Repeated Lipopolysaccharide Injections Research Articles

Analysis of correlations between behavioral parameters in the elevated plus maze and the levels of interleukin-1beta in blood plasma in rats

Peripheral cytokines may influence psychoemotional behavior, but the role of interleukin-1beta (IL-1beta) in altering anxiety and motor activity in response to inflammatory activation remains unclear. To clarify this issue, correlations between behavioral parameters in the elevated plus maze (EPM) test and plasma levels of IL-1beta after administration of the proinflammatory stimulus lipopolysaccharide (LPS) in different modes were analyzed in adult male rats. LPS in doses of 0.5 or 5 mg/kg, as well as physiological solution (control), were administered to rats intraperitoneally. The most pronounced behavioral effect 24 hours after a single injection was an endotoxin dose-dependent inhibition of the animals’ motor activity. After a dose of 5 mg/kg, increased anxious behavior was also noted every other day. The behavioral changes caused by the high dose of endotoxin were completely normalized after a week. The behavior of the animals one day after the end of repeated injections of LPS at a lower dose for a week (0.5 mg/kg; once every two days) also did not differ from the control. The inhibition of motor activity after LPS could be due to an increase in the level of IL-1beta in the blood plasma, as indicated by the identified significant negative correlations between IL-1beta and the corresponding behavioral parameters. No significant correlation was found between the peripheral level of IL-1beta and such a classic indicator of anxiety as the percentage of entries into the open arms of the maze. In general, the obtained results allow us to conclude that IL-1beta is an undoubted participant in the mechanism of the transient inhibitory effect of LPS on motor activity.

Dysfunction of NRG1/ErbB4 Signaling in the Hippocampus Might Mediate Long-term Memory Decline After Systemic Inflammation.

Accumulating evidence has suggested that a great proportion of sepsis survivors suffer from long-term cognitive impairments after hospital discharge, leading to decreased life quality and substantial caregiving burdens for family members. However, the underlying mechanism remains unclear. In the present study, we established a mouse model of systemic inflammation by repeated lipopolysaccharide (LPS) injections. A combination of behavioral tests, biochemical, and in vivo electrophysiology techniques were conducted to test whether abnormal NRG1/ErbB4 signaling, parvalbumin (PV) interneurons, and hippocampal neural oscillations were involved in memory decline after repeated LPS injections. Here, we showed that LPS induced long-term memory decline, which was accompanied by dysfunction of NRG1/ErbB4 signaling and PV interneurons, and decreased theta and gamma oscillations. Notably, NRG1 treatment reversed LPS-induced decreases in p-ErbB4 and PV expressions, abnormalities in theta and gamma oscillations, and long-term memory decline. Together, our study demonstrated that dysfunction of NRG1/ErbB4 signaling in the hippocampus might mediate long-term memory decline in a mouse model of systemic inflammation induced by repeated LPS injections. Thus, targeting NRG1/ErbB4 signaling in the hippocampus may be promising for the prevention and treatment of this long-term memory decline.

Differential effects of lipopolysaccharide on cognition, corticosterone and cytokines in socially-housed vs isolated male rats

Social isolation is an established risk factor for mental illness and impaired immune function. Evidence suggests that neuroinflammatory processes contribute to mental illness, possibly via cytokine-induced modulation of neural activity. We examined the effects of lipopolysaccharide (LPS) administration and social home cage environment on cognitive performance in the 5-Choice Serial Reaction Time Task (5CSRTT), and their effects on corticosterone and cytokines in serum and brain tissue. Male Long-Evans rats were reared in pairs or in isolation before training on the 5CSRTT. The effects of saline and LPS (150 µg/kg i.p.) administration on sickness behaviour and task performance were then assessed. LPS-induced sickness behaviour was augmented in socially-isolated rats, translating to increased omissions and slower response times in the 5CSRTT. Both social isolation and LPS administration reduced impulsive responding, while discriminative accuracy remained unaffected. With the exception of reduced impulsivity in isolated rats, these effects were not observed following a second administration of LPS, revealing behavioural tolerance to repeated LPS injections. In a separate cohort of animals, social isolation potentiated the ability of LPS to increase serum corticosterone and IL-6, which corresponded to increased IL-6 in the orbitofrontal and medial prefrontal cortices and the nucleus accumbens. Basal IL-4 levels in the nucleus accumbens were reduced in socially-isolated rats. These findings are consistent with the adaptive response of reduced motivational drive following immune challenge, and identify social isolation as an exacerbating factor. Enhanced IL-6 signalling may play a role in mediating the potentiated behavioural response to LPS administration in isolated animals.

