenge dampens the extent of demyelination during adulthood likely by reprogramming the local brain inflammatory response to demyelinating insults.

Androstenediol Reduces Demyelination-Induced Axonopathy in the Rat Corpus Callosum: Impact on Microglial Polarization.

Aims: We have previously shown that the neurosteroid androstenediol (ADIOL) promotes remyelination following gliotoxin-induced demyelination. However, the impact of this ADIOL on axonal recovery is not yet known. In the present study, we investigated the impact of ADIOL on axonal integrity following a focal demyelination in the corpus callosum.Methods: A 2 μl solution of either ethidium bromide (EB; 0.04%) or pyrogen-free saline were stereotaxically injected into the corpus callosum of Sprague Dawley rats. Each of these two rat groups was divided into two subgroups and received daily subcutaneous injections of either ADIOL (5 mg/kg) or vehicle. The brains were collected at 2, 7 and 14 days post-stereotaxic injection. Immunofluorescent staining was used to explore the impact of ADIOL on axonal integrity (neurofilament (NF)-M) and microglial activation (ionized calcium binding adapter molecule 1, Iba1). The inducible nitric oxide synthase (iNOS) and arginase-1 (arg-1), two major markers of microglial polarization towards the proinflammatory M1 and the regulatory M2 phenotypes respectively, were monitored using western blot.Results: ADIOL increased the density of NF fibers and decreased the extent of axonal damage in the vicinity of the demyelination lesion. ADIOL-induced decrease in axonal damage was manifested by decreased number of axonal spheroids at both 2 and 7 days post-demyelination insult. This reduced axonopathy was associated with decreased expression of iNOS and enhanced expression of arg-1 during the acute phase.Conclusion: These data strongly suggest that ADIOL reduces demyelination-induced axonal damage, likely by dampening the local inflammatory response in the white matter and shifting microglial polarization towards a reparative mode.

Environmental enrichment rescues the effects of early life inflammation on markers of synaptic transmission and plasticity

Environmental enrichment (EE) has been successful at rescuing the brain from a variety of early-life psychogenic stressors. However, its ability to reverse the behavioral and neural alterations induced by a prenatal maternal infection model of schizophrenia is less clear. Moreover, the specific interactions between the components (i.e. social enhancement, novelty, physical activity) of EE that lead to its success as a supportive intervention have not been adequately identified. In the current study, standard housed female Sprague-Dawley rats were administered either the inflammatory endotoxin lipopolysaccharide (LPS; 100μg/kg) or pyrogen-free saline (equivolume) on gestational day 15. On postnatal day 50, offspring were randomized into one of three conditions: EE (group housed in a large multi-level cage with novel toys, tubes and ramps), Colony Nesting (CN; socially-housed in a larger style cage), or Standard Care (SC; pair-housed in standard cages). Six weeks later we scored social engagement and performance in the object-in-place task. Afterwards hippocampus and prefrontal cortex (n=7–9) were collected and evaluated for excitatory amino acid transporter (EAAT) 1-3, brain-derived neurotrophic factor (BDNF), and neurotrophic tyrosine kinase, receptor type 2 (TrkB) gene expression (normalized to GAPDH) using qPCR methods. Overall, we show that gestational inflammation downregulates genes critical to synaptic transmission and plasticity, which may underlie the pathogenesis of neurodevelopmental disorders such as schizophrenia and autism. Additionally, we observed disruptions in both social engagement and spatial discrimination. Importantly, behavioral and neurophysiological effects were rescued in an experience dependent manner. Given the evidence that schizophrenia and autism may be associated with infection during pregnancy, these data have compelling implications for the prevention and reversibility of the consequences that follow immune activation in early in life.

