GDNF reduces oxidative stress in a 6-hydroxydopamine model of Parkinson's disease

Many current theories of Parkinson's disease (PD) suggest that oxidative stress is involved in the neurodegenerative process. Potential neuroprotective agents could protect neurons through inherent antioxidant properties or through the upregulation of the brain's antioxidant defenses. Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore dopamine neurons in experimental models of PD and to improve motor function in human patients. This study was designed to investigate GDNF's effect on oxidative stress in a model of PD. GDNF or vehicle was injected into the right striatum of male Fischer-344 rats. Three days later 6-OHDA or saline was injected into the same striatum. The striatum and substantia nigra from both sides of the brain were removed 24h after 6-OHDA or saline injection and analyzed for the oxidative stress markers protein carbonyls and 4-hydroxynonenal. Both markers were significantly reduced in GDNF+6-OHDA treated animals compared to vehicle+6-OHDA treated animals. In addition, in animals allowed to recover for 3.5-4 weeks after the 6-OHDA administration, the GDNF led to significant protection against loss of striatal and nigral tissue levels of dopamine. These results suggest that the protective effects of GDNF against 6-OHDA involve a reduction in oxidative stress.     

Effects of aerobic exercise training on large-conductance Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

Purpose

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels provide a negative feedback that regulates vascular tone in the brain circulation. This study investigated the effects of aerobic exercise on gating properties of BK_Ca channels in rat cerebral artery.

Methods

Rats were subjected to moderate-intensity exercise at low (EX-3d/w) and high (EX-5d/w) training volume on a motor-driven treadmill, and compared with age-matched sedentary animals (SED). Inside–out (I/O) patch clamp recording was performed to measure gating properties of the BK_Ca channel.

Results

Aerobic exercise induced a reduction in heart rate and body weight in both training groups. Exercise increased the channel activity, which was more pronounced in EX-5d/w than that in EX-3d/w group. Kinetic analysis revealed that (1) the contribution of short open states was elevated and the duration of both short and long open states were extended by exercising in EX-3d/w; (2) Ex-3d/w had no significant change on conformation of close states; (3) EX-3d/w increased the mean open time without changing mean closed time; (4) EX-5d/w increased both the contribution and duration of long open states; (5) EX-5d/w increased channel mean open time while decreased mean closed time.

Conclusion

The results suggest that regular aerobic exercise may enhance BK_Ca channel activity in cerebral arterial myocytes by changing its biophysical properties, and the electrical remolding induced by exercise may be training volume-dependent.     

Neuroinflammation mediated by IL-1β increases susceptibility of dopamine neurons to degeneration in an animal model of Parkinson's disease

Background

The etiology of Parkinson's disease (PD) remains elusive despite identification of several genetic mutations. It is more likely that multiple factors converge to give rise to PD than any single cause. Here we report that inflammation can trigger degeneration of dopamine (DA) neurons in an animal model of Parkinson's disease.

Methods

We examined the effects of inflammation on the progressive 6-OHDA rat model of Parkinson's disease using immunohistochemistry, multiplex ELISA, and cell counting stereology.

Results

We show that a non-toxic dose of lipopolysaccharide (LPS) induced secretion of cytokines and predisposed DA neurons to be more vulnerable to a subsequent low dose of 6-hydroxydopamine. Alterations in cytokines, prominently an increase in interleukin-1beta (IL-1β), were identified as being potential mediators of this effect that was associated with activation of microglia. Administration of an interleukin-1 receptor antagonist resulted in significant reductions in tumor necrosis factor-α and interferon-γ and attenuated the augmented loss of DA neurons caused by the LPS-induced sensitization to dopaminergic degeneration.

Conclusion

These data provide insight into the etiology of PD and support a role for inflammation as a risk factor for the development of neurodegenerative disease.     

Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson's disease

Background

Marrow stromal cells (MSC), the non-hematopoietic precursor cells in bone marrow, are being investigated for therapeutic potential in CNS disorders. Although in vitro studies have suggested that MSC may be immunologically inert, their immunogenicity following transplantation into allogeneic recipients is unclear. The primary objective of this study was to investigate the cellular immune response to MSC injected into the striatum of allogeneic recipients (6-hydroxydopamine [6-OHDA]-hemilesioned rats, an animal model of Parkinson's disease [PD]), and the secondary objective was to determine the ability of these cells to prevent nigrostriatal dopamine depletion and associated motor deficits in these animals.

