Systemic Lipopolysaccharide Challenge Induces Inflammatory Changes in Rat Dorsal Root Ganglia: An Ex Vivo Study.

Inflammatory processes within the peripheral nervous system (PNS) are associated with symptoms of hyperalgesia and allodynia. Pro-inflammatory mediators, such as cytokines or prostaglandins, modulate the excitability of nociceptive neurons, called peripheral sensitization. Here, we aimed to examine if previously reported effects of in vitro stimulation with lipopolysaccharide (LPS) on primary cell cultures of dorsal root ganglia (DRG) reflect changes in a model of LPS-induced systemic inflammation in vivo. Male rats were intraperitoneally injected with LPS (100 µg/kg) or saline. Effects of systemic inflammation on expression of inflammatory mediators, neuronal Ca2+ responses, and activation of inflammatory transcription factors in DRG were assessed. Systemic inflammation was accompanied by an enhanced expression of pro-inflammatory cytokines and cyclooxygenase-2 in lumbar DRG. In DRG primary cultures obtained from LPS-treated rats enhanced neuronal capsaicin-responses were detectable. Moreover, we found an increased activation of inflammatory transcription factors in cultured macrophages and neurons after an in vivo LPS challenge compared to saline controls. Overall, our study emphasizes the role of inflammatory processes in the PNS that may be involved in sickness-behavior-associated hyperalgesia induced by systemic LPS treatment. Moreover, we present DRG primary cultures as tools to study inflammatory processes on a cellular level, not only in vitro but also ex vivo.

Norepinephrine Inhibits Lipopolysaccharide-Stimulated TNF-α but Not Oxylipin Induction in n-3/n-6 PUFA-Enriched Cultures of Circumventricular Organs.

Sensory circumventricular organs (sCVOs) are pivotal brain structures involved in immune-to-brain communication with a leaky blood-brain barrier that detect circulating mediators such as lipopolysaccharide (LPS). Here, we aimed to investigate the potential of sCVOs to produce n-3 and n-6 oxylipins after LPS-stimulation. Moreover, we investigated if norepinephrine (NE) co-treatment can alter cytokine- and oxylipin-release. Thus, we stimulated rat primary neuroglial sCVO cultures under n-3- or n-6-enriched conditions with LPS or saline combined with NE or vehicle. Supernatants were assessed for cytokines by bioassays and oxylipins by HPLC-MS/MS. Expression of signaling pathways and enzymes were analyzed by RT-PCR. Tumor necrosis factor (TNF)α bioactivity and signaling, IL-10 expression, and cyclooxygenase (COX)2 were increased, epoxide hydroxylase (Ephx)2 was reduced, and lipoxygenase 15-(LOX) was not changed by LPS stimulation. Moreover, LPS induced increased levels of several n-6-derived oxylipins, including the COX-2 metabolite 15d-prostaglandin-J2 or the Ephx2 metabolite 14,15-DHET. For n-3-derived oxylipins, some were down- and some were upregulated, including 15-LOX-derived neuroprotectin D1 and 18-HEPE, known for their anti-inflammatory potential. While the LPS-induced increase in TNFα levels was significantly reduced by NE, oxylipins were not significantly altered by NE or changes in TNFα levels. In conclusion, LPS-induced oxylipins may play an important functional role in sCVOs for immune-to-brain communication.

Gabapentinoids Suppress Lipopolysaccharide-Induced Interleukin-6 Production in Primary Cell Cultures of the Rat Spinal Dorsal Horn.

