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.

Leptin is involved in age-dependent changes in response to systemic inflammation in the rat.

Obesity contributes to a state of subclinical peripheral and central inflammation and is often associated with aging. Here we investigated the source and contribution of adipose tissue derived cytokines and the cytokine-like hormone leptin to age-related changes in lipopolysaccharide (LPS)-induced brain-controlled sickness-responses. Old (24 months) and young (2 months) rats were challenged with LPS or saline alone or in combination with a neutralizing leptin antiserum (LAS) or control serum. Changes in the sickness-response were monitored by biotelemetry. Additionally, ex vivo fat-explants from young and old rats were stimulated with LPS or saline and culture medium collected and analyzed by cytokine-specific bioassays/ELISAs. We found enhanced duration/degree of the sickness-symptoms, including delayed but prolonged fever in old rats. This response was accompanied by increased plasma-levels of interleukin (IL)-6 and IL-1ra and exaggerated expression of inflammatory markers in brain and liver analyzed by RT-PCR including inhibitor κBα, microsomal prostaglandin synthase and cyclooxygenase 2 (brain). Moreover, for the first time, we were able to show prolonged elevated plasma leptin-levels in LPS-treated old animals. Treatment with LAS in young rats tended to attenuate the early- and in old rats the prolonged febrile response. Fat-explants exhibited unchanged IL-6 but reduced IL-1ra and tumor necrosis factor (TNF)-α release from adipose tissue of aged compared to young animals. In addition, we found increased expression of the endogenous immune regulator microRNA146a in aged animals suggesting a role for these mediators in counteracting brain inflammation. Overall, our results indicate a role of adipose tissue and leptin in "aging-related-inflammation" and age-dependent modifications of febrile-responses.

Interleukin-10 modulates the synthesis of inflammatory mediators in the sensory circumventricular organs: implications for the regulation of fever and sickness behaviors.

BACKGROUND: Whereas the role played by interleukin (IL)-10 in modulating fever and sickness behavior has been linked to it targeting the production of pro-inflammatory cytokines in the circulation, liver and spleen, it is not known whether it could directly target the local production of pro-inflammatory cytokines within the sensory circumventricular organs (CVOs) situated within the brain, but outside the blood-brain barrier. Using inactivation of IL-10, we, therefore, investigated whether IL-10 could modulate the synthesis of pro-inflammatory cytokines within the sensory CVOs, in particular the organum vasculosum laminae terminalis (OVLT) and area postrema (AP). FINDINGS: Primary OVLT and AP microcultures were established from topographically excised rat pup brain tissue. The microcultures were pretreated with either IL-10 antibodies (AB) (10 µl/350 µl medium) or phosphate-buffered saline (PBS) (10 µl/350 µl medium) before being incubated with lipopolysaccharide (LPS) (100 µg/ml) or PBS in complete medium for 6 h. Supernatants were removed from the microcultures after 6 h of incubation with LPS and used for the determination of IL-6 and tumor necrosis factor (TNF)-α. Pre-treating the OVLT and AP microcultures with IL-10 antibodies significantly enhanced the LPS-induced increase in TNF-α and IL-6 in the supernatant obtained from the microcultures. CONCLUSIONS: Our results show for the first time that the LPS-induced release of pro-inflammatory cytokines in cells cultured from the AP and OVLT can be modulated in the presence of IL-10 antibodies. Thus, we have identified that the sensory CVOs may have a key role to play in both the initiation and modulation of neuroinflammation.

The viral mimetic polyinosinic:polycytidylic acid (poly I:C) induces cellular responses in primary cultures from rat brain sites with an incomplete blood-brain barrier.

Primary microcultures of the organum vasculosum laminae terminalis (OVLT) and the area postrema (AP), brain sites with an incomplete blood-brain barrier, were established from topographically excised rat pup tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, we showed for the first time that polyinosinic:polycytidylic acid (poly I:C) can induce calcium signalling in single OVLT and AP cells. Poly I:C stimulation caused fast, transient rises in intracellular calcium in about 5% of neurons and astrocytes and some microglial cells. Frequently, the responses of astrocytes and microglial cells showed a shorter onset-latency compared to neurons. In addition, exposure to poly I:C led to a time dependent release of bioactive tumour necrosis factor (TNF) and interleukin-6 (IL-6) into the supernatants of OVLT and AP cultures. The demonstration of direct cellular responses of OVLT- and AP-intrinsic cells to stimulations with poly I:C is in agreement with the discovered existence of Toll-like receptor 3 (TLR3), the cognate receptor for poly I:C, in the brain.