Methods

5-Bromo-2-deoxyuridine (BrdU) – labeled MSC from two allogeneic sources (Wistar and ACI rats) were implanted into the striatum of adult Wistar rats at the same time as 6-OHDA was administered into the substantia nigra. Behavioral tests were administered one to two weeks before and 16–20 days after 6-OHDA lesioning and MSC transplantation. Immunocytochemical staining for T helper and T cytotoxic lymphocytes, microglia/macrophages, and major histocompatibility class I and II antigens was performed on post-transplantation days 22–24. MSC were detected with an anti-BrdU antibody.

Results

Tissue injury due to the transplantation procedure produced a localized cellular immune response. Unexpectedly, both sources of allogeneic MSC generated robust cellular immune responses in the host striatum; the extent of this response was similar in the two allograft systems. Despite these immune responses, BrdU⁺ cells (presumptive MSC) remained in the striatum of all animals that received MSC. The numbers of remaining MSC tended to be increased (p = 0.055) in rats receiving Wistar MSC versus those receiving ACI MSC. MSC administration did not prevent behavioral deficits or dopamine depletion in the 6-OHDA-lesioned animals.

Conclusion

MSC, when implanted into the striatum of allogeneic animals, provoke a marked immune response which is not sufficient to clear these cells by 22–24 days post-transplantation. In the experimental paradigm in this study, MSC did not prevent nigrostriatal dopamine depletion and its associated behavioral deficits. Additional studies are indicated to clarify the effects of this immune response on MSC survival and function before initiating trials with these cells in patients with PD or other neurodegenerative disorders.     

Effects of propofol on early and late cytokines in lipopolysaccharide-induced septic shock in rats

Objective

It has been reported that the intravenous anesthetic propofol (PPF) has anti-inflammatory effects in vitro and in patients. The purpose of this study was to investigate whether PPF has anti-inflammatory effects in lipopolysaccharide (LPS)-induced septic shock by inhibiting the induction of pro-inflammatory cytokines [interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)] and high mobility group box 1 (HMGB1) in rats.

Methods

Thirty six male Wistar rats were randomly assigned to one of three groups (control group, PPF + LPS group and LPS group; n = 12 per group). Control group rats received a 0.9% NaCl solution (NS) by the tail vein. The PPF + LPS group rats received PPF (10 mg/kg bolus, followed by infusion at 10 mg/(kg·h) through a femoral vein catheter) 1 h before LPS (7.5 mg/kg) administration via the tail vein. The LPS group rats received injection of LPS (7.5 mg/kg) via the tail vein. Hemodynamic effects were recorded as well as mortality rates, and plasma cytokine con-centrations (TNF-α, IL-6, HMGB1) were measured for the 24-h observation period.

Results

The mean arterial pressure and heart rate of the PPF + LPS group were more stable than those of the LPS group. The mortality at 24 h after the administration of the LPS injection was much higher in the LPS group (58.3%) compared to the PPF + LPS group (25.0%). Plasma concentrations of cytokines (IL-6 and TNF-α) and HMGB1 were significantly reduced in the PPF + LPS group compared to the LPS group (P < 0.05).

Conclusion

Pretreatment with PPF reduced the mortality rate of rats and attenuated the pro-inflammatory cytokine responses in an endotoxin shock model through an anti-inflammatory action inhibiting induction of HMGB1.     

Lipopolysaccharide-induced failure of the gut barrier is site-specific and inhibitable by growth hormone

Background

Gut barrier failure caused by endotoxemia is a life-threatening problem. The present study aimed to determine whether any specific intestinal site is highly correlated with gut barrier failure, and whether recombinant human growth hormone (rhGH) can ameliorate gut barrier failure in a rat model of endotoxemia.

Methods

Enterostomy tubes were surgically placed in adult male Sprague–Dawley rats three days before induction of endotoxemia by lipopolysaccharide (LPS) injection. Controls received no LPS. Rats were then randomly assigned to receive subcutaneous injections of rhGH (experimental, n = 30) or 0.9 % saline (control, n = 15) at 24, 48, or 72 h after LPS injection. Escherichia coli labeled with green fluorescent protein (GFP) were injected into the intestinal segment of all rats through the enterostomy tubes. The number of GFP-labeled E. coli detected in mesenteric lymph nodes was examined after 96 h. Apoptosis and proliferation rates of intestinal epithelial cells, and intestinal permeability were measured.

