Mark C Flynn

Pro-inflammatory and anti-inflammatory cytokine mRNA induction in the periphery and brain following intraperitoneal administration of bacterial lipopolysaccharide

Gram-negative bacteria-derived lipopolysaccharide (LPS or endotoxin) is known to play an important role in immune and neurological manifestations during bacterial infections. LPS exerts its effects through cytokines, and peripheral or brain administration of LPS activates cytokine production in the brain. In this study, we investigated cytokine and neuropeptide mRNA profiles in specific brain regions and peripheral organs, as well as serum tumor necrosis factor (TNF)-alpha protein levels, in response to the intraperitoneal administration of LPS. For the first time, the simultaneous analysis of interleukin (IL)-1beta system components (ligand, signaling receptor, receptor accessory proteins, receptor antagonist), TNF-alpha, transforming growth factor (TGF)-beta1, glycoprotein 130 (IL-6 receptor signal transducer), OB protein (leptin) receptor, neuropeptide Y, and pro-opiomelanocortin (opioid peptide precursor) mRNAs was done in samples from specific brain regions in response to peripherally administered LPS. The same brain region/organ sample was assayed for all cytokine mRNA components. Peripherally administered LPS up-regulated pro-inflammatory cytokine (IL-1beta and/or TNF-alpha) mRNAs within the cerebral cortex, cerebellum, hippocampus, spleen, liver, and adipose tissue. LPS also increased plasma levels of TNF-alpha protein. LPS did not up-regulate inhibitory (anti-inflammatory) cytokine (IL-1 receptor antagonist and TGF-beta1) mRNAs in most brain regions (except for IL-1 receptor antagonist in the cerebral cortex and for TGF-beta1 in the hippocampus), while they were increased in the liver, and IL-1 receptor antagonist was up-regulated in the spleen and adipose tissue. Overall, peripherally administered LPS modulated the levels of IL-1beta system components within the brain and periphery, but did not affect the neuropeptide-related components studied. The data suggest specificity of transcriptional changes induced by LPS and that cytokine component up-regulation in specific brain regions is relevant to the neurological and neuropsychiatric manifestations associated with peripheral LPS challenge.

Kindling modulates the IL-1β system, TNF-α, TGF-β1, and neuropeptide mRNAs in specific brain regions

Cytokines and neuropeptides may be involved in seizure-associated processes. Following amygdala kindling in rats, we determined alterations of IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-1 receptor type I (IL-1RI), IL-1 receptor accessory proteins (IL-1R AcPs) I and II, TNF-α, TGF-β1, neuropeptide Y (NPY), glycoprotein 130 (gp 130) and pro-opiomelanocortin (POMC) mRNA levels in the parietal, prefrontal and piriform cortices, amygdala, hippocampus and hypothalamus. Messenger RNAs expression in all brain regions was determined 2 h or 3 weeks following the last generalized convulsive seizure triggered from the ipsilateral kindled amygdala. The same brain region sample was used to assay for changes of all mRNA components. The results show that the 2 h-kindled group exhibited a significant up-regulation of IL-1β, IL-1RI, TNF-α and TGF-β1 mRNAs in all three cortical brain regions, amygdala and hippocampus. The largest up-regulation occurred in the prefrontal cortex (about 30-fold induction for IL-1β and TNF-α mRNAs). IL-1R AcP I and II mRNA levels were also up-regulated in the cortical regions. No changes in IL-1β, IL-1RI or TNF-α mRNA levels occurred in the 3 week-kindled group. NPY mRNA levels increased in the hippocampus, prefrontal and piriform cortices in the 2 h-kindled group, while IL-1Ra, gp 130, or POMC mRNA levels did not change in any group. The overall profile of mRNA changes shows specificity of transcriptional modulation induced by amygdala kindling. The data support a role of cytokines and NPY in the adaptive mechanisms associated with generalized seizure activity, with implications for neuroprotection, neuronal dysfunction and vulnerability associated with epileptic activity.