Beneficial effects of QTC-4-MeOBnE in an LPS-induced mouse model of depression and cognitive impairments: The role of blood-brain barrier permeability, NF-κB signaling, and microglial activation

Clinical and preclinical investigations have suggested a possible biological link betweenmajor depressive disorder (MDD) and Alzheimer’s disease (AD). Therefore, a pharmacologic approach to treating MDD could be envisioned as a preventative therapy for some AD cases. In line with this, 1-(7-chloroquinolin-4-yl)-N-(4-methoxybenzyl)-5-methyl-1H-1,2,3-triazole-4 carboxamide (QTC-4-MeOBnE) is characterized as an inhibitor of β-secretase, glycogen synthase kinase 3β, and acetylcholinesterase and has also shown secondary effects underlying the modulation of neurogenesis and synaptic plasticity pathways. Therefore, we investigated the effects of QTC-4-MeOBnE treatment (0.1 or 1 mg/kg) on depressive-like behavior and cognitive impairments elicited by repeated injections of lipopolysaccharide (LPS; 250 μg/kg) in mice. Injections of LPS for seven days led to memory impairments and depressive-like behavior, as evidenced in the Y-maze/object recognition test and forced swimming/splash tests, respectively. However, these impairments were prevented in mice that, after the last LPS injection, were also treated with QTC-4-MeOBnE (1 mg/kg). This effect was associated with restoring blood-brain barrier permeability, reducing oxidative/nitrosative biomarkers, and decreasing neuroinflammation mediated NF-κB signaling in the hippocampus and cortex of the mice. To further investigate the involvement with NF-κB signaling, we evaluated the effects of QTC-4-MeOBnE on microglial cell activation through canonical and non-canonical pathways and the modulation of the involved components. Together, our findings highlight the pharmacological benefits of QTC-4-MeOBnE in a mouse model of sickness behavior and memory impairments, supporting the novel concept that since this molecule produces anti-depressant activity, it could also be beneficial for preventing AD onset and related dementias in subjects suffering from MDD through inflammatory pathway modulation.

Effect of Systemic Inflammation on Rat Attentional Function and Neuroinflammation: Possible Protective Role for Food Restriction.

Background: Aging is characterized by subtle cognitive decline, which correlates with increased peripheral inflammation. Acute activation of the peripheral immune system, via lipopolysaccharide (LPS) injection, elicits deficits in hippocampal-dependent spatial memory. Little is known concerning the effect of chronic inflammation on prefrontal cortex (PFC)-dependent vigilance. We examined the impact of repeated LPS injections in young and middle-age rats on the 5-choice serial reaction time task (5-CSRTT), expecting repeated LPS treatment to induce attentional deficits with greater disruption in middle-age.Methods: Male Fischer-344 rats, 4- and 12-months-old, were food restricted and trained on the 5-CSRTT. Once rats reached criterion, they were injected with LPS (1 mg/kg, i.p.) weekly for 4 weeks and testing started 48 h after each injection. To examine the possibility that mild food restriction inherent to the behavioral task influenced inflammation markers, a second group of food-restricted or ad-lib-fed rats was assessed for cytokine changes 48 h after one injection.Results: Performing LPS-treated rats exhibited a sickness response, manifesting as reduced initiated and completed trials during the first week but recovered by the second week of testing. After the first week, LPS-treated rats continued to exhibit longer response latencies, despite no change in food retrieval latency, suggestive of LPS-induced cognitive slowing. Similarly, LPS-induced impairment of attention was observed as increased omissions with heightened cognitive demand and increased age. Repeated LPS-treatment increased the level of PFC IL-1α, and PFC IL-6 was marginally higher in middle-age rats. No effect of age or treatment was observed for plasma cytokines in performing rats. Histological examination of microglia indicated increased colocalization of Iba1+ and CD68+ cells from middle-age relative to young rats. Examination of food restriction demonstrated an attenuation of age- and LPS-related increases in plasma cytokine levels.Conclusions: Systemic inflammation, induced through LPS treatment, impaired attentional function, which was independent of sickness and exacerbated by increased cognitive demand and increased age. Additional studies revealed that food restriction, associated with the task, attenuated markers of neuroinflammation and plasma cytokines. The results emphasize the need for improved methods for modeling low-level chronic systemic inflammation to effectively examine its impact on attention during aging.