Results

Endotoxemia led to high mortality, compared with the control group, and rhGH treatment did not improve survival. Intestinal permeability, reflected by translocation rates of GFP-labeled E. coli, and apoptosis rates in the LPS-induced endotoxemia group were higher than those in the non-endotoxemia control group, and the endotoxemia ileum group had the highest rates of both bacterial translocation and apoptosis. The LPS+GH group had less bacterial translocation and apoptosis than the LPS-induced endotoxemia group. In contrast, the proliferation rates were lower in the LPS group compared to the LPS+GH group.

Conclusions

Endotoxemia can induce gut barrier failure in rats, and the ileum is the site of greatest risk. The GH can reduce the incidence of endotoxemia-induced gut barrier failure, but not the associated mortality.     

Intrastriatal administration of erythropoietin protects dopaminergic neurons and improves neurobehavioral outcome in a rat model of Parkinson's disease

Erythropoietin (EPO), a hematopoietic cytokine, has recently been demonstrated to protect nigral dopaminergic neurons in a mouse model of Parkinson's disease (PD). In the present study, we tested the hypothesis that recombinant human erythropoietin (rhEPO) could protect dopaminergic neurons and improve neurobehavioral outcome in a rat model of PD. rhEPO (20 units in 2 microl of vehicle) was stereotaxically injected into one side of the striatum. 6-hydroxydopamine (6-OHDA) was injected into the same side 1 day later. Another group of rats received rhEPO (5000 u/kg, i.p.) daily for 8 days, and unilateral injection of 6-OHDA in the striatum 3 days after systemic administration of rhEPO. We observed that intrastriatal administration, but not systemic administration of rhEPO significantly reduced the degree of rotational asymmetry. The rhEPO-treated rats also showed an improvement in skilled forelimb use when compared with control rats. The number of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons in the ipsilateral substantia nigra (SN) was significantly larger in intrastriatal rhEPO-treated rats than that in control rats. TH-IR fibers in the 6-OHDA-lesioned striatum were also increased in the intrastriatal rhEPO-treated rats when compared with control rats. In addition, there were lower levels of expression of major histocompatibility complex (MHC) class II antigens and a smaller number of activated microglia in the ipsilateral SN in intrastriatal rhEPO-treated rats than that in control rats at 2 weeks, suggesting that intrastriatal injection of rhEPO attenuated 6-OHDA-induced inflammation in the ipsilateral SN. Our results suggest that intrastriatal administration of rhEPO can protect nigral dopaminergic neurons from cell death induced by 6-OHDA and improve neurobehavioral outcome in a rat model of PD. Anti-inflammation may be one of mechanisms responsible for rhEPO neuroprotection.     

Neuroprotective effects of ginsenosides Rh1 and Rg2 on neuronal cells

Background

The present study investigates the effects of ginsenosides Rh₁ and Rg₂ against 6-hydroxydopamine (6-OHDA), a neurotoxin on SH-SY5Y cells and PC-12 cells. The effects of these two ginsenosides on neuronal differentiation are also examined.

Methods

LDH assay was used to measure cell viability after exposure to 6-OHDA and ginsenosides. Neuronal differentiation was evaluated by changes in cell morphology and density of neurite outgrowths. Western blotting was used to determine the ginsenosides' effects on activation of extracellular signal-regulated protein kinases (ERKs).

Results

Rh₁ and Rg₂ attenuated 6-OHDA toxicity in SH-SY5Y cells and induced neurite outgrowths in PC-12 cells. 6-OHDA-induced ERK phosphorylation was decreased by Rh₁ and Rg₂. 20(R)-form and 20(S)-form of the ginsenosides exerted similar effects in inducing neurite outgrowths in PC-12 cells.

Conclusion

The present study demonstrates neuroprotective effects of ginsenosides Rh₁ and Rg₂ on neuronal cell lines. These results suggest potential Chinese medicine treatment for neurodegenerative disorders (eg Parkinson's disease).     