INTRODUCTION: Gabapentin and pregabalin are drugs to treat neuropathic pain. Several studies highlighted effects on presynaptic terminals of nociceptors. Via binding to α2δ subunits of voltage-gated calcium channels, gabapentinoids modulate the synaptic transmission of nociceptive information. However, recent studies revealed further properties of these substances. Treatment with gabapentin or pregabalin in animal models of neuropathic pain resulted not only in reduced symptoms of hyperalgesia but also in an attenuated activation of glial cells and decreased production of pro-inflammatory mediators in the spinal dorsal horn. METHODS: In the present study, we aimed to investigate the impact of gabapentinoids on the inflammatory response of spinal dorsal horn cells, applying the established model of neuro-glial primary cell cultures of the superficial dorsal horn (SDH). We studied effects of gabapentin and pregabalin on lipopolysaccharide (LPS)-induced cytokine release (bioassays), expression of inflammatory marker genes (RT-qPCR), activation of transcription factors (immunocytochemistry), and Ca2+ responses of SDH neurons to stimulation with substance P and glutamate (Ca2+-imaging). RESULTS: We detected an attenuated LPS-induced expression and release of interleukin-6 by SDH cultures in the presence of gabapentinoids. In addition, a significant main effect of drug treatment was observed for mRNA expression of microsomal prostaglandin E synthase 1 and the inhibitor of nuclear factor kappa B. Nuclear translocation of inflammatory transcription factors in glial cells was not significantly affected by gabapentinoid treatment. Moreover, both substances did not modulate neuronal responses upon stimulation with substance P or glutamate. CONCLUSION: Our results provide evidence for anti-inflammatory capacities of gabapentinoids on the acute inflammatory response of SDH primary cultures upon LPS stimulation. Such effects may contribute to the pain-relieving effects of gabapentinoids.

SK-Channel Activation Alters Peripheral Metabolic Pathways in Mice, but Not Lipopolysaccharide-Induced Fever or Inflammation.

PURPOSE: Previously, we have shown that CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine), a pharmacological small-conductance calcium-activated potassium (SK)-channel positive modulator, antagonizes lipopolysaccharide (LPS)-induced cytokine expression in microglial cells. Here, we aimed to test its therapeutic potential for brain-controlled sickness symptoms, brain inflammatory response during LPS-induced systemic inflammation, and peripheral metabolic pathways in mice. METHODS: Mice were pretreated with CyPPA (15 mg/kg IP) 24 hours before and simultaneously with LPS stimulation (2.5 mg/kg IP), and the sickness response was recorded by a telemetric system for 24 hours. A second cohort of mice were euthanized 2 hours after CyPPA or solvent treatment to assess underlying CyPPA-induced mechanisms. Brain, blood, and liver samples were analyzed for inflammatory mediators or nucleotide concentrations using immunohistochemistry, real-time PCR and Western blot, or HPLC. Moreover, we investigated CyPPA-induced changes of UCP1 expression in brown adipose tissue (BAT)-explant cultures. RESULTS: CyPPA treatment did not affect LPS-induced fever, anorexia, adipsia, or expression profiles of inflammatory mediators in the hypothalamus or plasma or microglial reactivity to LPS (CD11b staining and CD68 mRNA expression). However, CyPPA alone induced a rise in core body temperature linked to heat production via altered metabolic pathways like reduced levels of adenosine, increased protein content, and increased UCP1 expression in BAT-explant cultures, but no alteration in ATP/ADP concentrations in the liver. CyPPA treatment was accompanied by altered pathways, including NFκB signaling, in the hypothalamus and cortex, while circulating cytokines remained unaltered. CONCLUSION: Overall, while CyPPA has promise as a treatment strategy, in particular according to results from in vitro experiments, we did not reveal anti-inflammatory effects during severe LPS-induced systemic inflammation. Interestingly, we found that CyPPA alters metabolic pathways inducing short hyperthermia, most likely due to increased energy turnover in the liver and heat production in BAT.

Neuroinflammation in Primary Cultures of the Rat Spinal Dorsal Horn Is Attenuated in the Presence of Adipose Tissue-Derived Medicinal Signalling Cells (AdMSCs) in a Co-cultivation Model.