Anti-Inflammatory Effect of the <i>Isatis tinctoria</i> L. Root Extract on Lipopolysaccharide-Induced Periodontitis in Rats

Objectives: Isatis tinctoria L., clary, is an herbal plant traditionally used in folk medicine for the treatment of various diseases and conditions. Although it has been primarily used as an antimicrobial and antifungal, there are data on traditional use of I. tinctoria as an agent against antiallergic, anti-thrombocytosis. The aim of the study was to examine the effect of the I. tinctoria root extract on the lipopolysaccharide (LPS)-induced periodontitis in rats on osteoclast associated bone resorptive activity, cell death including apoptosis, and inflammation in a rat of disease model of periodontitis. Materials and Methods: Periodontitis, acute or chronic inflammatory status in periodontal tissue in rats could be induced by repeated injections of LPS from Escherichia coli into the periodontal pocket area between the first and second right maxillary molars. Eighteen male rats were distributed among the following treatment groups: 1) I. tinctorial root extract (Antifect) 200 mg/kg body weight, 2) acetylsalicylic acid (ASA), 20 mg/kg body weight and 3) Phosphate buffered saline (PBS) treatment used as a control. After 15 days, maxilla, alveolar bone, molar teeth and associated periodontal tissues were harvested. Inflammatory alveolar bone resorption was analyzed by microcomputerized tomography (μCT) (microcomputer tomography). Tissues fixed with paraformaldehyde and formalin for 2 days, after that paraffin embedded histological sections were stained with haematoxylin and eosin (H/E) for the assessment of histopathological changes or tested to immunohistochemistry for detecting TRAP (tartrate resistant acid phosphatase) positive cells and caspase 3. Cell death and Apoptosis were analyzed in the periodontal tissues by tunnel assay. The inflammatory status was assessed by the measurements of proinflammatory cytokines interleukin-Iβ (IL-Iβ), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) of gingival tissues and descriptive analysis of histological sections of periodontal. Results: Treatment with Antifect, compared to the control group, significantly diminished the process of inflammation decreasing the levels of IL-Iβ, IL-6 and TNF-α, reducing the gingival tissue lesions and preserving bone alveolar resorption. Considerably a smaller number of inflammatory cells and a larger number of fibroblasts were noticed. Also, μCT analysis showed that only Antifect treated group reduced bone resorption and the number of TRAP-positive multinucleated cells (osteoclasts), also, significantly reduced the number of apoptotic cells in the gingival tissues and of osteocytes in the alveolar bone crest. Conclusion: Antifect manifested anti-inflammatory elect and reducing alveolar bone resorption in LPS-induced periodontitis suggest that it may have a role as a therapeutic agent in periodontal diseases.

The detrimental effects of lipopolysaccharide-induced neuroinflammation on adult hippocampal neurogenesis depend on the duration of the pro-inflammatory response.

Adult hippocampal neurogenesis is a finely tuned process regulated by extrinsic factors. Neuroinflammation is a hallmark of several pathological conditions underlying dysregulation of neurogenesis. In animal models, lipopolysaccharide (LPS)-induced neuroinflammation leads to a neurogenic decrease mainly associated to the early inflammatory response. However, it is not well understood how the neuroinflammatory response progresses over time and if neurogenesis continues to be diminished during the late neuroinflammatory response. Moreover, it is unknown if repeated intermittent administration of LPS along time induces a greater reduction in neurogenesis. We administered one single intraperitoneal injection of LPS or saline or four repeated injections (one per week) of LPS or saline to young-adult mice. A cohort of new cells was labeled with three 5-bromo-2-deoxyuridine injections (one per day) 4 days after the last LPS injection. We evaluated systemic and neuroinflammation-associated parameters and compared the effects of the late neuroinflammatory response on neurogenesis induced by each protocol. Our results show that 1) a single LPS injection leads to a late pro-inflammatory response characterized by microglial activation, moderate astrocytic reaction and increased interleukin-6 levels. This response correlates in time with decreased neurogenesis and 2) a repeated intermittent injection of LPS does not elicit a late pro-inflammatory response although activated microglia persists. The latter profile is not accompanied by a continued long-term hippocampal neurogenic decrease. Hereby, we provide evidence that the neuroinflammatory response is a dynamic process that progresses in a milieu-dependent manner and does not necessarily lead to a neurogenic decrease, highlighting the complex interaction between the immune system and neurogenesis.