NREM sleep is increased in high-aggressive, but not in non-aggressive rats by an immune challenge

Sleep is altered in response to an immune challenge: NREM sleep is increased and fragmented, REM sleep is inhibited. Different levels of trait aggressiveness are associated with specific patterns of neuroendocrine and autonomic stress responsiveness. Sleep and immune response are affected by stress. Aim of the present study was to test the hypothesis that sleep patterns are differentially affected by an immune challenge in animals that differ widely for aggression. High-aggressive (HA, n = 7) and non-aggressive (NA, n = 8) rats were selected on the basis of their latency time to attack a male intruder in the resident-intruder test. Afterwards, they were instrumented for chronic polygraphic recording of sleep-wake patterns and kept on a 12:12 h light:dark cycle. At dark onset, on different days, each rat was intraperitoneally injected with pyrogen free saline (vehicle, VEH) and 250 microgram/kg lipopolysaccharide. In HA rats, lipopolysaccharide administration induced a significant and long lasting (from hour 3 to hour 8 post-injection) increase (in comparison to VEH administration) in NREM sleep, and a biphasic fever. No changes in NREM sleep, and only a short lasting fever were induced by lipopolysaccharide administration in NA rats. REM sleep was inhibited in both HA and NA rats, but with a different time course. Results show that sleep and febrile responses to an immune challenge are different in HA and NA rats.

Tracing the trajectory of behavioral impairments and oxidative stress in an animal model of neonatal inflammation

Exposure to early-life inflammation results in time-of-challenge-dependent changes in both brain and behavior. The consequences of this neural and behavioral reprogramming are most often reported in adulthood. However, the trajectory for the expression of these various changes is not well delineated, particularly between the juvenile and adult phases of development. Moreover, interventions to protect against these neurodevelopmental disruptions are rarely evaluated. Here, female Sprague–Dawley rats were housed in either environmental enrichment (EE) or standard care (SC) and their male and female offspring were administered 50μg/kg i.p. of lipopolysaccharide (LPS) or pyrogen-free saline in a dual-administration neonatal protocol. All animals maintained their respective housing assignments from breeding until the end of the study. LPS exposure on postnatal days (P) 3 and 5 of life resulted in differential expression of emotional and cognitive disruptions and evidence of oxidative stress across development. Specifically, social behavior was reduced in neonatal-treated (n)LPS animals at adolescence (P40), but not adulthood (P70). In contrast, male nLPS rats exhibited intact spatial memory as adolescents which was impaired in later life. Moreover, these males had decreased prefrontal cortex levels of glutathione at P40, which was normalized in adult animals. Notably, EE appeared to offer some protection against the consequences of inflammation on juvenile social behavior and fully prevented reduced glutathione levels in the juvenile prefrontal cortex. Combined, these time-dependent effects provide evidence that early-life inflammation interacts with other developmental variables, specifically puberty and EE, in the expression (and prevention) of select behavioral and molecular programs.

Comparison of Different Irrigants in the Removal of Endotoxins and Cultivable Microorganisms from Infected Root Canals.

This study was conducted to compare the effectiveness of different irrigants used to remove endotoxins and cultivable microorganisms during endodontic therapy. Forty root canals were contaminated and divided into groups according to the irrigant: 2% NaOCl + surfactant, 2% CHX, 2.5% NaOCl, and pyrogen-free saline solution (control). Samples were collected after root canal contamination (S1), after instrumentation (S2), and 7 days after instrumentation (S3). Microorganisms and endotoxins were recovered from 100% of the contaminated root canals (S1). At S2, 2% NaOCl + surfactant, 2% CHX, and 2.5% NaOCl were able to completely eliminate cultivable microorganisms. At S3, both 2% CHX and 2.5% NaOCl were effective in preventing C. albicans and E. coli regrowth, but E. faecalis was still detected. No microorganism species was recovered from root canals instrumented with 2% NaOCl + surfactant. At S2, a higher percentage value of endotoxin reduction was found for 2% NaOCl + surfactant (99.3%) compared to 2% CHX (98.9%) and 2.5% NaOCl (97.18%) (p < 0.05). Moreover, at S3, 2% NaOCl + surfactant (100%) was the most effective irrigant against endotoxins. All irrigants tested were effective in reducing microorganisms and endotoxins from root canals. Moreover, 2% NaOCl + surfactant was the most effective irrigant against endotoxins and regrowth of microorganisms.