Neuroinflammation within the superficial dorsal horn (SDH) of the spinal cord induces inflammatory pain with symptoms of hyperalgesia and allodynia. Glial activation and production of inflammatory mediators (e.g. cytokines) is associated with modulation of nociceptive signalling. In this context, medicinal signalling cells, e.g. obtained from adipose tissue (AdMSCs), gained attention due to their capacity to modulate the inflammatory response in several diseases, e.g. spinal cord injury. We applied the recently established mixed neuroglial primary cell culture of the rat SDH to investigate effects of AdMSCs on the inflammatory response of SDH cells. Following establishment of a co-cultivation system, we performed specific bioassays for tumour necrosis factor alpha (TNFα) and interleukin (IL)-6, RT-qPCR and immunocytochemistry to detect changes in cytokine production and glial activation upon inflammatory stimulation with lipopolysaccharide (LPS). LPS-induced expression and release of pro-inflammatory cytokines (TNFα, IL-6) by SDH cells was significantly attenuated in the presence of AdMSCs. Further evidence for anti-inflammatory capacities of AdMSCs derived from a blunted LPS-induced TNFα/IL-10 expression ratio and suppressed nuclear translocation of the inflammatory transcription factor nuclear factor kappa B (NFκB) in SDH microglial cells. Expression of IL-10, transforming growth factor beta (TGF-ß) and TNFα-stimulated gene-6 (TSG-6) was detected in AdMSCs, which are putative candidates for anti-inflammatory capacities of these cells. We present a novel co-cultivation system of AdMSCs with neuroglial primary cultures of the SDH to investigate immunomodulatory effects of AdMSCs at a cellular level.

Sensitization of primary cultures from rat dorsal root ganglia with lipopolysaccharide (LPS) requires a robust inflammatory response.

OBJECTIVE: We investigated whether it is possible to induce a state of "LPS-sensitization" in neurons of primary cultures from rat dorsal root ganglia by pre-treatment with ultra-low doses of LPS. METHODS: DRG primary cultures were pre-treated with low to ultra-low doses of LPS (0.001-0.1 µg/ml) for 18 h, followed by a short-term stimulation with a higher LPS-dose (10 µg/ml for 2 h). TNF-α in the supernatants was measured as a sensitive read out. Using the fura-2 340/380 nm ratio imaging technique, we further investigated the capsaicin-evoked Ca2+-signals in neurons from DRG, which were pre-treated with a wide range of LPS-doses. RESULTS: Release of TNF-α evoked by stimulation with 10 µg/ml LPS into the supernatant was not significantly modified by pre-exposure to low to ultra-low LPS-doses. Capsaicin-evoked Ca2+-signals were significantly enhanced by pre-treatment with LPS doses being above a certain threshold. CONCLUSION: Ultra-low doses of LPS, which per se do not evoke a detectable inflammatory response, are not sufficient to sensitize neurons (Ca2+-responses) and glial elements (TNF-α-responses) of the primary afferent somatosensory system.

Microbubble-mediated sonothrombolysis with BR38 of a venous full blood thrombus in a rat embolic stroke model.

BACKGROUND: Early recanalization of an occluded vessel is associated with a better clinical outcome in acute ischemic stroke. Intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) is only available in a minority of patients and often fails to reopen the occluded vessel. Mechanical recanalization is more effective in this matter but only available for selected patients when a thrombectomy centre can be reached. Therefore, sonothrombolysis might represent an alternative or complementary approach. Here, we tested microbubble-mediated sonothrombolysis (mmSTL) in a thromboembolic stroke model for middle cerebral artery occlusion (MCAO) in rats. METHODS: Sixty-seven male Wistar rats underwent MCAO using an autologous full blood thrombus and were randomly assigned to four groups receiving rt-PA, mmSTL, a combination of both, or a placebo. Diagnostic workup included neurological examination, assessment of infarct size, and presence of intracerebral haemorrhage by magnetic resonance imaging (MRI) and presence of microbleedings in histological staining. RESULTS: Neurological examination revealed no differences between the treatment groups. In all treatment groups, there was a reduction in infarct size 24 hours after MCAO as compared to the placebo (P≤0.05), but there were no differences between the active treatment groups (P>0.05) (placebo 0.75±0.10 cm3; mmSTL 0.43±0.07 cm3; rt-PA 0.4±0.07 cm3; mmSTL + rt-PA 0.27±0.08 cm3). Histological staining displayed intracerebral microbleedings in all animals. The frequency of gross bleeding detected by MRI did not differ between the groups (placebo 3; mmSTL 4; rt-PA 2; mmSTL + rt-PA 2; P>0.05) and was not associated with worse performance in clinical testing (P>0.05). There were no statistical differences in the mortality between the groups (P>0.05). CONCLUSIONS: Our study showed the efficacy and safety of mmSTL with or without rt-PA in an embolic rat stroke model using a continuous full blood thrombus. Sonothrombolysis might be useful for patients who need to be transported to a thrombectomy centre or for those with distal vessel occlusion.