LPS response pattern of inflammatory adipokines in an in vitro 3T3-L1 murine adipocyte model

Objective

In vitro 3T3-L1 mouse cells represent a reliable model to investigate the inflammatory phenotype of adipocytes activated by bacteria-derived lipopolysaccharide (LPS). In this study we have evaluated the differential expression of adipokines in response to increasing doses of LPS and various incubation times.

Methods

3T3-L1 mouse adipocytes were treated with E. coli LPS (from 0 to 10 μg/ml) for a time course ranging from 4 to 24 h, 4 h each. A time point at 2 h was also included to highlight early activation by LPS. mRNA expression by RT-PCR on cell lysates and ELISA assays on cell culture supernatants were performed.

Results

Cells activated by increasing doses of LPS upregulated TNF-α expression in the first 2 h, but this expression slowed down within 6–8 h, while IL-6 expression was increasing. This reduction was also observed for CXCL12/SDF1α. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-α and CXCL12 gene expression occurred early in the time-course and exhibited a second increase following the first 4–6 h of incubation with LPS. Optimal expression of most adipokines needed 6–8 h of a prolonged treatment with LPS at 37 °C. The chemokines MIP-1α/CCL3 and MIP-1β/CCL4 were maximally expressed within the first 8 h, then significantly reduced in the following times. IL-10 expression was upregulated by low doses of LPS and downregulated by prolonging time with the bacterial endotoxin. ELISA analysis of released products generally confirmed the result from gene expression experiments.

Conclusion

These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte.     

Urinary trypsin inhibitor suppresses excessive generation of superoxide anion radical, systemic inflammation, oxidative stress, and endothelial injury in endotoxemic rats

Objective and design

The protective effects of ulinastatin, a human urinary trypsin inhibitor (UTI), against superoxide radical (O ₂ ^?· ) generation, systemic inflammation, lipid peroxidation, and endothelial injury were investigated in endotoxemic rats.

Materials and treatment

Twenty-one Wistar rats were allocated to a control group, a UTI group, and a sham group. A bolus of lipopolysaccharide (LPS; 3 μg/g) was administered intravenously to the control group, a bolus of LPS and UTI (5 U/g) to the UTI group, and a bolus of saline to the sham group.

Methods

The O ₂ ^?· generated was measured as the current in the right atrium using an electrochemical O ₂ ^?· sensor. Plasma nitrite, high mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-α, inteleukin (IL)-6, malondialdehyde, and soluble intercellular adhesion molecule-1 (sICAM-1) were measured 360 min after LPS administration.

Results

The O ₂ ^?· current increased in the control group and was significantly attenuated in the UTI group after 55 min (P < 0.05 at 55–60 min, P < 0.01 at 65–360 min). Plasma nitrite, HMGB1, TNF-α, IL-6, malondialdehyde, and sICAM-1 were attenuated in the UTI group.

Conclusions

UTI suppressed excessive O ₂ ^?· generation, systemic inflammation, lipid peroxidation, and endothelial injury in endotoxemic rats.     

Effect of Toll-like receptor 4 inhibitor on LPS-induced lung injury

Objective and design

Toll-like receptor 4 (TLR4) plays important roles in the recognition of lipopolysaccharide (LPS) and the activation of inflammatory cascade. In this study, we evaluated the effect of TAK-242, a selective TLR4 signal transduction inhibitor, on acute lung injury (ALI).

Materials and methods

C57BL/6J mice were intravenously treated with TAK-242 15 min before the intratracheal administration of LPS or Pam3CSK4, a synthetic lipopeptide. Six hours after the challenge, bronchoalveolar lavage fluid was obtained for a differential cell count and the measurement of cytokine and myeloperoxidase levels. Lung permeability and nuclear factor-κB (NF-κB) DNA binding activity were also evaluated.

Results

TAK-242 effectively attenuated the neutrophil accumulation and activation in the lungs, the increase in lung permeability, production of inflammatory mediators, and NF-κB DNA-binding activity induced by the LPS challenge. In contrast, TAK-242 did not suppress inflammatory changes induced by Pam3CSK4.

Conclusion

TAK-242 may be a promising therapeutic agent for ALI, especially injuries associated with pneumonia caused by Gram-negative bacteria.     