Maternal Exercise before Pregnancy Alleviates Periventricular Leukomalacia by Lipopolysaccharide-Induced Maternal Intrauterine Inflammation in Infant Rat Brain

In the present study, we investigated the effects of maternal exercise before pregnancy on lipopolysaccharide (LPS)-induced hypomyelination and astrogliosis in neonatal rats. Female Sprague-Dawley rats (180 ± 10 g, 8 weeks old, n = 45) were used for maternal rats in this experiment. Before pregnancy, rats were randomly divided into three groups: the non-ex-control (CON) group, non-ex-LPS (LPS) group, and the Pre-ex-LPS (Ex-LPS) group (n = 15 in each group). The rats in the Ex-LPS group were forced to run on a motorized treadmill for 30 min once a day for 4 weeks. After the completion of exercise, all rats were allowed to mate with male rats for a period of 24 h. After conforming of pregnancies (n = 5 in each group), the maternal rats were treated with 0.15 mg/kg LPS and/or the same amount of pyrogen-free saline (PFS) on the 15th, 17th, and 20th day of pregnancy. The expression of Cyclooxygenasee-2 (COX-2) and Tumor necrosis factor-α (TNF-α) in brain was analyzed by RT-PCR. Immunohistochemistry for the glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) in the corpus callosum were conducted. In results, the expressions of COX-2, TNF-α and GFAP was significantly increased while the expression of MBP was significantly decreased in the neonatal rat brain of the LPS group compared to those of the CON group (p<.05). This result showed that LPS might induce toxic effect in neonatal rat brain such as hypomyelination and astrogliosis. In contrast, the expressions of COX-2, TNF-α and GFAP was significantly decreased while the expression of MBP was significantly increased in the neonatal rat brain of the Ex-LPS group compared to those of the LPS group (p<.05). In conclusion, the present results revealed that regular maternal exercise before pregnancy might be effective to ameliorate the symptoms of PVL such as astrogliosis and hypomyelination in neonatal rat brain and it can be used as a therapeutic strategy for preventing the detrimental effects of CP.

Evaluation of active carbon fibers used in cell biocompatibility and rat cystitis treatment

Escherichia coli is the predominant pathogen in catheter-associated urinary tract infection (CAUTI) and cystitis. CAUTI treated with prophylactic antibiotics induces antibiotic resistance in hospitals and nursing homes. Therefore, we developed a physical method to treat cystitis by using activated carbon (AC) fibers to remove lipopolysaccharide (LPS) from the bladder. An in vitro assay showed that AC had remarkable LPS adsorption capability (20.24EU per mg AC for 60min) and good biocompatibility, as revealed by morphological observation through field-emission scanning electron microscopy and MTS assay in SV-HUC-1 cell line. In an in vivo assay in rats, 3 groups were formed: control (pyrogen-free saline), LPS, and AC (n=10, each group). The LPS and AC groups were pretreated with protamine sulfate (10mg/mL), and E. coli LPS (5mg/kg) was administered to induce cystitis. Subsequently, the AC group received 0.4mg/kg AC suspension. After 24h, histologic analysis revealed increased urinary bladder weight (edema) and marked increase in vascular congestion and hemorrhage in the LPS group. In contrast, the AC group showed significant reduction in degree of edema, inflammatory cell infiltration, and hemorrhage (p<0.05). Thus, AC has the potential to act as an auxiliary agent for cystitis amelioration.

The efficacy of intravenous immunoglobulin on lipopolysaccharide-induced fetal brain inflammation in preterm rats

Interleukin-1 is accepted as one of the major cytokines; it is involved in inflammatory processes and systemic fetal inflammatory response that is triggered by maternal lipopolysaccharide (LPS) injection. Because it is an antiinflammatory agent, we investigated (in the brain damage of rat pups) the role of intravenous immunoglobulin (IVIG) in decreasing interleukin-1 beta (IL-1β) expression and caspase 3 activity that was induced by maternal LPS administration. Dams were divided into 3 groups. Pyrogen-free saline solution (NS) was administered intraperitoneally to group 1; LPS (0.3mg/kg) suspension in NS was administered to groups 2 and 3 at 19 days of gestation. Two hours after the first injection, a second injection of NS was administered intravenously to group 1 (NS+ NS), of IVIG was administered intravenously to group 2 (LPS+ IVIG), and of NS was administered intravenously to group 3 (LPS+ NS). Hysterectomy was performed in one-half of the dams 2 hours after the second injection and in the other one-half of the dams 22 hours after the second injection. Pups were delivered, and the brains were extracted just after delivery. IL-1β expression and caspase 3 activity were determined in brain tissues. For the pups at 4 hours, the IL-1β expression of group 2was significantly lower than groups 1 and 3. For the pups at 24hours, the IL-1β expression of group 2 was significantly lower than group 3 but was similar to group 1. For the pups at 24 hours, caspase 3 activity of groups 1 and 2 were significantly lower than group3. Maternal IVIG administration decreased IL-1β expression and caspase 3 activity in the brain tissue of rat pups, which had been induced by maternal LPS-administration.