LPS Primes Brain Responsiveness to High Mobility Group Box-1 Protein.

High mobility group box (HMGB)1 action contributes to late phases of sepsis, but the effects of increased endogenous plasma HMGB1 levels on brain cells during inflammation are unclear. Here, we aimed to further investigate the role of HMGB1 in the brain during septic-like lipopolysaccharide-induced inflammation in rats (LPS, 10 mg/kg, i.p.). HMGB-1 mRNA expression and release were measured in the periphery/brain by RT-PCR, immunohistochemistry and ELISA. In vitro experiments with disulfide-HMGB1 in primary neuro-glial cell cultures of the area postrema (AP), a circumventricular organ with a leaky blood-brain barrier and direct access to circulating mediators like HMGB1 and LPS, were performed to determine the direct influence of HMGB1 on this pivotal brain structure for immune-to-brain communication. Indeed, HMGB1 plasma levels stayed elevated after LPS injection. Immunohistochemistry of brains and AP cultures confirmed LPS-stimulated cytoplasmatic translocation of HMGB1 indicative of local HMGB1 release. Moreover, disulfide-HMGB1 stimulation induced nuclear factor (NF)-κB activation and a significant release of interleukin-6, but not tumor necrosis factor α, into AP culture supernatants. However, only a few AP cells directly responded to HMGB1 with increased intracellular calcium concentration. Interestingly, priming with LPS induced a seven-fold higher percentage of responsive cells to HMGB1. We conclude that, as a humoral and local mediator, HMGB1 enhances brain inflammatory responses, after LPS priming, linked to sustained sepsis symptoms.

Manifestation of lipopolysaccharide-induced tolerance in neuro-glial primary cultures of the rat afferent somatosensory system.

OBJECTIVE: Bacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed LPS tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model of LPS tolerance within these structures. METHODS: Tolerance was induced by pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-term stimulation with a higher LPS dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures). Supernatants from both cultures were assayed for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by highly sensitive bioassays. RESULTS: At the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-α and enhanced IL-10/TNF-α expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS tolerance. SDH cultures further showed reduced release of TNF-α into the supernatants and attenuated TNF-α immunoreactivity in microglial cells. In the state of LPS tolerance macrophages from DRG and microglial cells from SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFκB and NF-IL6. Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG and astrocytes from SDH in LPS tolerant cultures. CONCLUSION: A state of LPS tolerance can be induced in primary cultures from the afferent somatosensory system, which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study the effects of LPS tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns upon inflammatory stimulation.

Mitochondrial pyruvate carrier as a key regulator of fever and neuroinflammation.

The mitochondrial pyruvate carrier (MPC) is an inner-membrane transporter that facilitates pyruvate uptake from the cytoplasm into mitochondria. We previously reported that MPC1 protein levels increase in the hypothalamus of animals during fever induced by lipopolysaccharide (LPS), but how this increase contributes to the LPS responses remains to be studied. Therefore, we investigated the effect of UK 5099, a classical MPC inhibitor, in a rat model of fever, on hypothalamic mitochondrial function and neuroinflammation in LPS-stimulated preoptic area (POA) primary microcultures. Intracerebroventricular administration of UK 5099 reduced the LPS-induced fever. High-resolution respirometry revealed an increase in oxygen consumption and oxygen flux related to ATP synthesis in the hypothalamic homogenate from LPS-treated animals linked to mitochondrial complex I plus II. Preincubation with UK 5099 prevented the LPS-induced increase in oxygen consumption, ATP synthesis and spare capacity only in complex I-linked respiration and reduced mitochondrial H2O2 production. In addition, treatment of rat POA microcultures with UK 5099 reduced the secretion of the proinflammatory and pyrogenic cytokines TNFα and IL-6 as well as the immunoreactivity of inflammatory transcription factors NF-κB and NF-IL6 four hours after LPS stimulation. These results suggest that the regulation of mitochondrial pyruvate metabolism through MPC inhibition may be effective in reducing neuroinflammation and fever.