Hypertonic saline solution reduces the inflammatory response in endotoxemic rats

OBJECTIVE:

Volume replacement in septic patients improves hemodynamic stability. This effect can reduce the inflammatory response. The objective of this study was to evaluate the effect of 7.5% hypertonic saline solution versus 0.9% normal saline solution for volume replacement during an inflammatory response in endotoxemic rats.

METHODS:

We measured cytokines (serum and gut), nitrite, and lipid peroxidation (TBARS) as indicators of oxidative stress in the gut. Rats were divided into four groups: control group (C) that did not receive lipopolysaccharide; lipopolysaccharide injection without treatment (LPS); lipopolysaccharide injection with saline treatment (LPS +S); and lipopolysaccharide injection with hypertonic saline treatment (LPS +H). Serum and intestine were collected. Measurements were taken at 1.5, 8, and 24 h after lipopolysaccharide administration.

RESULTS:

Of the four groups, the LPS +H group had the highest survival rate. Hypertonic saline solution treatment led to lower levels of IL-6, IL-10, nitric oxide, and thiobarbituric acid reactive substances compared to 0.9% normal saline. In addition, hypertonic saline treatment resulted in a lower mortality compared to 0.9% normal saline treatment in endotoxemic rats. Volume replacement reduced levels of inflammatory mediators in the plasma and gut.

CONCLUSION:

Hypertonic saline treatment reduced mortality and lowered levels of inflammatory mediators in endotoxemic rats. Hypertonic saline also has the advantage of requiring less volume replacement.     

Combined effects of a neutrophil elastase inhibitor (sivelestat sodium) and a free radical scavenger (edaravone) on lipopolysaccharide-induced acute lung injury in rats

Objective and design

The present study aimed to investigate the combined effects of a neutrophil elastase inhibitor, sivelestat sodium, with a free radical scavenger, edaravone, on lipolysaccharide (LPS)-induced acute lung injury (ALI).

Materials and methods

Adult male Sprague–Dawley rats were anesthetized and instilled intratracheally with 2?mg/kg LPS. Sivelestat sodium (10?mg/kg, i.p.) and/or edaravone (8?mg/kg, i.p.) were administered 1?h after LPS instillation. The severity of pulmonary injuries was evaluated 12?h after inducing acute lung injury.

Results

In lung tissues, either sivelestat or edaravone treatment alone showed significant protective effects against neutrophil infiltration and tissue injury, as demonstrated by myeloperoxidase activity and histopathological analysis. Sivelestat or edaravone treatment also attenuated the LPS-induced production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in rat lungs. However, the LPS-induced elevation of malondialdehyde levels in rat lungs was reduced only by edaravone, but not by sivelestat. In addition, combined treatment with both sivelestat and edaravone demonstrated additive protective effects on LPS-induced lung injury, compared with single treatments.

Conclusions

Combination of sivelestat and edaravone shows promise as a new treatment option for ALI/acute respiratory distress syndrome patients.     

Effect of chlorogenic acid on LPS-induced proinflammatory signaling in hepatic stellate cells

Objective and design

This study was aimed at investigating the effect of chlorogenic acid (CGA) on lipopolysaccharide (LPS)-induced proinflammatory signaling in hepatic stellate cells (HSCs).

Methods

An immortalized rat HSC line was cultured in vitro and treated with LPS in the absence or presence of CGA. Reactive oxygen species (ROS) production in the HSCs was monitored by flow cytometer using DCFH-DA. The protein expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor-κB (NF-κB), and p-IκB-α were determined by Western blot. The mRNA expression levels of TLR4, MyD88, monocyte chemotactic protein 1(MCP-1), and interleukin 6 (IL-6) were detected by RT-PCR. The levels of MCP-1 and IL-6 in the culture supernatant of HSCs were measured by ELISA.

Results

CGA had no effect on expression of TLR4 and MyD88. However, the treatment of CGA can inhibit LPS-induced production of ROS in HSCs. Meanwhile, CGA can inhibit LPS-induced nuclear translocation of NF-κB and IκB-α phosphorylation in HSCs, as well as NAC (a ROS scavenger). The mRNA expression and the levels of MCP-1 and IL-6 in the culture supernatant of the HSCs in this study were elevated by LPS stimulation and inhibited by CGA treatment, as well as NAC and PDTC (a NF-κB inhibitor).