Lipopolysaccharide-preconditioning protects against endotoxin-induced white matter injury in the neonatal rat brain

BackgroundExposing the brain to a sub-damaging stimulus can protect against a subsequent lethal insult, a phenomenon termed preconditioning. The aim of this study was to investigate the neuroprotective effect of low dose LPS (lipopolysaccharide) pretreatment in endotoxin induced periventricular leukomalacia (PVL) in a rat model. Methods: Wistar rats with dated pregnancies were allocated to 5 groups: (i) no LPS administered, intraperitoneally (i.p.) pyrogen-free saline injected (Control group), (ii) 500μg/kg LPS administrated i.p. on days 18 and 19 (PVL group), (iii) 50μg/kg LPS administrated i.p. on day 17 followed by 500μg/kg LPS i.p. on days 18 and 19 (PC-PVL group), (iv) 50μg/kg LPS administrated on day 17 (PC only), and (v) i.p. pyrogen-free saline injected control group on day 17. Results: LPS-preconditioning given 24h before potent LPS exposure significantly reduced the number of apoptotic cell deaths and prevented hypomyelination. Antioxidant enzyme gene expression levels (Superoxide Dismutase-SOD1, SOD2, and SOD3) were increased and Tumor Necrosis Factor (TNF)α expression levels were decreased in the PC+PVL group when compared with the PVL group. Conclusion: Low-dose LPS given one day before potent doses of LPS reduces antepartum LPS-induced brain damage. The mechanisms of protection might involve oxidation and inflammation.

Prenatal Immune Stress in Rats Dampens Fever during Adulthood

Fever is a major component of the host’s defense against infection. Inadequate febrile response can predispose an individual to the deleterious effects of infection. Neonatal exposure to infectious agents such as bacterial lipopolysaccharide (LPS) permanently dampens the adult febrile response. Whether prenatal immune challenge alters febrile response during adulthood is still not known. In the present study, LPS (100 µg/kg, i.p.) or pyrogen-free saline was administered to pregnant rats on either gestation day (GD) 12, 15 or 19 and the febrile response of their respective adult offspring was monitored. During adulthood (>70 days old), the rats born to LPS-injected dams on GD15 displayed a significantly attenuated febrile response to LPS (50 µg/kg, i.p.) compared to their control counterparts born to dams given saline on GD15. Immune challenge during either early (GD12) or late (GD19) pregnancy did not have a significant impact on fever in the adult offspring. Immune challenge on GD15, but not on GD12 or 19, heightened the plasma corticosterone response to a subsequent LPS injection to the adult offspring but did not have a significant effect on their basal plasma corticosterone levels. Finally, LPS-induced COX-2 in the fever-controlling regions of the hypothalamus was significantly reduced in the adult rats born to dams given LPS on GD15 compared to their counterparts born to dams given saline on GD15. Such COX-2 reduction was not observed in the adult offspring born to dams given LPS on either GD12 or 19. Taken together, these data suggest that a single immune challenge during a critical window of pregnancy alters the neuroimmune response in adult offspring.

Dectin1 activation of β-(1-3)/(1-6)-d-glucan produces an anti-mastitis effect in rats

A lipopolysaccharide (LPS)-induced mastitis model in rats was used to study the protective effect of β-glucan. On gestation day 10, β-glucan was administered to rats daily by gavage at either 0 (control), 0.1 mg/kg BW, 1 mg/kg BW and 10 mg/kg BW until parturition. LPS or pyrogen-free physiological saline was inoculated into the mammary gland 72 h after parturition and the rats were euthanized at 12 h post-infection. LPS increased dectin1 and toll-like receptor 4 (TLR4) mRNA and protein expression significantly in mammary tissues. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, N-acetyl-β-D-glucosaminidase (NAGase), inducible nitric oxide synthase (iNOs) in mammary tissues and serum, and myeloperoxidase (MPO) in mammary tissues were significantly increased 12 h after LPS infusion. β-glucan administration could enhance dectin1 mRNA and protein expression while downregulating the expression of TLR4. Level of TNF-α, IL-1β in mammary tissues and serum, MPO, NAGase and iNOs activity in mammary tissues was decreased, but the level of IL-2 in serum was increased by β-glucan. The results indicate that β-glucan may protect mammary tissue against LPS-induced rat acute mastitis.