Association of bovine uterine involution disturbances with serum neuropeptide concentrations.

BACKGROUND AND AIM: Puerperal diseases influence fertility and should be diagnosed as soon as possible. This study aimed to evaluate the applicability of serum concentrations of substance P (SP), vasoactive intestinal polypeptide (VIP), and interleukin (IL)1ß in the early diagnosis of uterine involution disturbances. MATERIALS AND METHODS: Blood serum samples of 86 dairy cows from six different farms were harvested within the first 20 days after calving from cows with uterine involution disturbances and healthy controls, respectively. Serum concentrations for SP, VIP, and IL-1ß were determined using commercially available ELISA test kits. Statistical analyses included timely changes in blood serum levels and group comparisons of healthy cows and cows with uterine disease. RESULTS: SP concentrations increased significantly within 20 days after calving (p<0.04) with no significant difference observed between the groups. Moreover, no significant differences were found between VIP and log IL-1ß. CONCLUSION: Results showed that none of the examined serum parameters seems suitable as indicator of uterine involution disorders. Due to the timely changes in serum concentrations of SP after calving, a correlation to diseases might not be precluded. Further research is needed as regards the establishment of normative values concerning this parameter.

Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the afferent somatosensory system.

One maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 µg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 µg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

Age-Dependent Changes of Adipokine and Cytokine Secretion From Rat Adipose Tissue by Endogenous and Exogenous Toll-Like Receptor Agonists.

White adipose tissue but recently also brown adipose tissue have emerged as endocrine organs. Age-associated obesity is accompanied by prolonged and elevated lipopolysaccharide (LPS)-induced sickness symptoms and increased cytokine and adipokine levels in the circulation partially originating from adipose tissue. In the present study, ex vivo fat explants were used to investigate how the exogenous pathogen-associated molecular pattern (PAMP) LPS or the endogenous danger-associated molecular patterns (DAMPs) high mobility group box-1 protein (HMGB1) and biglycan modulate the release of cytokines and adipokines/batokines and, thus, could influence systemic and/or local inflammation. The response of adipose tissue (epididymal, retroperitoneal, subcutaneous, and brown) was compared between young lean and old obese rats (2 vs. 24 months old). LPS induced a strong interleukin (IL)-6 and tumor necrosis factor (TNF) alpha release into the supernatant of all adipose tissue types investigated. HMGB1 (subcutaneous) and biglycan (retroperitoneal) led to an increased release of IL-6 and TNFalpha (HMGB1) and decreased visfatin and adiponectin (biglycan) secretion from epididymal adipose tissue (young rats). Visfatin was also decreased by HMGB1 in retroperitoneal adipose tissue of old rats. We found significantly higher leptin (all fat pads) and adiponectin (subcutaneous) levels in supernatants of adipose tissue from old compared to young rats, whereas visfatin secretion showed the opposite. The expression of the biglycan receptor Toll-like receptor (TLR) 2 as well as the LPS and HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE) were reduced with age (TLR4/RAGE) and by stimulation with their ligands (subcutaneous). Overall, we revealed that adipokines/adipose-tissue released cytokines show some modulation of their release caused by mediators of septic (batokines) and sterile inflammation with potential implication for acute and chronic disease. Moreover, aging may increase or decrease the release of fat-derived mediators. These data show that DAMPS and LPS locally modulate cytokine secretion while only DAMPS but not LPS can locally alter adipokine secretion during inflammation.

Effects of gabapentinoids on responses of primary cultures from rat dorsal root ganglia to inflammatory or somatosensory stimuli.

