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.

Expression of adipokines and adipocytokines by epidural adipose tissue in cauda equina syndrome in dogs.

BACKGROUND: Compression of epidural adipose tissue (EAT) within the scope of cauda equina syndrome (CES) could lead to an enhanced expression of inflammatory mediators, possibly contributing to pain amplification in dogs. OBJECTIVES: To analyze expression of inflammatory adipo(-cyto)kines within the EAT of dogs with CES. ANIMALS: Client-owned dogs: 15 dogs with CES and 9 dogs euthanized for unrelated medical reasons (controls). METHODS: Prospective, experimental study. Epidural adipose tissue and subcutaneous adipose tissue were collected during dorsal laminectomy and used for real-time quantitative polymerase chain reaction. Tissue explants were cultured for measurements of inflammation-induced release of cytokines. RESULTS: Results show a CES-associated upregulation of the cytokines tumor necrosis factor alpha (TNFα: mean ± SD: 18.88 ± 11.87, 95% CI: 10.90-26.86 vs 9.66 ± 5.22, 95% CI: 5.29-14.02, *: P = .04) and interleukin- (IL-) 10 (20.1 ± 9.15, 95% CI: 14.82-25.39 vs 11.52 ± 6.82, 95% CI: 5.82-17.22, *: P = .03), whereas the expression of the adipokine leptin was attenuated in EAT of dogs with CES (3.07 ± 2.29, 95% CI: 1.80-3.34 vs 9.83 ± 8.42, 95% CI: 3.36-16.30, **: P = .007). Inflammatory stimulation of EAT explant cultures resulted in an enhanced release of IL-6 (LPS: 5491.55 ± 4438, 95% CI: 833.7-10 149; HMGB1: 1001.78 ± 522.2, 95% CI: 518.8-1485; PBS: 310.9 ± 98.57, 95% CI: 228.5-393.3, ***: P < .001). CONCLUSION AND CLINICAL IMPORTANCE: Expression profile of inflammatory adipo(-cyto)kines by EAT is influenced from compressive forces acting in dogs with CES and might contribute to amplification of pain.

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.

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.

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.

Tick-borne encephalitis virus (TBEV) prevalence in field-collected ticks (Ixodes ricinus) and phylogenetic, structural and virulence analysis in a TBE high-risk endemic area in southwestern Germany.

BACKGROUND: Tick-borne encephalitis (TBE) is the most common viral CNS infection with incidences much higher than all other virus infections together in many risk areas of central and eastern Europe. The Odenwald Hill region (OWH) in southwestern Germany is classified as a TBE risk region and frequent case numbers but also more severe infections have been reported within the past decade. The objective of the present study was to survey the prevalence of tick-borne encephalitis virus (TBEV) in Ixodes ricinus and to associate TBEV genetic findings with TBE infections in the OWH. METHODS: Ticks were collected by the flagging methods supported by a crowdsourcing project implementing the interested public as collectors to cover completely and collect randomly a 3532 km2 area of the OWH TBE risk region. Prevalence of TBEV in I. ricinus was analysed by reversed transcription quantitative real-time PCR. Phylogeographic analysis was performed to classify OWH TBEV isolates within a European network of known TBEV strains. Mutational sequence analysis including 3D modelling of envelope protein pE was performed and based on a clinical database, a spatial association of TBE case frequency and severity was undertaken. RESULTS: Using the crowd sourcing approach we could analyse a total of 17,893 ticks. The prevalence of TBEV in I. ricinus in the OWH varied, depending on analysed districts from 0.12% to 0% (mean 0.04%). Calculated minimum infection rate (MIR) was one decimal power higher. All TBEV isolates belonged to the European subtype. Sequence analysis revealed a discontinuous segregation pattern of OWH isolates with two putative different lineages and a spatial association of two isolates with increased TBE case numbers as well as exceptional severe to fatal infection courses. CONCLUSIONS: TBEV prevalence within the OWH risk regions is comparatively low which is probably due to our methodological approach and may more likely reflect prevalence of natural TBEV foci. As for other European regions, TBEV genetics show a discontinuous phylogeny indicating among others an association with bird migration. Mutations within the pE gene are associated with more frequent, severe and fatal TBE infections in the OWH risk region.