In vitro evaluation of the action of irrigating solutions associated with intracanal medications on Escherichia coli and its endotoxin in root canals

ObjectiveThe purpose of this study was to evaluate the efficacy of auxiliary chemical substances and intracanal medications on Escherichia coli and its endotoxin in root canals.Material and MethodsTeeth were contaminated with a suspension of E. coli for 14 days and divided into 3 groups according to the auxiliary chemical substance used: G1) 2.5% sodium hypochlorite (NaOCl); G2) 2% chlorhexidine gel (CLX); G3) pyrogen-free solution. After, these groups were subdivided according to the intracanal medication (ICM): A) Calcium hydroxide paste (Calen®), B) polymyxin B, and C) Calcium hydroxide paste+2% CLX gel. For the control group (G4), pyrogen-free saline solution was used without application of intracanal medication. Samples of the root canal content were collected immediately after biomechanical preparation (BMP), at 7 days after BMP, after 14 days of intracanal medication activity, and 7 days after removal of intracanal medication. The following aspects were evaluated for all collections: a) antimicrobial activity; b) quantification of endotoxin by the Limulus Amebocyte Lysate test (LAL). Results were analyzed by the Kruskal-Wallis and Dunn’s tests at 5% significance level.ResultsThe 2.5% NaOCl and CLX were able to eliminate E. coli from root canal lumen and reduced the amount of endotoxin compared to saline.ConclusionsIt was concluded that 2.5% NaOCl and CLX were effective in eliminating E. coli. Only the studied intracanal medications were to reduce the amount of endotoxin present in the root canals, regardless of the irrigant used.

Adjunctive dexamethasone affects the expression of genes related to inflammation, neurogenesis and apoptosis in infant rat pneumococcal meningitis.

Streptococcus pneumoniae is the most common pathogen causing non-epidemic bacterial meningitis worldwide. The immune response and inflammatory processes contribute to the pathophysiology. Hence, the anti-inflammatory dexamethasone is advocated as adjuvant treatment although its clinical efficacy remains a question at issue. In experimental models of pneumococcal meningitis, dexamethasone increased neuronal damage in the dentate gyrus. Here, we investigated expressional changes in the hippocampus and cortex at 72 h after infection when dexamethasone was given to infant rats with pneumococcal meningitis. Nursing Wistar rats were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis or were sham-infected with pyrogen-free saline. Besides antibiotics, animals were either treated with dexamethasone or saline. Expressional changes were assessed by the use of GeneChip® Rat Exon 1.0 ST Arrays and quantitative real-time PCR. Protein levels of brain-derived neurotrophic factor, cytokines and chemokines were evaluated in immunoassays using Luminex xMAP® technology. In infected animals, 213 and 264 genes were significantly regulated by dexamethasone in the hippocampus and cortex respectively. Separately for the cortex and the hippocampus, Gene Ontology analysis identified clusters of biological processes which were assigned to the predefined categories “inflammation”, “growth”, “apoptosis” and others. Dexamethasone affected the expression of genes and protein levels of chemokines reflecting diminished activation of microglia. Dexamethasone-induced changes of genes related to apoptosis suggest the downregulation of the Akt-survival pathway and the induction of caspase-independent apoptosis. Signalling of pro-neurogenic pathways such as transforming growth factor pathway was reduced by dexamethasone resulting in a lack of pro-survival triggers. The anti-inflammatory properties of dexamethasone were observed on gene and protein level in experimental pneumococcal meningitis. Further dexamethasone-induced expressional changes reflect an increase of pro-apoptotic signals and a decrease of pro-neurogenic processes. The findings may help to identify potential mechanisms leading to apoptosis by dexamethasone in experimental pneumococcal meningitis.