Background Gabapentinoids are known to reduce neuropathic pain. The aim of this experimental study was to investigate whether gabapentinoids exert anti-inflammatory and/or anti-nociceptive effects at the cellular level using primary cultures of rat dorsal root ganglia (DRG). Methods Cells from rat DRG were cultured in the presence of gabapentin or pregabalin, and we tested the effects of subsequent stimulation with lipopolysaccharide (LPS) on the expression of genes (real-time polymerase chain reaction) and production of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) by specific bioassays. Using Ca2+ imaging, we further investigated in neurons the effects of gabapentinoids upon stimulation with the TRPV-1 agonist capsaicin. Results There is a small influence of gabapentinoids on the inflammatory response to LPS stimulation, namely, a significantly reduced expression of IL-6. Pregabalin and gabapentin further seem to exert a moderate inhibitory influence on capsaicin-induced Ca2+ signals in DRG neurons. Conclusions Although the single inhibitory effects of gabapentinoids on inflammatory and nociceptive responses are moderate, a combination of both effects might provide an explanation for the proposed function of these substances as an adjuvant for the reduction of neuropathic pain.

Neurons and astrocytes of the chicken hypothalamus directly respond to lipopolysaccharide and chicken interleukin-6.

In 4-5-month-old chicken, intravenous injections of bacterial lipopolysaccharide (LPS) induced a dose-dependent fever response and a pronounced increase of circulating interleukin-6 (IL-6). To assess a possible role for IL-6 in the brain of birds, a hypothalamic neuro-glial primary culture from 1-day-old chicken was established. Each well of cultured hypothalamic cells contained some 615 neurons, 1350 astrocytes, and 580 microglial cells on average. Incubation of chicken hypothalamic primary cultures with 10 or 100 µg/ml LPS induced a dose-dependent release of bioactive IL-6 into the supernatant. Populations of hypothalamic neurons (4%) and astrocytes (12%) directly responded to superfusion with buffer containing 10 µg/ml LPS with a transient increase of intracellular calcium, a sign of direct cellular activation. Stimulation of hypothalamic cultures with buffer containing 50 ng/ml chicken IL-6 induced calcium signaling in 11% of neurons and 22% of astrocytes investigated. These results demonstrate that IL-6 is produced in the periphery and in the hypothalamus in response to LPS in chicken. The observed cellular responses of hypothalamic cells to chicken IL-6 indicate that this cytokine may readily be involved in the manifestation of fever in the avian hypothalamus.

Visualizing and Profiling Lipids in the OVLT of Fat-1 and Wild Type Mouse Brains during LPS-Induced Systemic Inflammation Using AP-SMALDI MSI.

Lipids, including omega-3 polyunsaturated fatty acids (n-3-PUFAs), modulate brain-intrinsic inflammation during systemic inflammation. The vascular organ of the lamina terminalis (OVLT) is a brain structure important for immune-to-brain communication. We, therefore, aimed to profile the distribution of several lipids (e.g., phosphatidyl-choline/ethanolamine, PC/PE), including n-3-PUFA-carrying lipids (esterified in phospholipids), in the OVLT during systemic lipopolysaccharide(LPS)-induced inflammation. We injected wild type and endogenously n-3-PUFA producing fat-1 transgenic mice with LPS (i.p., 2.5 mg/kg) or PBS. Brain samples were analyzed using immunohistochemistry and high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization orbital trapping mass spectrometry imaging (AP-SMALDI-MSI) for spatial resolution of lipids. Depending on genotype and treatment, several distinct distribution patterns were observed for lipids [e.g., lyso(L)PC (16:0)/(18:0)] proposed to be involved in inflammation. The distribution patterns ranged from being homogeneously disseminated [LPC (18:1)], absent/reduced signaling within the OVLT relative to adjacent preoptic tissue [PE (38:6)], either treatment- and genotype-dependent or independent low signal intensities [LPC (18:0)], treatment- and genotype-dependent [PC 38:6)] or independent accumulation in the OVLT [PC (38:7)], and accumulation in commissures, e.g., nerve fibers like the optic nerve [LPE (18:1)]. Overall, screening of lipid distribution patterns revealed distinct inflammation-induced changes in the OVLT, highlighting the prominent role of lipid metabolism in brain inflammation. Moreover, known and novel candidates for brain inflammation and immune-to-brain communication were detected specifically within this pivotal brain structure, a window between the periphery and the brain. The biological significance of these newly identified lipids abundant in the OVLT and the adjacent preoptic area remains to be further analyzed.