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.

Impact of Sensitization and Inflammation on the Interaction of Mast Cells With the Intestinal Epithelium in Rats.

The density of intestinal mast cells has been reported to increase during inflammatory bowel disease (IBD). As mast cell mediators are known to increase the permeability of epithelial tight junctions, we hypothesized that antigen responses in sensitized animals might be enhanced under inflammatory conditions. This would contribute to a vicious circle by further enhancing the entry of luminal antigens into the colonic wall and thereby continuing the inadequate immune response during IBD. Therefore, one group of rats was sensitized against ovalbumin. In a second group of animals additionally a colitis was induced by rectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in ethanol. Specimens from distal colon and jejunum (as intestinal segment located distantly from the inflamed area) were mounted in Ussing chambers to measure tissue conductance, short-circuit current (Isc) induced by antigen exposure and paracellular permeability (fluorescein flux). This was paralleled by determination of mast cell markers and tight junction proteins with immunofluorescence and qPCR. In contrast to the initial hypothesis, antigen-induced Isc was not upregulated, but tended to be downregulated in the tissues from the colitis animals, both in colon and in jejunum. Only in the jejunum mast cell degranulation evoked an increase in fluorescein flux. Mast cell density was not altered significantly in the colon of the colitis animals. In the jejunum, sensitization induced a strong increase in mast cell density, which was unaffected by additional induction of colitis. Expression of sealing tight junction components claudin-3 and -4 were increased on the protein level in the sensitized animals in comparison to non-sensitized animals. Additional induction of colitis evoked a downregulation of claudin-3 in both intestinal segments and an upregulation of claudin-4 in the jejunum. Consequently, these data indicate segment differences in mast cell - epithelium interaction, but no enhancement of ion secretion in the TNBS/ethanol model of acute colitis after prior sensitization.

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.

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.

Effects of thermal stimulation on neurons and astrocytes cultured from the rat median preoptic nucleus.

Warming or cooling of the median preoptic nucleus (MnPO) in-vivo evokes appropriate thermoregulatory responses. In the present study, we aimed to investigate whether single neurons (and astrocytes) of primary rat MnPO cell cultures maintain properties, which are consistent with their putative role within the central thermoregulatory structures. Using the fura-2 ratio imaging technique, we therefore measured changes of intracellular Ca concentrations ([Ca]i) in neurons of rat MnPO primary cultures stimulated by rapid cooling from 37 to 25°C, or warming from 37 to 45°C, or glutamate, the transmitter which transfers thermal information to MnPO neurons. In the first experiment, we tested the responses to external cooling in a group of 212 neurons. Overall, 165 of these neurons were responsive to stimulation with glutamate; just four of them responded to the cold-stimulus with an increase of [Ca]i, and only one of these neurons was responsive to stimulation with menthol. In the second experiment, 24 of 327 neurons and 23 of 241 astrocytes responded to external warming with quick and pronounced Ca signals. Another 33 (10%) neurons showed a moderate and slowly developing increase of [Ca]i during the warming, which reflected the temperature changes in the chamber. These data correspond to properties of MnPO neurons upon thermal stimulation obtained by other experimental approaches. Primary cultures derived from the rat MnPO can thus be used to investigate neuronal thermosensitive properties and their possible modulation by other stimuli.

Clinical outcome and cerebrospinal fluid profiles in patients with tick-borne encephalitis and prior vaccination history.

