Acute and chronic administration of immunomodulators induces anorexia in Zucker rats.

To investigate the possible involvement of leptin signaling in lipopolysaccharide (LPS) anorexia, we compared the anorectic effect of LPS in genetically obese (fa/fa) Zucker rats and in their lean (Fa/?) counterparts. The effects of interleukin-1beta (IL-1beta) and muramyl dipeptide (MDP) were also tested. LPS [100 microg/kg body weight (BW)], IL-1beta (2 microg/kg BW) and MDP (2.2 mg/kg BW) injected intraperitoneally (i.p.) at lights out reduced food intake similarly in obese and lean rats. LPS injection at 500 or 1000 microg/kg BW (i.p.) also reduced food intake and BW similarly in obese and lean rats, but obese regained BW faster than lean rats. LPS (2.45 microg or 9.8 microg/h/rat) administered chronically with i.p. implanted osmotic pumps reduced food intake similarly on experimental day 1, regardless of the genotype. After day 3, the lean rats' anorectic response and recovery were dose-dependent, whereas the anorectic response in obese rats was minimally affected by dose (significant dose effect only on day 3). Again, obese rats regained lost BW faster than lean rats. These results do not support a role for leptin as the sole mediator of anorexia induced by bacterial products (LPS and MDP) and IL-1beta.

Immunoregulators in the nervous system.

The nervous system, through the production of neuroregulators (neurotransmitters, neuromodulators and neuropeptides) can regulate specific immune system functions, while the immune system, through the production of immunoregulators (immunomodulators and immunopeptides) can regulate specific nervous system functions. This indicates a reciprocal communication between the nervous and immune systems. The presence of immunoregulators in the brain and cerebrospinal fluid is the result of local synthesis--by intrinsic and blood-derived macrophages, activated T-lymphocytes that cross the blood-brain barrier, endothelial cells of the cerebrovasculature, microglia, astrocytes, and neuronal components--and/or uptake from the peripheral blood through the blood-brain barrier (in specific cases) and circumventricular organs. Acute and chronic pathological processes (infection, inflammation, immunological reactions, malignancy, necrosis) stimulate the synthesis and release of immunoregulators in various cell systems. These immunoregulators have pivotal roles in the coordination of the host defense mechanisms and repair, and induce a series of immunological, endocrinological, metabolical and neurological responses. This review summarizes studies concerning immunoregulators--such as interleukins, tumor necrosis factor, interferons, transforming growth factors, thymic peptides, tuftsin, platelet activating factor, neuro-immunoregulators--in the nervous system. It also describes the monitoring of immunoregulators by the central nervous system (CNS) as part of the regulatory factors that induce neurological manifestations (e.g., fever, somnolence, appetite suppression, neuroendocrine alterations) frequently accompanying acute and chronic pathological processes.

Insulin in the cerebrospinal fluid.

The presence, distribution and specific localization of insulin and its receptors in the central nervous system (CNS) have been described in numerous reports. Insulin in the CNS appears to be similar to pancreatic insulin by biochemical and immunological criteria. While the presence of insulin in the cerebrospinal fluid (CSF)--an essential neurohumoral transport system--has been widely reported, the available information is fragmented and therefore it is difficult to determine the significance of insulin in the CSF and to establish future research directions. This paper presents an integrative view of the studies concerning insulin in the CSF of various species including the human. Evidence suggests that insulin in the CSF and brain may be the result of local synthesis in the CNS, and uptake from the peripheral blood through the blood-brain barrier and circumventricular organs. The passage of insulin from the peripheral blood through the blood-brain barrier may be mediated by a specific transport system coupled to insulin receptors in cerebral microvessels. The transfer of insulin from the peripheral blood through the circumventricular organs is not specific and may depend on simple diffusion. Slow access of insulin to brain interstitial fluid adjacent to the blood-brain barrier and circumventricular organs may be followed by selective transport to other brain sites and into the ventricular-subarachnoideal CSF. It has been hypothesized that the choroid plexuses, which constitute the blood-CSF interface, might be a nonspecific pathway for rapid insulin transport into the CSF. Insulin may also pass from the CSF into the peripheral blood via absorption into the arachnoid villi. This evidence indicates that insulin may be transported in both directions between the CSF-brain and the peripheral blood. Evidence also suggests that the presence of insulin in the CSF is of pivotal importance for its neurophysiological or neuropathophysiological significance.