Conclusion

Our results indicate that CGA can efficiently inhibit LPS-induced proinflammatory responses in HSCs and the anti-inflammatory effect may be due to the inhibition of LPS/ROS/NF-κB signaling pathway.     

Long-term treatment with methanandamide attenuates LPS-induced periodontitis in rats

Objective

Evidence exists of the anti-inflammatory and immunological properties of endocannabinoids in various tissues; the aim of the present study was therefore to assess the effect of long-term treatment with the synthetic cannabinoid methanandamide (Meth-AEA) on the progression of periodontitis in rats.

Materials and methods

Periodontitis was induced by injecting LPS (1?mg/ml) into the gingiva around the neck of the first upper and lower molars, and into the inter-dental space between the first and second molars. This protocol was repeated for 6?weeks on days 1, 3, and 5 of each week.

Results

Long-term treatment with topical Meth-AEA (500?ng/ml), applied daily to gingival tissue of rats induced with periodontitis, significantly diminished the alveolar bone loss, measured as the distance between the cemento-enamel junction and the alveolar crest, in both maxillary and mandibular first molars, compared to rats without treatment (P?P?P?Conclusion These results demonstrate the beneficial effects of treatment with Meth-AEA on gingival tissue of rats with periodontitis.     

Effects of exogenous annexin-1 on lipopolysaccharide-induced proliferation and reactive oxygen species production partially through modulation of CRAC channels but independent of NF-κB pathway

Objective

To investigate the effects of exogenous annexin-1 (ANXA1) on lipopolysaccharide(LPS)-induced proliferation, reactive oxygen species (ROS) production, and calcium signal transduction in RAW264.7 macrophages.

Methods

RAW264.7 macrophages were treated with or without LPS in the absence or presence of ANXA1. The proliferation effects were detected by Cell Counting Kit-8 assay. ROS were quantified by flow cytometry and fluorescence microscopy. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was analyzed by laser confocal scanning microscopy. IκBα degradation and NF-κB translocation were tested by Western blot.

Results

Exogenous ANXA1 inhibited LPS-induced proliferation and ROS production in a dose-dependent manner. LPS evoked [Ca²⁺]_i increase through CRAC channels, and ANXA1 suppressed LPS-induced [Ca²⁺]_i increase in a dose-dependent manner. The CRAC channels were associated with LPS-induced proliferation and ROS production. Exogenous ANXA1 had no effect on LPS-induced IκB degradation and NF-κB translocation.

Conclusions

ANXA1 inhibited LPS-induced proliferation and ROS production in RAW264.7 macrophages partially through modulation of CRAC channels but independent of the NF-κB pathway.     

Effects of anti-inflammatory vagus nerve stimulation in endotoxemic rats on blood and spleen lymphocyte subsets

Background

Anti-inflammatory cytokine effects of vagus nerve stimulation in sepsis syndromes are well established. Effects on immune cells are less clear. Therefore, we studied changes in peripheral and spleen leukocyte subsets in an endotoxic rat sepsis model.

Methods

Ventilated and sedated adult male SD rats received 5 mg/kg b.w. lipopolysaccharide intravenously to induce endotoxic sepsis. Controls and a group with both-sided vagotomy were compared to animals with both sided vagotomy and left distal vagus nerve stimulation. 4.5 h after sepsis induction immune cell counts and types in the peripheral blood and spleen were determined [T-lymphocytes (CD3+), T-helper cells (CD3+ CD4+), activated T-helper cells (CD3+ CD4+ CD134+), cytotoxic T-cells (CD3+ CD8+), activated cytotoxic T-cells (CD3+ CD8+ CD134+), B-lymphocytes (CD45R+ CD11cneg-dim), dendritic cells (CD11c+ OX-62 +), natural killer cells (CD161+ CD3neg) and granulocytes (His48 +)] together with cytokine and chemokine plasma levels (IL10; IFN-g, TNF-a, Cxcl5, Ccl5).

Results

Blood cell counts declined in all LPS groups. However, vagus nerve stimulation but not vagotomy activated cytotoxic T-cells. Vagotomy also depleted natural killer cells. In the spleen, vagotomy resulted in a strong decline of all cell types which was not present in the other septic groups where only granulocyte numbers declined.