Increased Glucocorticoid Receptor Expression and Activity Mediate the LPS Resistance of SPRET/EI Mice

SPRET/Ei mice are extremely resistant to acute LPS-induced lethal inflammation when compared with C57BL/6. We found that in vivo SPRET/Ei mice exhibit strongly reduced expression levels of cytokines and chemokines. To investigate the role of the potent anti-inflammatory glucocorticoid receptor (GR) in the SPRET/Ei phenotype, mice were treated with the GR antagonist RU486 or bilateral adrenalectomy. Under such conditions, both C57BL/6 and SPRET/Ei mice were strongly sensitized to LPS, and the differences in LPS response between SPRET/Ei and C57BL/6 mice were completely gone. These results underscore the central role of GR in the LPS hyporesponsiveness of SPRET/Ei mice. Compared with C57BL/6, SPRET/Ei mice were found to express higher GR levels, which were reflected in increased GR transactivation. Using a backcross mapping strategy, we demonstrate that the high GR transcription levels are linked to the Nr3c1 (GR) locus on chromosome 18 itself. Unexpectedly, SPRET/Ei mice exhibit a basal overactivation of the hypothalamic-pituitary-adrenal axis, namely strongly increased corticosterone levels, ACTH levels, and adrenocortical size. As a consequence of the excess of circulating glucocorticoids (GCs), levels of hepatic gluconeogenic enzymes are increased, and insulin secretion from pancreatic β-cells is impaired, both of which result in hyperglycemia and glucose intolerance in SPRET/Ei mice. We conclude that SPRET/Ei mice are unique as they display an unusual combination of elevated GR expression and increased endogenous GC levels. Hence, these mice provide a new and powerful tool for the study of GR- and GC-mediated mechanisms, including immune repressive functions, neuroendocrine regulation, insulin secretion, and carbohydrate metabolism.

Expression of myelin basic protein, glial fibrillary acidic protein, interleukin-1β and tumor necrosis factor-α in neonatal rats brain after intrauterine exposure on lipopolysaccharide

Objective To probe into the expression of inflammatory factrors and white matter damage after intrauterine lipopolysaccharide(LPS) exposure in neonatal rats brain. Methods LPS (0. 4 μg) was administrated into the intrauterine cavity between every two embryo sacs of SD rats at day 19 of gestation in LPS group(n= 14) , while pyrogen-free saline was administrated in control group (n=10). Neonatal brain tissues were collected at postnatal day 1,3,7 and 11. Reverse transcription-polymerase chain reaction analysis was used to examine mRNA expression of interleukin-1βand tumor necrosis factor-a, and immunohistochemistry was used to evaluate the expression of myelin basic protein and glial fibrillary acidic protein in neonatal brain tissues. Statistical analysis was performed using t test. Results Placental hematoxylin-eosin staining in the LPS group showed distinct inflammatory cell infiltration, prominent hyperplasia of interstitial tissue and narrowed capillary. Myelin basic protein in cerebellum and cerebrum of neonatal rats at postnatal 11 days in LPS group was much weaker than in control group (cerebellum: 1. 29 ±0. 76 vs 2. 43 ±0.79, t =2. 038,P=0.045; cerebrum: 1. 71 ±0. 49 vs 2.50±0.76, t= 2. 420,P = 0.032). Glial fibrillary acidic protein of neonatal rats brain at postnatal 11 days in LPS group was much stronger than in control group (hippocampus: 2.71±0.49 vs 1.75±0.50, (t=-3.029, P = 0.026. corpus callus: 2.40±0.55 vs 1. 50±0. 58, t= -2. 646,P = 0. 019. cerebrum: 1. 50±0. 55 vs 1. 00±0. 00, t= -2. 236, P=0. 049. cerebellum: 2. 80 ±0. 45 vs 1.60 ±0.55, t = -3.08,P =0.009). But there was no statistical difference between LPS group and control group in the expressions of IL-1β and TNF-α mRNA in neonatal rats brain at each time point. Conclusions Intrauterine LPS exposure may decrease the placental blood flow resulting in neonatal low birth weight and white matter damage, which characterized by astrogliosis and hypomyelination. Key words: Pregnancy complications, infectious) Lipopolysaccharides; Animals, newborn