Cytokines and anorexia: a brief overview.

Cytokine immunotherapy with interferon-alpha, interleukin-1, interleukin-2, or tumor necrosis factor-alpha is consistently associated with anorexia and other neurologic and neuropsychiatric manifestations. Preclinical and clinical studies have shown that cytokines induce anorexia when administered peripherally or into the brain. Cytokine-induced anorexia involves both peripheral and central nervous system mechanisms. Cytokines modulate gastrointestinal activities, cause metabolic changes, affect the endocrine system, and modulate the normal neurotransmitter/neuropeptide profile of the hypothalamus, all of which can influence eating behavior. The direct effects of interferon-alpha, interleukin-1beta, and tumor necrosis factor-alpha on glucose-sensitive neurons in two specific regions of the hypothalamus could cause profound changes in eating patterns in animals and humans. Cytokine immunotherapy also induces production of a large array of endogenous cytokines, which may act synergistically to cause anorexia, taste aversions, and other neuropsychiatric manifestations. A variety of approaches have been proposed for ameliorating cytokine-induced anorexia. These include dietary supplementation with specific fatty acids, megestrol acetate, and specific neuropeptides or peptide hormones that have the ability to modulate cytokine production or activity.

Regulation of brain interleukin-1 beta (IL-1 beta) system mRNAs in response to pathophysiological concentrations of IL-1 beta in the cerebrospinal fluid.

Interleukin-1 beta (IL-1 beta) is released during pathophysiological processes. IL-1 beta induces neurological manifestations when administered into the cerebrospinal fluid (CSF) at pathophysiological concentrations detected during central nervous system (CNS) infections and other neurological disorders. In the present study, we investigated the regulation of the IL-1 beta system in the CNS in response to the chronic intracerebroventricular (icv) microinfusion of IL-1 beta at estimated pathophysiological concentrations in the CSF. IL-1 receptor type I (IL-1RI), IL-1 receptor antagonist (IL-1Ra), and IL-1 beta mRNAs were determined by sensitive RNase protection assays in brain target regions for IL-1 beta (cerebellum, parieto-frontal cortex, hippocampus, and midbrain). The results show that chronic icy microinfusion of IL-1 beta induced significant anorexia, increased the cerebellar IL-1RI mRNA content, increased IL-1Ra and IL-1 beta mRNAs levels in the cerebellum > midbrain > cortex > hippocampus, and induced profiles of IL-1RI mRNA, IL-1Ra mRNA, and IL-1 beta mRNA that were highly intercorrelated. On the other hand, levels of rat glyceraldehyde 3-phosphate dehydrogenase mRNA and 18S rRNA were fairly constant, and heat-inactivated IL-1 beta had no effect on food intake or on IL-1RI, IL-1Ra, and IL-1 beta mRNAs levels in any brain region. The data suggest the operation of an IL-1 beta feedback system (IL-1 beta/ IL-1Ra/IL-1RI) in brain regions. Dysregulation of the CNS IL-1 beta feedback system may have pathophysiological significance. This may be reflected, for example, in the pathogenicity and severity of neurological diseases, such as CNS infections.

1998 Curt P. Richter Award. Brain mechanisms in cytokine-induced anorexia.

Our research focuses on the mechanisms underlying cytokine action in the central nervous system (CNS) using an integrative and multidisciplinary strategy organized through supracellular (behavioral analysis by computerized monitoring systems), cellular (extracellular and intracellular neurophysiological recording), and molecular (patch-clamp recording, and DNA, RNA and protein analyses) approaches. An integrative strategy that combines computerized meal pattern analyses with cellular and molecular biology approaches allows the study of underlying brain mechanisms in cytokine- and disease-associated anorexia. This paper presents a comprehensive discussion of our laboratory's previously published data on brain mechanisms involved in cytokine-induced anorexia including the relevance of meal pattern analysis (meal size, meal duration, meal frequency, intermeal intervals), modulation of hypothalamic neuronal activity, molecular processes involving ionic conductances, cytokine-cytokine and cytokine-peptide interactions, and modulation of cytokine and peptide/neuropeptide system components (ligands, endogenous inhibitors, receptor subtypes, signal transduction molecules, intracellular mediators) and cytokine feedback systems.

Molecular regulation of the brain interleukin-1 beta system in obese (fa/fa) and lean (Fa/Fa) Zucker rats.