Diverse Protein Profiles in CNS Myeloid Cells and CNS Tissue From Lipopolysaccharide- and Vehicle-Injected APPSWE/PS1ΔE9 Transgenic Mice Implicate Cathepsin Z in Alzheimer's Disease.

Neuroinflammation, characterized by chronic activation of the myeloid-derived microglia, is a hallmark of Alzheimer’s disease (AD). Systemic inflammation, typically resulting from infection, has been linked to the progression of AD due to exacerbation of the chronic microglial reaction. However, the mechanism and the consequences of this exacerbation are largely unknown. Here, we mimicked systemic inflammation in AD with weekly intraperitoneal (i.p.) injections of APPSWE/PS1ΔE9 transgenic mice with E. coli lipopolysaccharide (LPS) from 9 to 12 months of age, corresponding to the period with the steepest increase in amyloid pathology. We found that the repeated LPS injections ameliorated amyloid pathology in the neocortex while increasing the neuroinflammatory reaction. To elucidate mechanisms, we analyzed the proteome of the hippocampus from the same mice as well as in unique samples of CNS myeloid cells. The repeated LPS injections stimulated protein pathways of the complement system, retinoid receptor activation and oxidative stress. CNS myeloid cells from transgenic mice showed enrichment in pathways of amyloid-beta clearance and elevated levels of the lysosomal protease cathepsin Z, as well as amyloid precursor protein, apolipoprotein E and clusterin. These proteins were found elevated in the proteome of both LPS and vehicle injected transgenics, and co-localized to CD11b+ microglia in transgenic mice and in primary murine microglia. Additionally, cathepsin Z, amyloid precursor protein, and apolipoprotein E appeared associated with amyloid plaques in neocortex of AD cases. Interestingly, cathepsin Z was expressed in microglial-like cells and co-localized to CD68+ microglial lysosomes in AD cases, and it was expressed in perivascular cells in AD and control cases. Taken together, our results implicate systemic LPS administration in ameliorating amyloid pathology in early-to-mid stage disease in the APPSWE/PS1ΔE9 mouse and attract attention to the potential disease involvement of cathepsin Z expressed in CNS myeloid cells in AD.

Alcohol intake in two different mouse drinking models after recovery from the lipopolysaccharide-induced sickness reaction.

Neuroinflammation may play an important role in the development of alcohol addiction. Recent preclinical reports suggest that enhanced innate immune system signaling increases consumption of alcohol. Our aim was to study whether consequences of lipopolysaccharide (LPS)-induced sickness reaction increase long-term alcohol intake. Adult male C57BL/6J mice, housed in individually ventilated cages, were injected with LPS intraperitoneally (i.p.) and allowed to recover from an acute sickness reaction for 1 week before analysis of their alcohol intake in two different drinking models. Effects of LPS challenge were tested in a continuous two-bottle free choice test with increasing concentrations of alcohol and in a drinking in the dark (DID) binge model. In addition, the effect of repeatedly administered LPS during abstinence periods between binge drinking was analyzed in the DID model. In addition, the DID model was used to study the effects of the microglia inhibitor minocycline (50mg/kg/day, 4 days) and purinergic P2X7 receptor antagonist Brilliant Blue G (75mg/kg/day, 7 days) on alcohol intake. In contrast to previous findings, pretreatment with a 1-mg/kg dose of LPS did not significantly increase ethanol consumption in the continuous two-bottle choice test. As a novel finding, we report that increasing the LPS dose to 1.5mg/kg reduced consumption of 18 and 21% (v/v) ethanol. In the DID model, pretreatment with LPS (0.2-1.5mg/kg) did not significantly alter 15% or 20% ethanol consumption. Neither did repeated LPS injections affect binge alcohol drinking. Minocycline reduced alcohol, but also water, intake regardless of LPS pretreatment. No data on effects of P2X7 antagonists on alcohol consumption have been previously published; therefore, we report here that subchronic Brilliant Blue G had no effect on alcohol intake in the DID model. As a conclusion, further studies are needed to validate this LPS model of the interaction between immune system activation and alcohol consumption.