Mode of action of OB protein (leptin) on feeding

OB protein (leptin) decreases food intake in a variety of species. Here we investigated the effects of the intracerebroventricular administration of recombinant murine OB protein on food consumption and meal parameters in Wistar rats maintained ad libitum. The intracerebroventricular administration of OB protein (0.56-3.5 μg/rat) decreased feeding in a dose-dependent manner. Computer analysis of meal parameters demonstrated that OB protein (3.5 μg/rat, n = 10) decreased nighttime meal size by 42%, whereas meal frequency and meal duration were unaffected. Derived analyses for the nighttime also showed that OB protein decreased the feeding rate (meal size/meal duration) by 30%, whereas the satiety ratio (intermeal intervals/meal size) increased by 100%. A similar profile was observed during the daytime and total daily periods. The intracerebroventricular administration of heat-inactivated OB protein (3.5 μg/rat, n = 10) had no effect on any meal parameter. The results show that OB protein administered intracerebroventricularly inhibits feeding through a specific reduction of meal size.

Neither acute nor chronic exposure to a naturalistic (predator) stressor influences the interleukin-1β system, tumor necrosis factor-α, transforming growth factor-β1, and neuropeptide mRNAs in specific brain regions

Physical (neurogenic) stressors may influence immune functioning and interleukin-1β (IL-1β) mRNA levels within several brain regions. The present study assessed the effects of an acute or repeated naturalistic, psychogenic stressor (predator exposure) on brain cytokine and neuropeptide mRNAs. Acute predator (ferret) exposure induced stress-like behavioral effects, including elicitation of a startle response and reduced exploratory behaviors; these responses diminished after 30 sessions. Moreover, acute and repeated predator exposure, like acute restraint stress, increased plasma corticosterone levels measured 5 min later, but not 2 h after stressor exposure. In contrast, none of the stressors used influenced IL-1β, IL-1 receptor antagonist, IL-1 receptor type I, IL-1 receptor accessory proteins I and II, or tumor necrosis factor-α mRNA levels in the prefrontal cortex, amygdala, hippocampus, or hypothalamus. Likewise, there were no stressor effects on transforming growth factor-β1, neuropeptide Y, glycoprotein 130, or leptin receptor mRNAs in brain regions. Thus, the naturalistic/psychogenic stressor used does not affect any of the brain cytokine component mRNAs studied. It is suggested that this type of stressor activates homeostatic mechanisms (e.g., glucocorticoid release), which act to preclude brain cytokine alterations that would otherwise favor neuroinflammatory/neuroimmunological responses and the consequent increase of brain sensitivity to neurotoxic and neurodegenerative processes.

Interleukin-1β System (Ligand, Receptor Type I, Receptor Accessory Protein and Receptor Antagonist), TNF-α, TGF-β1 and Neuropeptide Y mRNAs in Specific Brain Regions During Bacterial LPS-Induced Anorexia

Abstract Bacterial lipopolysaccharide (LPS) or endotoxin induces neurological manifestations including anorexia. It is proposed that LPS-induced cytokine production is involved in the generation of neurological manifestations and in neuroinflammatory/immunological responses during Gram-negative infections. For example, LPS-induced effects can be blocked or ameliorated by the interleukin-1 receptor antagonist (IL-1Ra). Here, sensitive and specific RNase protection assays were used to investigate the effects of the intracerebroventricular (i.c.v.) administration of LPS on mRNA levels of interleukin-1β (IL-1β) system components, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and neuropeptide Y (NPY) in the cerebellum, hippocampus, and hypothalamus. The same brain region sample was analyzed with all of the antisense probes. The data show simultaneous local induction of multiple cytokine components messenger ribonucleic acids (mRNAs) within specific brain regions in anorectic rats responding to i.c.v. administered LPS (500 ng/rat). Interleukin-1β and IL-1Ra had a similar mRNA induction profile (hypothalamus > cerebellum > hippocampus). Interleukin-1 receptor type I (IL-1RI) mRNA also increased in all three brain regions examined, and the soluble form of IL-1 receptor accessory protein (IL-1R AcP II) mRNA was induced in the hypothalamus. Tumor necrosis factor-α mRNA levels increased in the hypothalamus > hippocampus > cerebellum. Levels of membrane bound IL-1R AcP, TGF-β1, and NPY mRNAs did not change significantly in any brain region. The results suggest that: (1) endogenous up-regulation of IL-1β and TNF-α in the hypothalamus contribute to LPS-induced anorexia; and (2) the ratio IL-1Ra/IL-1β, and IL-1β ↔ TNF-α interactions may have implications for Gram-negative infections associated with high levels of LPS in the brain-cerebrospinal fluid.