Interleukin-1 beta (IL-1 beta) induces anorexia when administered acutely or chronically into the cerebrospinal fluid (CSF) at doses that yield estimated pathophysiological concentrations. Enhanced sensitivity to IL-1 beta-induced anorexia has been observed in animal models of obesity, including the obese (fa/fa) Zucker rat. Obesity is also associated with increased tumor necrosis factor-alpha mRNA expression in adipose tissue. This suggests that obese individuals may have dissimilar sensitivity to cytokine action and differential regulation of cytokine production. In this study, we investigated the regulation of the IL-1 beta system (IL-1 beta, IL-1 receptor type I (IL-1RI) and IL-1 receptor antagonist (IL-1Ra)) in the central nervous system (CNS) in response to the chronic intracerebroventricular (i.c.v.) microinfusion (via osmotic minipumps) of 8 ng IL-1 beta/24 h/72 h-a dose that yields estimated pathophysiological concentrations in the CSF. IL-1 beta, IL-1RI and IL-1Ra mRNAs were determined by sensitive RNase protection assays in brain target regions for IL-1 beta (cerebellum, parieto-frontal cortex, hippocampus, hypothalamus and midbrain). The results show that chronic i.c.v. microinfusion of IL-1 beta increased the IL-1 beta mRNA, IL-1R1 mRNA and IL-1Ra mRNA levels in the hypothalamus > cerebellum in both obese (fa/fa) and lean (Fa/Fa) Zucker rats. IL-1 beta mRNA levels also increased in the cortex, hippocampus and midbrain of obese (fa/fa) rats. The profiles of IL-1 beta mRNA, IL-1RI mRNA and IL-1Ra mRNA in the same hypothalamic samples obtained from obese or lean rats were highly intercorrelated. However, no significant differences in the level of IL-1 beta system mRNAs induction were observed in any brain region between obese and lean rats. On the other hand, levels of rat glyceraldehyde 3-phosphate dehydrogenase mRNA were fairly constant, and heat-inactivated IL-1 beta (8 ng/24 h/72 h) had no effect on IL-1 beta, IL-1RI and IL-1Ra mRNAs levels in any brain region. The data suggest: (1) the operation of an IL-1 beta feedback system (IL-1 beta/IL-1Ra/IL-1RI) in brain regions; (2) that enhanced sensitivity of obese rats to IL-1 beta-induced anorexia is not dependent on changes in the brain IL-1 beta system at the mRNA level; and (3) that the present novel approach can be used to investigate the molecular basis of cytokine action in the CNS.

Creatine kinase-B mRNA levels in brain regions from male and female rats.

The creatine kinase-B (CKB) enzyme is proposed to have a pivotal role in the regeneration of ATP in the nervous system. In the present study, the steady-state levels of CKB mRNA were determined by RNase protection assay in seventeen separate brain regions obtained from rats during the initial interval of the light period or period of inactivity in rats. The antisense probe used specifically hybridizes to CKB mRNA and discriminates CKB from CKM mRNA. The results show that brain regions from Wistar rats differ in CKB mRNA content. Highest levels of CKB mRNA were detected in the male and female cerebellum. High levels of CKB mRNA were observed in the spinal cord, brain stem and its structures (medulla, pons and midbrain) and olfactory bulb of the male rats. Female rats also contained high levels of CKB mRNA in the brain stem. In both male and female rats, the frontal cortex, occipital cortex, hippocampus and striatum exhibited lower levels of CKB mRNA relative to the complete brain. Statistical analyses demonstrated a significant difference between the male and female CKB mRNA profiles. However, CKB mRNA levels in brain regions with estrogen receptors (hypothalamus, hippocampus) were similar in male and female rats. Differential CKB mRNA levels in various brain regions may suggest diverse physiological significance of the CKB system in the regulation of brain energy metabolism.

In vivo IL-1beta-induced modulation of G-protein alphaO subunit subclass in the hypothalamic ventromedial nucleus: implications to IL-1beta-associated anorexia.