Parthenolide attenuates LPS-induced fever, circulating cytokines and markers of brain inflammation in rats.

Parthenolide, a sesquiterpene lactone, has been reported to exhibit a variety of anti-inflammatory and immunomodulatory effects. To test the effect of parthenolide on brain inflammatory responses, brain oxidative stress and fever, we treated rats with parthenolide (1 mg/kg), simultaneously or 1 h prior to a systemic (i.p.) challenge with a moderate dose (100 µg/kg) of lipopolysaccharide (LPS). The initial hypothermia was exaggerated; the second phase of the biphasic LPS-induced fever and circulating interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were significantly attenuated only in parthenolide-pretreated animals. In the hypothalamus, markers of NFκB/NF-IL6 pathway activation (inhibitor κBα, NF-IL6 and the serin/threonin kinase-like protein mRNA expression) and markers of oxidative stress (including nuclear respiratory factor 1) and NFκB immunoreactivity were significantly reduced while NF-IL6 immunoreactivity and suppressor of cytokine signaling 3 mRNA expression remained unaltered, 8 h after LPS-stimulation with parthenolide-pretreatment. Importantly, this response was accompanied by decreased mRNA expression of the rate limiting enzyme in prostaglandin synthesis, cyclooxygenase 2 (COX2), known for its critical role in fever induction pathways. A direct action of parthenolide on brain cells was also confirmed in a primary neuro-glial cell culture of the vascular organ of the lamina terminalis a pivotal brain structure for fever manifestation with a leaky blood-brain barrier. In summary, pretreatment with parthenolide attenuates the febrile response during LPS-induced systemic inflammation by reducing circulating IL-6 and TNFα and decreasing hypothalamic NFκB/NF-IL6 activation, oxidative stress and expression of COX2. Thus parthenolide appears to have the potential to reduce brain inflammation.

Spatiotemporal nuclear factor interleukin-6 expression in the rat brain during lipopolysaccharide-induced fever is linked to sustained hypothalamic inflammatory target gene induction.

Rats injected with lipopolysaccharide (LPS) show brain-controlled sickness symptoms, including fever. In these animals, early genomic activation of brain cells was previously monitored by immunohistochemical detection of transcription factors such as nuclear factor (NF)-κB or signal transducer and activator of transcription (STAT)3 and was linked to the initiation or maintenance of the febrile response. To investigate whether NF-IL6 might be another important transcription factor implicated in this kind of immune-to-brain signaling, rats were injected with LPS (100 µg/kg, intraperitoneally) or phosphate-buffered saline, and brains were analyzed by immunohistochemistry, real-time PCR, or Western blot 4, 6, 8, and 10 hours later. Moderate to strong LPS-induced nuclear NF-IL6 immunoreactivity (IR) occurred in a time-dependent manner within circumventricular organs, namely, the vascular organ of the lamina terminalis, the subfornical organ, the area postrema, and the median eminence, brain structures with a leaky blood-brain barrier. Furthermore, nuclear NF-IL6-IR was observed in the pituitary gland, the choroid plexus, and the meninges as well as blood vessels throughout the entire brain. Endothelial, microglial, and ependymal cells, astrocytes, perivascular macrophages, and neurons exhibited LPS-induced nuclear NF-IL6-IR; mRNA levels of NF-IL6, responsive inflammatory genes, and NF-IL6 protein levels were significantly elevated. As opposed to observations on STAT3 or NFκB, the percentage of NF-IL6-reactive cells increased in parallel to late phases of the febrile response. In conclusion, these results suggest a potential role for NF-IL6 in the maintenance or possibly the termination of LPS-induced fever. Moreover, we propose NF-IL6 to be a delayed brain cell activation marker.