Conclusion

Vagotomy strongly declines immune cell counts in the septic spleen. This could not be explained by an evasion or apoptosis of cells. A marginalisation of spleen immune cells into the peripheral microcirculation might be therefore most likely. Further studies are warranted to clear this issue.     

Serum response factor modulates neuron survival during peripheral axon injury

Background

Systemic inflammation might cause neuronal damage and sustain neurodegenerative diseases and behavior impairment, with the participation of pro-inflammatory cytokines, like tumor necrosis factor (TNF)-?? and interleukin (IL)-18. However, the potential contribution of these cytokines to behavioral impairment in the long-term period has not been fully investigated.

Methods

Wistar rats were treated with a single intraperitoneal injection of LPS (5?mg/kg) or vehicle. After 7?days and 10?months, the animal behavior was evaluated by testing specific cognitive functions, as mnesic, discriminative, and attentional functions, as well as anxiety levels. Contextually, TNF-?? and IL-18 protein levels were measured by ELISA in defined brain regions (that is, frontal cortex, hippocampus, striatum, cerebellum, and hypothalamus).

Results

Behavioral testing demonstrated a specific and persistent cognitive impairment characterized by marked deficits in reacting to environment modifications, possibly linked to reduced motivational or attentional deficits. Concomitantly, LPS induced a TNF-?? increase in the hippocampus and frontal cortex (from 7?days onward) and cerebellum (only at 10?months). Interestingly, LPS treatment enhanced IL-18 expression in these same areas only at 10?months after injection.

Conclusions

Overall, these results indicate that the chronic neuroinflammatory network elicited by systemic inflammation involves a persistent participation of TNF-?? accompanied by a differently regulated contribution of IL-18. This leads to speculation that, though with still unclear mechanisms, both cytokines might take part in long-lasting modifications of brain functions, including behavioral alteration.     

The expression pattern of Nischarin after lipopolysaccharides (LPS)-induced neuroinflammation in rats brain cortex

Objective

To investigate whether Nischarin participated in neuronal apoptosis induced by neuroinflammation and via the phosphatidylinositol 3-kinase (PI3K) and PKB-dependent pathway.

Material

Use of male Sprague–Dawley rats, rat pheochromocytoma (PC12), and murine microglial cells (BV-2). Treatment lipopolysaccharides (LPS) were injected into the brain lateral ventricle of the rat. The BV-2 cells were treated by LPS. The PC12 cells were pretreated by or not pretreated by conditioned media and siRNA.

Methods

Western blotting was used for analyzing the expression level of Nischarin, pAKT, BAD and Bcl-2. Immunohistochemistry and immunofluorescence were used to perform the morphology and localization of Nischarin. The siRNA could down-regulate the protein level of endogenous Nischarin.

Results

The expression level of Nischarin was elevated after LPS injection; meanwhile, Nischarin was located in the neuron. Nischarin was involved in regulating the PI3K/PKB patway.

Conclusion

Nischarin might be involved in mediating the process of PI3K/PKB pathway-dependent neuronal apoptosis. After the silencing of Nischarin in cultured PC12 (pheochromocytoma) by siRNA, these results showed that it would induce a reduction of pAKT and Bcl-2 proteins expression; meanwhile, it induces an increase of BAD and active caspase-3.     

Over-expression of caveolin-1 aggravate LPS-induced inflammatory response in AT-1 cells via up-regulation of cPLA2/p38 MAPK

Objective and design

The aim of this study was to study the effect of caveolin-1 on the cytosolic phospholipase A2 (cPLA2), p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor κB (NF-κB) in mouse lung alveolar type-1 cells' (AT-1 cells) inflammatory response induced by LPS.

Materials and methods

Gene clone technique was used to over-express caveolin-1 in AT-1 cells by lentivirus vector. The level of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), cPLA2, p38 MAPK and NF-κB was measured by ELISA, western blotting and EMSA.

Treatment

AT-1 cells were treated with LPS.

Results

Over-expression of caveolin-1 not only increased the production of pro-inflammatory cytokine TNF-α and IL-6, but also enhanced the expression of the cPLA2, p38 MAPK, and NF-κB.

Conclusions

Our data demonstrated that over-expression of caveolin-1 aggravates the AT-1 injury induced by LPS, involving in modulation of the cPLA2 mediated by the cPLA2/p38 MAPK pathway.