BACKGROUND: Tick-borne encephalitis (TBE) is endemic in southern and eastern districts of Germany. Approximately 10-14% of the infected individuals suffer from long-term disability and in 1.5-3.6% the course is fatal. Two well-tolerated vaccines are available, which provide high protection and which have been confirmed in several field studies. Here we investigate clinical course, long-term outcome and cerebrospinal fluid (CSF) characteristics of TBE cases with a prior history of any vaccination as well as real vaccination breakthrough (VBT). METHODS: A case series of 11 patients with a prior history of vaccination, part of a recently published lager cohort of 111 TBE cases. Evaluation included clinical data, degree of disability (modified RANKIN scale, mRS) and analysis of CSF and serum samples. Furthermore, metadata for extended analysis on clinical outcome of TBE with VBT were analysed. RESULTS: One patient had a clear VBT and ten of them had irregular vaccinations schedules (IVS). Infection severity did not differ in patients with IVS as compared to a non-vaccinated control cohort (median mRS: both 3.0) but these patients showed a stronger cellular immune response as measured by CSF pleocytosis (IVS, 205 cells/µL versus non-vaccinated control, 114 cell/µL, P < 0.05) and by differential pattern of CSF (intrathecal) immunoglobulin synthesis. However, shift analysis of VBT metadata using linear-by-linear association revealed a more serious course of TBE in patients with VBT than in a non-vaccinated control cohort (χ2 = 9.95, P = 0.002). Furthermore, ordinal logistic regression analysis showed that VBT patients had an age-corrected, 2.65 fold (CI: 1.110-6.328; χ2 = 4.813; p = 0.028) significant higher risk to suffer from moderate or severe infections, respectively. CONCLUSION: A history of IVS surprisingly seems to have no impact on the clinical course of TBE but may leave marks in the specific brain immune response. VBT patients, however, carry an age-independent, significant risk to experience a severe infection.

The relevance of kalikrein-kinin system via activation of B2 receptor in LPS-induced fever in rats.

PURPOSE: This study evaluated the involvement of endogenous kallikrein-kinin system and the bradykinin (BK) B1 and B2 receptors on LPS- induced fever and the POA cells involved in this response. MATERIAL AND METHODS: Male Wistar rats received either i.v. (1 mg/kg), i.c.v. (20 nmol) or i.h. (2 nmol) injections of icatibant (B2 receptor antagonist) 30 or 60 min, respectively, before the stimuli. DALBK (B1 receptor antagonist) was given either 15min before BK (i.c.v.) or 30 min before LPS (i.v.). Captopril (5 mg/kg, sc.,) was given 1 h prior LPS or BK. Concentrations of BK and total kininogenon CSF, plasma and tissue kallikrein were evaluated. Rectal temperatures (rT) were assessed by telethermometry. Ca++ signaling in POA cells was performed in rat pup brain tissue microcultures. RESULTS: Icatibant reduced LPS fever while, captopril exacerbated that response, an effect abolished by icatibant. Icatibant (i.h.) reduced fever to BK (i.h.) but not that induced by LPS (i.v.). BK increased intracellular calcium concentration in neurons and astrocytes. LPS increased levels of bradykinin, tissue kallikrein and total kininogen. BK (i.c.v.) increased rT and decreased tail skin temperature. Captopril potentiated BK-induced fever an effect abolished by icatibant. DALBK reduced the fever induced by BK. BK (i.c.v.) increased the CSF PGE2concentration. Effect abolished by indomethacin (i.p.). CONCLUSIONS: LPS activates endogenous kalikrein-kinin system leading to production of BK, which by acting on B2-receptors of POA cells causes prostaglandin synthesis that in turn produces fever. Thus, a kinin B2-receptor antagonist that enters into the brain could constitute a new and interesting strategy to treat fever.

Functional expression of P2 purinoceptors in a primary neuroglial cell culture of the rat arcuate nucleus.