Anti-inflammatory effect of the Salvia sclarea L. ethanolic extract on lipopolysaccharide-induced periodontitis in rats

Ethnopharmacological relevanceSalvia sclarea L., clary, is an aromatic plant traditionally used in folk medicine for the treatment of various diseases and conditions. Although it has been primarily used as a stomachic, there are data on traditional use of S. sclarea as an agent against gingivitis, stomatitis and aphthae. Aim of the studyThe aim of the study was to examine the effect of the S. sclarea ethanolic extract on the lipopolysaccharide (LPS)-induced periodontitis in rats from the immunological and histopathological standpoint. Material and methodsPeriodontal inflammation in rats was induced by repeated injections of LPS from Escherichia coli into the interdental papilla between the first and second right maxillary molars. The extract was administered two times a day by oral gavage (200mg/kg body weight). The inflammatory status was assessed by the measurements of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) of gingival tissues and descriptive analysis of histological sections of periodontium. Chemical characterization of the extract was determined using high performance liquid chromatography system (HPLC). Antioxidant activity of the extract was estimated with two in vitro complementary methods: 2,2-diphenyl-1-picrylhydrazyl and β-carotene/linoleic acid models. ResultsTreatment with S. sclarea extract, compared to the untreated group of the rats, significantly diminished the process of inflammation decreasing the levels of IL-1β, IL-6 and TNF-α, reducing the gingival tissue lesions and preserving bone alveolar resorption. Considerably smaller number of inflammatory cells and larger number of fibroblasts was noticed. The administration of the extract three days earlier did not have significant preventive effects. Rosmarinic acid was the predominant compound in the extract. The extract showed strong antioxidant effects in both test systems. ConclusionsS. sclarea extract manifested anti-inflammatory effect in LPS-induced periodontitis suggesting that it may have a role as a therapeutic agent in periodontal diseases. Having in mind that overproduction of reactive oxygen species is connected to periodontitis, the strong antioxidant capacity may be contributable to anti-inflammatory properties of the extract.

Abstract # 1859 A new LPS-based mouse model of chronic inflammation and depression

Lipopolysaccharide (LPS) exposure is widely used to model depression in animals, but the transient nature of its effect and an occurrence of LPS tolerance limit its use for longitudinal studies. We tested a new schedule of injections designed to avoid tolerance and to induce neurobiological changes relevant for depression. Mice received intraperitoneal injections of escalating doses of LPS (from 0.33 to 0.83 mg/kg) once a week for 6 weeks. The results showed that LPS induced sickness behaviour every week. The peripheral blood cytokine response to LPS was modified by repeated injections. Some cytokines which were elevated by acute injection showed a lower magnitude of elevation (interleukin 6, tumor necrosis factor alpha), while others showed an increased response to repeated exposure. Interleukin 2 was only elevated by repeated LPS injections, while C-reactive protein was decreased. Anti-inflammatory cytokines were not elevated by chronic LPS exposure. This cytokine profile suggests a distinct mode of immune activation with minor signs of LPS tolerance. Assessment of adult hippocampal neurogenesis, relevant for depression neurobiology, found that exposure to escalating doses of LPS led to a reduction of neuroblasts maturation in the dentate gyrus. We conclude that minimal signs of tolerance and changes observed in hippocampal neurogenesis suggest that schedule of LPS injections used might be a suitable model to utilise in future studies looking into immunological aspects of mood disorders.

Repeated LPS Injection Induces Distinct Changes in the Kynurenine Pathway in Mice.