Neurons and glial cells of the rat organum vasculosum laminae terminalis directly respond to lipopolysaccharide and pyrogenic cytokines.

During systemic immune challenge, the organum vasculosum laminae terminalis (OVLT) with its dense vascularization by fenestrated capillaries lacking blood-brain barrier function allows direct access of circulating pyrogens to brain tissue located in close vicinity to the preoptic area. We aimed to analyze direct responses of OVLT cells to exposure to lipopolysaccharide (LPS) and fibroblast-stimulating lipopeptide-1 (FSL-1) or the cytokines tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6. A primary microculture of the OVLT was established from topographically excised rat pup brain tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, pyrogen-induced calcium signaling in single OVLT cells could be characterized. LPS--as opposed to FSL-1--stimulation caused fast, transient rises in intracellular calcium concentration in 17% of neurons, 9% of astrocytes, and <5% of microglial cells investigated. LPS additionally led to enhanced expression of TNF-α and IL-1ß exclusively in microglial cells, as well as a time-dependent release of TNF-α and IL-6 from OVLT microcultures. TNF-α evoked calcium signals in 11% of neurons, 22% of astrocytes, and 5% of microglial cells tested. A considerable population of neurons (11%) but only few astrocytes and microglial cells responded to IL-6, whereas 8% of microglial cells and 3% of astrocytes or neurons were activated by IL-1ß. The demonstration of direct cellular responses of OVLT-intrinsic cells to stimulations with LPS or cytokines reinforces the suggested role of this brain structure as a responsive brain site to circulating pyrogens.

Nuclear translocation of the transcription factor STAT5 in the rat brain after systemic leptin administration.

Leptin binding to its functional receptor stimulates JAK-STAT-signaling pathway, which finally results in activation and nuclear translocation of transcription factors of the signal transducer and activator of transcription (STAT) family, namely of STAT3. Here we report for the first time that systemic treatment with leptin (5 mg/kg; intraperitoneal injection) also increased the number of nuclear STAT5 signals in the hypothalamus. In particular, the entire arcuate nucleus (ARC), the ventral premammilary nucleus (PMV), and the supraoptic nucleus (SO) showed an enhanced nuclear STAT5 translocation in response to leptin when compared to saline, 120 min after the respective injection. Co-localization studies revealed that a high percentage of those STAT5-responsive cells proved to be neurons. In addition, some astrocytes within the ARC showed nuclear STAT5 signals. The functional relevance of leptin-induced nuclear STAT5 activation in hypothalamic cells still has to be determined.

Interleukin-6 mediates lipopolysaccharide-induced nuclear STAT3 translocation in astrocytes of rat sensory circumventricular organs.

Pyrogenic treatment with lipopolysaccharide (LPS) or interleukin-6 (IL-6) induces nuclear STAT3 translocation in the vascular organ of the laminae terminalis (OVLT) and the subfornical organ (SFO). STAT3 immunohistochemistry was combined with the detection of marker proteins (glial, neuronal, endothelial) and the nuclear DAPI stain to determine the phenotype of responding cells. At time points with high pyrogen-induced IL-6 plasma levels, nuclear STAT3 signals were co-localized with an astrocytic cell marker. IL-6 might therefore mediate genomic activation of OVLT/SFO astrocytes during LPS-induced fever.

Influence of the hypothalamus on the midbrain tonic inhibitory mechanism on metabolic heat production in rats.

Influence of the hypothalamus on increased body temperature was examined in male rats. Body temperature was increased by removing the midbrain tonic inhibitory mechanism (TIM) on heat production from brown adipose tissue (BAT) by microinjections of a local anesthetic, procaine, into the midbrain. Procaine microinjections in unanesthetized rats increased rectal temperature that was followed by a strong tail skin temperature rise. Procaine microinjections in unanesthetized and decerebrated rats also increased rectal temperature but without skin temperature rise. These decerebrated animals fatally developed hyperthermia. In anesthetized rats, procaine microinjections increased temperature of the interscapular BAT (IBAT) higher with shorter onset for temperature rise than rectal temperature. Increased IBAT temperature by procaine microinjections in anesthetized rats was attenuated during hypothalamic warming, and enhanced during hypothalamic cooling when compared with that observed during thermoneutral hypothalamic temperature. These results suggest that the midbrain TIM is able to function in unanesthetized conscious rats, and that the integrity of the midbrain mechanism to tonically inhibit metabolic heat production does not require the presence of intact hypothalamus. These results also suggest that the hypothalamus modulates directly or indirectly IBAT heat production that was induced by removal of the midbrain TIM.