The arcuate nucleus (ARC) plays an important role in the hypothalamic control of energy homeostasis. Expression of various purinoceptor subtypes in the rat ARC and physiological studies suggest a modulatory function of P2 receptors within the neuroglial ARC circuitry. A differentiated mixed neuronal and glial microculture was therefore established from postnatal rat ARC, revealing neuronal expression of ARC-specific transmitters involved in food intake regulation (neuropeptide Y (NPY), proopiomelanocortin (POMC), tyrosine hydroxylase (TH)). Some NPYergic neurons cosynthesized TH, while POMC and TH expression proved to be mutually exclusive. Stimulation with the general purinoceptor agonists 2-methylthioadenosine-5'triphosphate (2-MeSATP) and ATP but not the P2X1/P2X3 receptor subtype agonist α,ß-methyleneadenosine-5'triphosphate (α,ß-meATP) induced intracellular calcium signals in ARC neurons and astrocytes. Some 5-10% each of 2-MeSATP responsive neurons expressed POMC, NYP or TH. Supporting the calcium imaging data, radioligand binding studies to hypothalamic membranes showed high affinity for 2-MeSATP, ATP but not α,ß-meATP to displace [α-(35)S]deoxyadenosine-5'thiotriphosphate ([(35)S]dATPαS) from P2 receptors. Repetitive superfusion with equimolar 2-MeSATP allowed categorization of ARC cells into groups with a high or low (LDD) degree of purinoceptor desensitization, the latter allowing further receptor characterization. Calcium imaging experiments performed at 37°C vs. room temperature showed further reduction of desensitization. Agonist-mediated intracellular calcium signals were suppressed in all LDD neurons but only 25% of astrocytes in the absence of extracellular calcium, suggestive of metabotropic P2Y receptor expression in the majority of ARC astrocytes. The highly P2Y1-selective receptor agonists MRS2365 and 2-methylthioadenosine-5'diphosphate (2-MeSADP) activated 75-85% of all 2-MeSATP-responsive ARC astrocytes. Taking into consideration the high potency to dose-dependently stimulate ARC cells of the LDD group, the high affinity for rat P2X(1-3) and low affinity for rat P2X4, P2X7 and P2Y receptor subtypes except P2Y1 and P2Y13, the agonist 2-MeSATP primarily acted upon P2X2 and P2Y1 purinoceptors to trigger intracellular calcium signaling in ARC neurons and astrocytes.

Predictors, Neuroimaging Characteristics and Long-Term Outcome of Severe European Tick-Borne Encephalitis: A Prospective Cohort Study.

BACKGROUND AND OBJECTIVES: Tick-borne encephalitis (TBE) still represents a considerable medical and health economic problem in Europe and entails a potential threat to travellers. The aim of this study was to characterise the conditions of severe TBE by precisely recording its clinical variants, the related neuroimaging features, and the variant-specific long-term outcome and by identifying predictors for severe courses. METHODS: A cohort of 111 TBE patients (median age 51, range 17-75 years; 42% females) was analysed prospectively. Data were acquired from the department of neurology, University Hospital Heidelberg, and the infectious diseases registry of the Robert-Koch institute Berlin. Neurological status was ascertained by protocol at admission and discharge and the degree of disability was scored using the modified RANKIN Scale (mRS; clinical score addressing neurological disability, range from 0, healthy to 6, dead) at admission and at follow-up. Follow-up examination was conducted by means of a telephone interview. To identify independent predictors for severe TBE and functional outcome, modelled logistic regression was performed. MRI changes were correlated with infection variants. To assess alpha-motor neuron injury patterns, we used high-resolution magnetic resonance neurography (hrMRN). Analyses were performed at the Department of Neurology, University Hospital, University of Heidelberg from April 2004 through September 2014. RESULTS: Acute course: 3.6% of patients died during the acute infection. All patients with a lethal course suffered from meningoencephaloradiculitis (MER, 14.4% of the cohort), which is associated with a significantly higher risk of requiring intensive care (p = 0.004) and mechanical ventilation (p<0.001) than menigoencephalitis (ME, 27.9% of the cohort). At admission, both MER and ME groups were severely affected, with the MER group having a statistically higher mRS score (median of 5 in the MER groups versus 4 in the ME group; p<0.001). Long-term outcome: outcome for MER was considerably worse (median mRS = 4) than for ME (mRS = 1, p<0.0001) and meningitis (mRS = 0, 57.7% of the cohort). RISK FACTORS: advanced age (p<0.001) and male gender (p = 0.043) are independent risk factors for a severe infection course. Furthermore, we identified pre-existing diabetes mellitus (p = 0.024) as an independent risk factor for MER. In MER, alpha-motor neuron injury accounts for the poor prognosis confirmed by hrMRN. CONCLUSION AND RELEVANCE: These data provide critical information for neurologists and other health professionals to use in evaluating TBEV patients who live in or travel to endemic areas. This information can be used to classify clinical presentation and estimate infection-associated complications and individual prognosis. Furthermore, the risk for severe, disabling infections in older patients should prompt general practitioners to recommend and encourage vaccination to those patients living in or travelling to endemic areas.