The intracerebroventricular (i.c.v.) administration of interleukin-1beta (IL-1beta) induces anorexia in rats at doses that yield estimated pathophysiological concentrations in the cerebrospinal fluid. IL-1beta also induces anorexia when administered into the hypothalamic ventromedial nucleus (VMN), an important brain site for the control of feeding. A variety of guanine nucleotide binding protein (G-protein) coupled receptors (e. g., for neurotransmitters and neuropeptides) participate in the integrative regulation of feeding. Our previous studies reported that the VMN G-protein alphaO common subunit subclass is involved in the control of normal feeding, and that IL-1beta modulates calcium channel currents via a pertussis toxin (PTX)-sensitive G-protein (GalphaO/Galphai). Here, we examined the profile of GalphaO protein expression in the hypothalamic VMN during IL-1beta-induced anorexia. Intracerebroventricular microinfusion of IL-1beta (0.5 to 8.0 ng/24 h for 72 h) into the third cerebral ventricle dose-dependently induced anorexia (p<0.001) and decreased the VMN GalphaO common protein levels (p<0.001). Heat-inactivated IL-1beta and IL-1beta plus IL-1 receptor antagonist (a competitive inhibitor of IL-1beta action) had no effect on food intake or on VMN GalphaO common protein content. RT-PCR analysis of VMN RNA from IL-1beta-treated rats generated an expression profile for GalphaO common subunit; however, no modulation at the mRNA level was observed. The results suggest that anorexia induced by the central administration of IL-1beta involves modification of G-protein alphaO common subunit profile in the central nervous system.

Antisense oligodeoxynucleotides to G-protein alpha-subunit subclasses identify a transductional requirement for the modulation of normal feeding dependent on G alpha OA subunit.

A variety of G-protein-coupled receptors are proposed to participate in the modulation of ingestive behavior and in the mode of action of antiobesity drugs. In the present study, we investigated the involvement of G-protein alpha-subunit subclasses (molecular transducers of multiple chemical signals) in the control of ingestive behavior. We report here that the chronic intracerebroventricular (i.c.v.) microinfusion for 72 h (via osmotic minipumps) with antisense phosphothio-oligodeoxynucleotides corresponding to G-protein alpha-subunitO common (to OA and OB) and OA subclasses decrease the nighttime food intake without affecting water intake in rats. Computerized analyses of the microstructure of feeding indicate that the G alpha OA antisense depresses feeding by reducing meal frequency, while meal size and meal duration increased slightly, but not significantly. The effects of G alpha O common and G alpha OA antisense on feeding are specific since the chronic i.c.v. microinfusion of sense to G alpha O common or G alpha OA, antisense to the related subclass G alpha OB, and antisense to other G-protein alpha-subunits (G alpha S, G alpha Q, G alpha 11 and G alpha i common) had no effect on food or water intake. The observed effects by G alpha O common and G alpha OA antisense imply a direct action in the central nervous system since the chronic subcutaneous microinfusion of G alpha O common and G alpha OA antisense in doses equivalent to two-fold higher relative to those administered centrally had no effect on food intake. The chronic microinfusion of G alpha O common antisense drastically decreased the levels of G alpha O protein detected in immunoblots of hypothalamic ventromedial nuclei. The results suggest that the G-protein alpha-subunit subclass G alpha OA is critical for the integrative modulation of normal feeding behavior, and that changes in its activity may be associated with modifications of feeding. These studies also show a novel approach to study the molecular basis of specific behaviors by manipulating elements of the transductional systems.

Basal and IL-1beta-stimulated cytokine and neuropeptide mRNA expression in brain regions of young and old Long-Evans rats.

Young and old Long-Evans rats respond with fevers of equal magnitude and duration to the brain administration of interleukin-1beta (IL-1beta). Here, we characterized brain regional mRNA expression of cytokine and neuropeptide components in response to the brain administration of IL-1beta. We used specific and highly sensitive RNase protection assays to determine mRNA changes for IL-1beta, IL-1 receptor type I (IL-1RI), IL-1R accessory proteins I and II (IL-1R AcP I and II), IL-1 receptor antagonist (IL-1Ra), transforming growth factor-beta1 (TGF-beta1), glycoprotein 130 (gp 130), leptin receptor (OB-R), neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) in the cerebellum, parieto-frontal cortex, hippocampus, hypothalamus, and midbrain of male young (3-5 months) and old (24-26 months) Long-Evans rats. In both young and old rats, IL-1beta induced a significant up-regulation of cerebellar IL-1Ra, IL-1RI, and TGF-beta1 mRNAs; hippocampal TGF-beta1 mRNA; hypothalamic IL-1beta, IL-1Ra, TGF-beta1, and gp 130 mRNAs; and midbrain IL-1beta and TGF-beta1 mRNAs. There were no age-related differences in any cytokine mRNA levels under basal or IL-1beta-stimulated conditions. Levels of hypothalamic POMC mRNA were different between age groups under basal and stimulated conditions. IL-1R AcP I and leptin receptor did not change in any brain region from either young or old rats, suggesting specificity of transcriptional changes. The data show that old Long-Evans rats are not defective in their capacity to develop an appropriate cytokine response to the brain administration of IL-1beta. The implications of these findings for neuroimmunological-neuroinflammatory and neurotoxic/neurodegenerative processes are discussed.