Lipopolysaccharide (LPS)- and muramyl dipeptide (MDP)-induced anorexia during refeeding following acute fasting: characterization of brain cytokine and neuropeptide systems mRNAs

We investigated the effectiveness of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) administered into the brain to induce anorexia in acutely fasted Wistar rats allowed to refeed. We also assayed for changes in mRNA levels of IL-1 system components, TNF-alpha, TGF-beta1, glycoprotein 130 (gp 130), leptin receptor (OB-R), pro-opiomelanocortin (POMC), neuropeptide Y (NPY), glucocorticoid receptor (GR), and CRF receptor (CRF-R) in selected brain regions. The data show that LPS and MDP induced anorexia differentially during refeeding. LPS-induced anorexia was of a stronger magnitude and duration than that of MDP. RNase protection assays showed that LPS and MDP significantly increased the expression of IL-1beta, IL-1 receptor type I, and TNF-alpha mRNAs in the cerebellum, hippocampus, and hypothalamus; LPS was more potent in all cases. MDP treatment, on the other hand, induced a stronger increase in hypothalamic levels of IL-1 receptor antagonist (IL-1Ra) and TGF-beta1 mRNAs relative to LPS. In addition, competitive RT-PCR analysis showed that LPS induced an eleven-fold increase in IL-1alpha mRNA in the hypothalamus relative to vehicle. These findings suggest that LPS and MDP mediate anorexia through different cytokine mechanisms. A stronger up-regulation of anti-inflammatory cytokines (IL-1Ra and TGF-beta1) mRNA expression by MDP may be involved in the weaker MDP-induced anorexia relative to LPS. No significant changes were observed in the peptide components examined except for an up-regulation in cerebellar gp 130 mRNA and down-regulation of hypothalamic GR mRNA expression in response to LPS or MDP. This study shows that LPS and MDP induce anorexia in fasted rats allowed to refeed, and suggests an important role for endogenous cytokine-cytokine interactions.

Feeding response to neuropeptide Y-related compounds in rats treated with Y5 receptor antisense or sense phosphothio-oligodeoxynucleotide.

Neuropeptide Y (NPY), NPY 3-36 and pancreatic polypeptide (PP) increase short-term (2-h) food intake to varying degrees when given intracerebroventricularly (i.c.v.). Various Y receptor subtypes are proposed to participate in Y receptor ligand-induced stimulation of food intake. Here, we used an antisense phosphothio-oligodeoxynucleotide sequence (-5 relative to the initiating ATG) to the Y5 receptor subtype, which has been suggested to mediate NPY-induced feeding. Rats were treated with i.c.v. antisense or sense phosphothio-oligodeoxynucleotide for 3.5 days before NPY, NPY 3-36, or PP i.c.v. administration. The results show that antisense to the Y5 receptor had no effect on either spontaneous 2-h or NPY-, NPY 3-36-, or PP-stimulated 2-h food intake. However, there was a significant decrease relative to the sense control group in 10-h food intake following the initial 2-h feeding response to NPY (n = 10, p < 0.0001) or NPY 3-36 (n = 10, p < 0.05). The data suggest that the Y5 receptor has a modulatory role in the maintenance of feeding, but not as the critical receptor to confer for NPY and NPY 3-36 action on food intake.