The immune system has been recognized as a potential contributor to psychiatric disorders. In animals, lipopolysaccharide (LPS) is used to induce inflammation and behaviors analogous to some of the symptoms in these disorders. Recent data indicate that the kynurenine pathway contributes to LPS-induced aberrant behaviors. However, data are inconclusive regarding optimal LPS dose and treatment strategy. Here, we therefore aimed to evaluate the effects of single versus repeated administration of LPS on the kynurenine pathway. Adult C57BL6 mice were given 0.83mg/kg LPS as a single or a repeated injection (LPS+LPS) and sacrificed after 24, 48, 72, or 120h. Mice receiving LPS+LPS had significantly elevated brain kynurenine levels at 24 and 48h, and elevated serum kynurenine at 24, 48 and 72h. Brain kynurenic acid and quinolinic acid were significantly increased at 24 and 48h in mice receiving LPS+LPS, whereas serum kynurenic acid levels were significantly decreased at 24h. The increase of brain kynurenic acid by LPS+LPS was likely unrelated to the higher total dose as a separate group of mice receiving 1.66mg/kg LPS as single injection 24h prior to sacrifice did not show increased brain kynurenic acid. Serum quinolinic acid levels were not affected by LPS+LPS compared to vehicle. Animals given repeated injections of LPS showed a more robust induction of the kynurenine pathway in contrast to animals receiving a single injection. These results may be valuable in light of data showing the importance of the kynurenine pathway in psychiatric disorders.

Immune system stimulation by repeated lipopolysaccharide injections alters longissimus dorsi sarcoplasmic protein profile in pigs

Immune system stimulation by repeated lipopolysaccharide injections alters longissimus dorsi sarcoplasmic protein profile in pigs

High ambient temperature alleviates the inflammatory response and growth depression in pigs challenged with Escherichia coli lipopolysaccharide

Pig production has increased in hot climate countries over recent years, but the effect of exposure to high temperatures on the health status of farm animals has not been investigated thoroughly. It is not clear how the ambient temperature (Ta) might influence responses to inflammatory challenge in pigs. The aim of the present study was to evaluate the effects of high Ta on performance and physiological parameters of growing pigs, subjected to repeated administration of Escherichia coli lipopolysaccharide (LPS).Thirty-seven pigs, each fitted with a jugular catheter, were assigned to one of two Ta conditions: thermo-neutral (TN, 24 °C) or high (HT, 30 °C). After a 14-day adaptation period, and a 7-day measurement period, pigs were administered five repeated injections of LPS at 48 h intervals. Irrespective of Ta, the LPS challenge reduced feed consumption and increased plasma pro-inflammatory cytokines, haptoglobin and cortisol. However, the extent of these responses was greater in pigs at TN than HT. In both groups, plasma thyroxine and triiodothyronine concentrations decreased, following the first LPS injection and thereafter returned to baseline, which occurred faster at HT than at TN. Moreover, the LPS challenge decreased growth and feed efficiency in pigs kept at TN, which was not observed in pigs kept at HT. The results suggest a greater capacity of pigs to limit the physiological and metabolic disturbances caused by inflammatory challenge, when kept at HT, compared to TN.

PI3K signaling pathway in leptin receptor expressing cells is not required for endotoxemia‐induced hypophagia (LB764)

Hypophagia and increased energy expenditure under inflammatory conditions, such as that observed after bacterial lipopolysaccharide (LPS) administration, are associated with leptin secretion and stimulation of JAK2‐STAT3 signaling in the CNS. Anorexic effect of leptin also depends on the activation of PI3K signaling pathway. However, the role of PI3K in the endotoxemia‐induced anorexia remains an open question. To investigate if PI3K in leptin receptor expressing cells has a role in the hypophagia and body weight (BW) loss induced by acute endotoxin treatment, mice expressing Cre‐recombinase driven by Lepr promoter were crossed with mice with loxP‐modified p110α allele (Pi3kca gene) and were treated with LPS (100ug/Kg, ip) (1 LPS). Since long term exposure to endotoxin induces desensitization of hypophagia and an inability of leptin to phosphorylate STAT3, we also tested PI3K role in feeding and BW gain in mice submitted to repeated LPS injections (6 LPS). Single LPS, but not 6 LPS control mice (p110α fl/fl), showed reduced food intake (1 LPS: 1.67 ± 0.18g, 6 LPS: 3.07 ± 0.34g), compared with saline injection (3.83 ± 0.25g). Acute LPS also reduced BW (‐0.97 ± 0.19g), compared with 6 LPS (0.49 ± 0.16g) and saline groups (0.31 ± 0.12g). The hypophagia and BW loss induced by 1 LPS were similar in p110α fl/fl‐LepR cre/cre mice; (1.72 ± 0.28g; ‐0.91 ± 0.14g BW), with no changes in 6 LPS (3.47 ± 0.12g; 0.92 ± 0.09g BW) and saline groups (3.25 ± 0.22g; 0.47 ± 0.18g BW). The data suggest that PI3K in leptin receptor expressing cells is not required for acute LPS induced anorexia and has no effect on food intake and BW gain during long term endotoxemia.Grant Funding Source: Supported by FAPESP and NIH: HD61539; HD 69702