Kindling modulates the IL-1beta system, TNF-alpha, TGF-beta1, 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-1beta, 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-alpha, TGF-beta1, 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-1beta, IL-1RI, TNF-alpha and TGF-beta1 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-1beta and TNF-alpha mRNAs). IL-1R AcP I and II mRNA levels were also up-regulated in the cortical regions. No changes in IL-1beta, IL-1RI or TNF-alpha 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.

Anorexia induced by activators of the signal transducer gp 130.

Cytokines [interleukin (IL-1, chemokines/intercrines, interferon, tumor necrosis factor-alpha (TNF-alpha)] induce anorexia when administered intracerebroventricularly (i.c.v.) at estimated pathophysiological concentrations. Here, the effects of the i.c.v. microinfusion (1.0, 20 and 100 ng) of activators of glycoprotein (gp) 130, a common signal transducer among receptors for members of the IL-6 subfamily [IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor (LIF), oncostatin M (OSM)], on short-term feeding in rats were investigated. Several members of the IL-6 subfamily induced anorexia, IL-11 was the most potent, LIF the most effective, and OSM had no effect. The results suggest that IL-6 subfamily members act centrally to decrease feeding and this effect may participate in the anorexia which frequently accompanies pathological processes. This action could interact with the anorexigenic effect of other cytokines released during disease.

Interleukin-2 modulates calcium currents in dissociated hippocampal CA1 neurons.

Interleukin-2 and its receptors are present in hippocampal structures, particularly the pyramidal cell layer. In the present study, we examined the interaction between recombinant human interleukin-2 (rhIL-2) and the voltage-dependent calcium (Ca2+) current using the whole-cell patch-clamp technique. RhIL-2 depressed a fraction of the inward Ca2+ current in acutely dissociated guinea-pig hippocampal CA1 neurons. This depression is rapid, dose-dependent with 0.001-1.0 ng rhIL-2/10 microliters (6.8 x 10(-12) to 6.8 x 10(-9) M) and reversible in > 80% of the neurons. Heat-inactivated rhIL-2 had no effect. The application of anti-rhIL-2 monoclonal antibody inhibited the rhIL-2-induced depression of the Ca2+ current. The depression of the Ca2+ current by IL-2 may play a role in the induction and/or progression of neuropathological processes of immunological origin, and in the induction of neurological manifestations during IL-2 immunotherapy.

Topiramate promotes neurite outgrowth and recovery of function after nerve injury.

Topiramate is a structurally novel neurotherapeutic agent with a unique combination of pharmacological properties and currently is available in most world markets for treating several seizure disorders. Because its pharmacological profile was suggestive of possible activity as a neuroprotectant, topiramate was evaluated and found to be active in several animal models of stroke or neuropathic pain. This prompted an evaluation of topiramate as a possible neurotrophic agent. In this study, topiramate enhanced the recovery of facial nerve function after injury when administered orally at therapeutically relevant doses, and significantly increased neurite outgrowth in cell cultures derived from fetal rat cortical and hippocampal tissues.

Epidermal growth factor and the nervous system.

Various growth factors and their receptors are present in the nervous system. This review focuses on the presence of epidermal growth factor (EGF) and its receptors in the central nervous system (CNS). Evidence indicates that EGF in the CNS is the result of local synthesis, by intrinsic and blood-derived macrophages, glial cells and neurons, and uptake from the peripheral blood through the circumventricular organs and probably also through the blood-brain barrier. Evidence is accumulating suggesting that EGF regulates a variety of CNS functions in a specific manner. EGF influences CNS growth, differentiation and maintenance (actions proposed to promote neural regeneration and cell survival following a variety of insults). EGF also induces neuromodulatory actions, affects the neuroendocrine system, and suppresses food intake and gastric acid secretion. Acute and chronic pathological processes, e.g., various cancers, stimulate the production and release of EGF in various cell systems. Monitoring of EGF by the CNS may participate in several neurological manifestations (e.g., appetite suppression, neuroendocrine alterations) frequently accompanying acute and chronic disease. EGF and transforming growth factor-alpha (TGF-alpha, a factor that binds to the EGF receptor with high affinity and induces the same biological signals as EGF) also may be involved in the promotion of malignancy in the CNS and in the neuropathogenesis of degenerative disorders. Thus evidence is accumulating concerning the neurophysiological or neuropathophysiological significance of EGF in the nervous system.