Interleukin-1 (IL-1) receptor type I mediates anorexia but not adipsia induced by centrally administered IL-1β

IL-1beta induces anorexia and adipsia. Here, we report that intracerebroventricular (ICV) pretreatment with an antisense (but not sense) phosphothio-oligodeoxynucleotide to the IL-1 receptor type I (IL-1RI, 1.28 microg or 239 pmol twice daily for 3.5 days before IL-1beta plus antisense) inhibits the anorexia, but not the adipsia induced by the ICV administration of 2.0 ng IL-1beta/rat (a dose that yields estimated pathophysiological concentrations in the cerebrospinal fluid). The mean 2 h food intake decrease in response to IL-1beta was 5.6% (n = 10) in the antisense- and 43% in the sense (n = 9)-treated groups; the mean 2 h water intake decrease was 40% in the antisense- and 39% in the sense-treated groups. The intraperitoneal administration of IL-1RI antisense, in doses equivalent to those administered centrally, had no effect on the anorexic effect induced by ICV administered IL-1beta; this indicates a direct action in the central nervous system. The results suggest that: i) IL-1RI is involved in the short-term anorexigenic, but not the adipsogenic effect induced by centrally administered IL-1beta; and ii) the approach presented using antisense strategies is applicable to study the molecular basis of IL-1 mediated behaviors.

Fiber-optic monitoring coupled with confocal microscopy for imaging gene expression in vitro and in vivo

Detection of fluorescent signals in living cells is a common and powerful technique used to monitor gene expression for multiple biomedical applications. A disadvantage of this approach in vivo, is the limited accessibility for long-term monitoring of the fluorescent signals within organs in living animals. Because of the multiple applications of gene expression monitoring through fluorescent signals, innovative methods for readout are required. We developed a strategy combining gene transfer, fiber-optic or endoscope monitoring, and confocal microscopy for the brain interstitial or cavitary endoscopic visualization of the efficacy of gene delivery and expression in vivo. The approach is also effective in vitro and can be applied to multiple organs in vivo. We show an example of the detection of green fluorescent protein (GFP)-emitted fluorescence following the administration of recombinant GFP-expressing adenovirus or implantation of rat C6 glioblastoma cells infected with the recombinant GFP adenovirus into the rat hippocampus of chronically cannulated rats. The results show that fiber-optic monitoring coupled with confocal microscopy in gene transfer studies is a practical approach that results in a direct, efficient, rapid, and sensitive visualization of fluorescent signals in the brain. This allows for the continuous real-time in vitro or in vivo brain monitoring of gene expression, accurate anatomical localization, multiple experimental manipulations in the same subject or preparation, while no sacrifice of the animal is required to monitor the efficacy of gene transfer and/or expression.

Neuropeptide Y-Related Compounds and Feeding

Neuropeptide Y (NPY) and related compounds increase short-term feeding. Previous studies have used different animal models, feeding schedules, sources of the compounds, and time and routes of administration. These differences in methodology are important in the variability reported on the potency of NPY-related compounds. To obtain reliable data on the relative efficacy, we tested NPY, NPY 3-36, and pancreatic polypeptide (PP) using an identical protocol and the same commercial source. These three NPY-related compounds were tested using the intracerebroventricular (i.c.v., into the third ventricle) administration, and the profile of the feeding enhancement including the dose response and potency was determined. Compounds were tested in parallel on at least 2 successive days. NPY, NPY 3-36, and PP exhibited different potencies in enhancing 2-h food intake. Comparison of their dose responses (using 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 microg/rat) demonstrated an overall potency of NPY 3-36 > NPY > PP for the high doses. To study ligand interactions, we examined the effects of various combinations of NPY-related compounds administered concomitantly. These combinations were justified based on the data obtained from the individual dose responses. The data show that the effects of NPY plus NPY 3-36 or NPY 3-36 plus PP were less than additive. When compared to the individual responses, the effects of NPY 3-36 were almost identical to those induced by the combinations using low doses of NPY plus NPY 3-36, or low and high doses of PP plus NPY 3-36. The results support the notion that NPY and its analogues induce a short-term feeding response by activating multiple receptor subtypes.