The development of endotoxin tolerance, and the role of hypothalamo-pituitary-adrenal function and glucocorticoids in Pekin ducks

Endotoxin tolerance represents a state of abated immunological responsiveness to pyrogens, which, in mammals, leads to the decline or abolition of the fever response. The development of endotoxin tolerance in birds is not well understood; consequently, the impact of repeated pathogenic exposure on the avian febrile response, and thus on the ability of birds to fight recurrent infection, is not known. We determined the effect of repeated injections of lipopolysaccharide (LPS) on the febrile response of Pekin ducks. We gave ducks five injections of LPS, spaced 1, 4 or 10 days apart, and recorded their core body temperature with abdominally implanted temperature data loggers. Once we established that Pekin ducks developed endotoxin tolerance, we investigated the effect of repeated injections of LPS on the central and peripheral segments of the hypothalamo-pituitary-adrenal (HPA) axis in an attempt to elucidate the role of glucocorticoids in the modulation of the febrile response during the tolerant period. When our ducks became tolerant to LPS, they had significantly higher basal levels of plasma corticosterone (CORT, the principal glucocorticoid in birds), and their HPA response to treatment with LPS was blunted. We propose that the augmented levels of basal plasma CORT resulted from sensitized HPA function, and this, in turn, contributed to the development of endotoxin tolerance. Regulation of the circulating level of CORT might be a possible target for the re-establishment of appropriate immune responses in birds.

Cytokine and chemokine responses in serum and brain after single and repeated injections of lipopolysaccharide: Multiplex quantification with path analysis

Administration of the proinflammatory molecule lipopolysaccharide (LPS) alters transport rates for many peptides across the blood-brain barrier (BBB). We and others have previously shown that effects of LPS on BBB transport are highly dependent on the injection paradigm used, and timing of the study. Cytokine expression in both brain and serum compartments influences the BBB response to an inflammatory stimulus, and mediates changes in BBB transport. Here, we used multianalyte technology to simultaneously determine the responses of 13 cytokines and chemokines (G-CSF, GM-CSF, IL-1α, IL-1β, IL-6, IL-10, IL-13, IP-10, KC, MCP-1, MIP-1α, RANTES, and TNF-α) in brain and blood to single and repeated injections of LPS and path analysis to determine the major relations among these analytes. Major findings are: (1) in comparison to measurements taken from a time course after a single injection of LPS, the three injection regimen of LPS produced significantly higher levels in brain for G-CSF, IL-1α, IL-6, MCP-1, MIP-1α, and TNF and in serum for G-CSF, IL-6, and GM-CSF and (2) path analysis distinguished direct from indirect correlations between analyte pairs, with MCP-1, IL-6, G-CSF, and KC mediating relations among these cytokines both within and between serum and brain compartments. These results suggest that potentiation of cytokine levels in brain and serum compartments could play important roles in the regulation of BBB transport, and that our novel application of an established statistical method can be used to assess direct correlations within multiplexed datasets.

Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation

BackgroundAlzheimer's disease (AD) is characterized by extensive loss of neurons in the brain of AD patients. Intracellular accumulation of beta-amyloid peptide (Aβ) has also shown to occur in AD. Neuro-inflammation has been known to play a role in the pathogenesis of AD.MethodsIn this study, we investigated neuro-inflammation and amyloidogenesis and memory impairment following the systemic inflammation generated by lipopolysaccharide (LPS) using immunohistochemistry, ELISA, behavioral tests and Western blotting.ResultsIntraperitoneal injection of LPS, (250 μg/kg) induced memory impairment determined by passive avoidance and water maze tests in mice. Repeated injection of LPS (250 μg/kg, 3 or 7 times) resulted in an accumulation of Aβ1–42 in the hippocampus and cerebralcortex of mice brains through increased β- and γ-secretase activities accompanied with the increased expression of amyloid precursor protein (APP), 99-residue carboxy-terminal fragment of APP (C99) and generation of Aβ1–42 as well as activation of astrocytes in vivo. 3 weeks of pretreatment of sulindac sulfide (3.75 and 7.5 mg/kg, orally), an anti-inflammatory agent, suppressed the LPS-induced amyloidogenesis, memory dysfunction as well as neuronal cell death in vivo. Sulindac sulfide (12.5–50 μM) also suppressed LPS (1 μg/ml)-induced amyloidogenesis in cultured neurons and astrocytes in vitro.ConclusionThis study suggests that neuro-inflammatory reaction could contribute to AD pathology, and anti-inflammatory agent could be useful for the prevention of AD.

Expression of hypothalamic neuropeptides and the desensitization of pituitary–adrenal axis and hypophagia in the endotoxin tolerance

Repeated exposure to lipopolysaccharide (LPS) induces desensitization of hypothalamus–pituitary–adrenal axis (HPA) responses and hypophagia. We investigated the interplay between the neural circuitries involved in the control of food intake and HPA axis activity following single or repeated LPS injections. Male Wistar rats received a single or repeated i.p. injection of LPS (100 μg/kg) for 6 days and were subdivided into four groups: 6 saline, 5 saline + 1 LPS, 5 LPS + 1 saline and 6 LPS. Animals with a single exposure to LPS showed increased plasma levels of ACTH, CORT, PRL, TNF-α and also CRF mRNA in the paraventricular nucleus of the hypothalamus. These animals exhibited a reduced food intake and body weight associated with an increase of CART expression in the arcuate nucleus (ARC). Leptin plasma levels were not altered. On the other hand, repeated LPS administration did not alter ACTH, CORT, PRL and TNF-α, but it reduced leptin level, compared to single LPS or saline treatment. Furthermore, repeated LPS administration did not increase CRF or CART mRNA expression. Food intake and weight gain after repeated LPS injections were not different from saline-treated animals. There was no change in NPY and POMC mRNA expression in the ARC after single or repeated injections of LPS. In conclusion, desensitization induced by repeated exposure to LPS involves the blockade of HPA axis activation and anorexigenic response, which are both associated with an unresponsiveness of TNF-α production and CRF and CART expression in the hypothalamus.

Bacterial Endotoxin: A Trigger Factor for Alcoholic Pancreatitis? Evidence From a Novel, Physiologically Relevant Animal Model

This study examined the possible role of endotoxinemia (from increased gut permeability) as an additional trigger factor for overt pancreatic disease and as a promoter of chronic pancreatic injury in alcoholics by using a rat model of chronic alcohol feeding and in vitro experiments with cultured pancreatic stellate cells (PSCs), the key mediators of pancreatic fibrosis. In the in vivo model, Sprague-Dawley rats fed isocaloric Lieber-DeCarli liquid diets +/- alcohol for 10 weeks were challenged with a single dose or 3 repeated doses of the endotoxin lipopolysaccharide (LPS) and the pancreas was examined. In the in vitro studies, rat PSCs were assessed for activation on exposure to LPS +/- ethanol. The expression of LPS receptors TLR4 and CD14 also was assessed in rat and human PSCs. In the in vivo model, single or repeated LPS challenge resulted in significantly greater pancreatic injury in alcohol-fed rats compared with rats fed the control diet without alcohol. Notably, repeated LPS injections caused pancreatic fibrosis in alcohol-fed rats, but not in rats fed the control diet. In the in vitro studies, PSCs were activated by LPS. Alcohol + LPS exerted a synergistic effect on PSC activation. Importantly, both rat and human PSCs expressed TLR4 and CD14. This study describes, for the first time, a clinically relevant animal model of alcohol-related pancreatic injury and provides strong in vivo and in vitro evidence that suggests that LPS is a trigger factor in the initiation and progression of alcoholic pancreatitis.