[Fever in acute illness: beneficial or harmful?]. / Fieber bei Akuterkrankung: Vorteilhaft oder maladaptiv?

Fever has had a long phylogenetic history: it occurs not only in infected birds and mammals, but also in infected reptiles, amphibians, fish and even insects. When these "cold-blooded" animals are prevented from adapting their body temperature to the risen thermoregulatory set-point by behavioral means, a more severe state of disease and a higher mortality are the consequences. It seems unlikely that an energy-dependent process, such as fever, would have been retained for hundreds of millions of years, in so many groups of organisms, if it provided no selective advantage. Fever may represent a leukocyte-based amplification mechanism to affect host challenge: enhanced motility of leukocytes, enhanced lymphocyte response to mitogens, increased production of interferon, enhanced immune response to viral antigens. Evidence for a beneficial effect of fever is also supported by the results of our animal experiments. Intraperitoneal injection of a high dose of bacterial lipopolysaccharide (LPS) in rats induces a septic shock like state which is accompanied by hypothermia on the day of LPS-administration and a robust fever on the following days. Co-injection of a neutralizing synthetic form of the soluble tumor necrosis factor (TNF) type 1 receptor completely neutralizes LPS-induced bioactive TNF in the lavage of the abdominal cavity and in blood plasma. Treatment with the TNF-antagonist results in much faster recovery from the hypothermic state. The rats develop pronounced fever already on the day of injection and there is significantly less reduction in body weight and food and water intake. Similar, but less pronounced effects can be induced by treatment with inhibitors of the inducible form of nitric oxide (NO)-synthase indicating that TNF-induced detrimental effects are, in part, mediated by excessive formation of NO. These results confirm that an accelerated onset of fever or a faster recovery from hypothermia in a septic state may have rather beneficial than maladaptive effects.

Selected contribution: role of IL-6 in LPS-induced nuclear STAT3 translocation in sensory circumventricular organs during fever in rats.

Interleukin-6 (IL-6) is regarded as an endogenous mediator of lipopolysaccharide (LPS)-induced fever. IL-6 is thought to act on the brain at sites that lack a blood-brain barrier, the circumventricular organs (CVOs). Cells that are activated by IL-6 respond with nuclear translocation of the signal transducer and activator of transcription 3 molecule (STAT3) and can be detected by immunohistochemistry. We investigated whether the LPS-induced release of IL-6 into the systemic circulation was accompanied by a nuclear STAT3 translocation within the sensory CVOs. Treatment with LPS (100 microg/kg) led to a slight (1 h) and then a strong increase (2-8 h) in plasma IL-6 levels, which started to decline at the end of the febrile response. Administration of both pyrogens LPS and IL-6 (45 microg/kg) induced a febrile response with IL-6, causing a rather moderate fever compared with the LPS-induced fever. Nuclear STAT3 translocation in response to LPS was observed within the vascular organ of the lamina terminalis (OVLT) and the subfornical organ (SFO) 2 h after LPS treatment. To investigate whether this effect was mediated by IL-6, the cytokine itself was systemically applied and indeed an identical pattern of nuclear STAT3 translocation was observed. However, nuclear STAT3 translocation already occurred 1 h after IL-6 application and proved to be less effective compared with LPS treatment when analyzing OVLT and SFO cell numbers that showed nuclear STAT3 immunoreactivity after the respective pyrogen treatment. Our observations represent the first molecular evidence for an IL-6-induced STAT3-mediated genomic activation of OVLT and SFO cells and support the proposed role of these brain areas as sensory structures for humoral signals created by the activated immune system and resulting in the generation of fever.