Cytoglobin Attenuates Neuroinflammation in Lipopolysaccharide-Activated Primary Preoptic Area Cells via NF-κB Pathway Inhibition.

Cytoglobin (Cygb) is a hexacoordinate protein, associated with the transport of oxygen, nitric oxide scavenging, tumor suppression and protection against oxidative stress and inflammation. This protein is expressed in brain areas including the preoptic area (POA) of the anterior hypothalamus, the region responsible for the regulation of body temperature. In this study, we show that Cygb is upregulated in the rat hypothalamus 2.5 h and 5 h after intravenous administration of lipopolysaccharide (LPS). We investigated the effect of treatment with Cygb in POA primary cultures stimulated with LPS for 4 h. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured and the results showed that Cygb reduced the concentrations of both cytokines. We further observed a decrease in immunoreactivity of the inflammatory transcription factor nuclear factor-κB (NF-κB), but not NF-IL6 and STAT3, in the nucleus of Cygb-treated POA cells. These findings suggest that Cygb attenuates the secretion of IL-6 and TNF-α in LPS-stimulated POA primary cultures via inhibition of the NF-κB signaling pathway, indicating that this protein might play an important role in the control of neuroinflammation and fever.

Serum Levels of Neuropeptides in Cows with Left Abomasal Displacement.

Abomasal displacement (AD) to the left is a common disease in high-yielding dairy cows after parturition. In view of the previously reported changes in tissue neuropeptide concentrations in cows with AD, the primary aim of this study was to evaluate the effect of AD and breed on serum neuropeptide concentrations. For this purpose, blood samples of 33 German Holstein (GH) cows with AD, 36 healthy controls (GH), and 32 healthy German Fleckvieh (GF) cows were collected, and concentrations of substance P (SP), vasoactive intestinal polypeptide (VIP), and interleukin1ß (IL-1ß) were measured via commercially available ELISA kits. To examine the effect of AD, we compared GH cows with and without AD and observed no significant effects of AD on SP, VIP, or Il-1 ß concentrations. To evaluate the effect of breed, we compared healthy GH with healthy GF cows and detected markedly higher VIP serum levels in the healthy GF cows (p < 0.01). No significant differences in SP or IL-1ß were detected. According to our results, there seems to be no effect of AD on the serum concentrations of SP, VIP, or IL-1 ß. In contrast, there seems to be a breed difference concerning serum VIP concentrations.

Primary Cultures from Rat Dorsal Root Ganglia: Responses of Neurons and Glial Cells to Somatosensory or Inflammatory Stimulation.

Primary cultures of rat dorsal root ganglia (DRG) consist of neurons, satellite glial cells and a moderate number of macrophages. Measurements of increased intracellular calcium [Ca2+]i induced by stimuli, have revealed that about 70% of DRG neurons are capsaicin-responsive nociceptors, while 10% responded to cooling and or menthol (putative cold sensors). Cultivation of DRG in the presence of a moderate dose of lipopolysaccharide (LPS, 1 µg/ml) enhanced capsaicin-induced Ca2+ signals. We therefore investigated further properties of DRG primary cultures stimulated with 10 µg/ml LPS for a short period. Exposure to LPS for 2 h resulted in pronounced release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) into the supernatants of DRG cultures, increased expression of both cytokines in the DRG cells and increased TNF immunoreactivity predominantly in macrophages. We further observed an accumulation of the inflammatory transcription factors NF-IL6 and STAT3 in the nuclei of LPS-exposed DRG neurons and macrophages. In the presence of the cytotoxic agent cisplatin (5 or 10 µg/ml), the number of macrophages was decreased significantly, the growth of satellite glial cells was markedly suppressed, but the vitality and stimulus-induced Ca2+ signals of DRG neurons were not impaired. Under these conditions the LPS-induced production and expression of TNF-α and IL-6 were blunted. Our data suggest a potential role for macrophages and satellite glial cells in the initiation of inflammatory processes that develop in sensory ganglia upon injury or exposure to pathogens.