Neuropeptide Y blocks and reverses interleukin-1 beta-induced anorexia in rats.

Neuropeptide Y (NPY) increases feeding by direct action in the central nervous system (CNS). Interleukin-1 beta (IL-1 beta), on the other hand, induces anorexia when administered ICV at estimated pathophysiological (e.g., yielded by 1.0 ng/rat dose) and pharmacological (> or = 4.0 ng) concentrations in the cerebrospinal fluid (CSF). In the present study, we investigated NPY/IL-1 beta interactions using the ICV administration. ICV microinfusion of NPY (5.0 micrograms) significantly increased 2-h food intake (by 89%), whereas IL-1 beta decreased 2-h food intake (32% decrease with 1.0 ng/rat; 53% with 4.0 ng/rat; and 51% with 8.0 ng/rat). NPY (5.0 micrograms) blocked the anorexic effect induced by all doses of IL-1 beta when both compounds were administered concomitantly. Central infusion of NPY was also able to induce feeding in IL-1 beta-pretreated rats exhibiting marked anorexia. The results show that ICV-administered NPY blocks and reverses the anorexia induced by estimated pathophysiological and pharmacological concentrations of IL-1 beta in rats. A second interpretation of a data subset is that IL-1 beta attenuates or blocks NPY-induced increase in feeding depending on the IL-1 beta dose used. Blockage and reversal of IL-1 beta-induced anorexia by NPY suggest the importance in studying cytokine-peptide interactions in the regulation of feeding behavior. Understanding these endogenous interactions may produce strategies with potential therapeutic implications for chronic diseases associated with long-term anorexia.

An efficient, reliable and inexpensive device for the rapid homogenization of multiple tissue samples by centrifugation.

Homogenization of tissue samples is a common first step in the majority of current protocols for RNA, DNA, and protein isolation. This report describes a simple device for centrifugation-mediated homogenization of tissue samples. The method presented is applicable to RNA, DNA, and protein isolation, and we show examples where high quality total cell RNA, DNA, and protein were obtained from brain and other tissue samples. The advantages of the approach presented include: (1) a significant reduction in time investment relative to hand-driven or individual motorized-driven pestle homogenization; (2) easy construction of the device from inexpensive parts available in any laboratory; (3) high replicability in the processing; and (4) the capacity for the parallel processing of multiple tissue samples, thus allowing higher efficiency, reliability, and standardization.

An approach to study molecular mechanisms involved in cytokine-induced anorexia.

Cytokines are released during pathophysiological processes. Cytokines (e.g., interleukin-1 beta or IL-1 beta) induce neurological manifestations including anorexia. Here, we show an integrative approach to investigate the cellular and molecular basis of cytokine-induced anorexia. In this approach: (1) the chronic intracerebroventricular (icv) microinfusion (via osmotic minipumps) of cytokines, at doses that will yield estimated pathophysiological concentrations reported in the cerebrospinal fluid, is used. (2) General and computerized behavioral monitoring characterizes the microstructure of behavioral modifications induced by a cytokine, and the time course for cytokine action. (3) Brain regions and subregions (nuclei/areas) from animals exhibiting significant anorexia in response to cytokine(s) are dissected, and RNA and protein are isolated. (4) The profile of cytokine subsystems (ligands, receptors, endogenous inhibitors; for example, IL-1 alpha and beta, IL-1 receptor types I and II, and IL-1 receptor antagonist) is characterized in the same brain samples with polymerase chain reaction, sensitive RNase protection assays and immunoblots. (5) The relationship between changes in cytokine subsystems at the molecular level and cytokine-induced anorexia within an animal is determined, and the general profile is analyzed with statistical methods. This approach is also pertinent to study neurotransmitter and neuropeptide profiles, and cytokine-cytokine, cytokine-neuropeptide and cytokine-neurotransmitter interactions in vivo. The results show that this integrative and novel strategy can be used to study the molecular basis of anorexia and other neurological manifestations (e.g., fever, sleep changes) induced by cytokines.