Pharmacological properties of S1RA, a new sigma-1 receptor antagonist that inhibits neuropathic pain and activity-induced spinal sensitization.

BACKGROUND AND PURPOSE: The sigma-1 (σ(1) ) receptor is a ligand-regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ(1) receptor ligands used as pharmacological tools are unclear and the demonstration that σ(1) receptor antagonists have efficacy in reversing central sensitization-related pain sensitivity is still missing. EXPERIMENTAL APPROACH: The pharmacological properties of a novel σ(1) receptor antagonist (S1RA) were first characterized. S1RA was then used to investigate the effect of pharmacological antagonism of σ(1) receptors on in vivo nociception in sensitizing conditions and on in vitro spinal cord sensitization in mice. Drug levels and autoradiographic, ex vivo binding for σ(1) receptor occupancy were measured to substantiate behavioural data. KEY RESULTS: Formalin-induced nociception (both phases), capsaicin-induced mechanical hypersensitivity and sciatic nerve injury-induced mechanical and thermal hypersensitivity were dose-dependently inhibited by systemic administration of S1RA. Occupancy of σ(1) receptors in the CNS was significantly correlated with the antinociceptive effects. No pharmacodynamic tolerance to the antiallodynic and antihyperalgesic effect developed following repeated administration of S1RA to nerve-injured mice. As a mechanistic correlate, electrophysiological recordings demonstrated that pharmacological antagonism of σ(1) receptors attenuated the wind-up responses in spinal cords sensitized by repetitive nociceptive stimulation. CONCLUSIONS AND IMPLICATIONS: These findings contribute to evidence identifying the σ(1) receptor as a modulator of activity-induced spinal sensitization and pain hypersensitivity, and suggest σ(1) receptor antagonists as potential novel treatments for neuropathic pain.

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.

A role for cyclooxygenase-2 in lipopolysaccharide-induced anorexia in rats.

Because nonselective cycloooxygenase (COX) inhibition attenuated anorexia after lipopolysaccharide (LPS) administration, we tested the ability of resveratrol (2.5, 10, and 40 mg/kg) and NS-398 (2.5, 10, and 40 mg/kg), selective inhibitors of the two COX isoforms COX-1 and -2, respectively, to attenuate LPS (100 microg/kg ip)-induced anorexia. NS-398 (10 and 40 mg/kg) administered with LPS at lights out attenuated LPS-induced anorexia, whereas resveratrol at all doses tested did not. Because prostaglandin (PG) E(2) is considered the major metabolite synthesized by COX, we measured plasma and cerebrospinal fluid (CSF) PGE(2) levels after LPS administration. LPS induced a time-dependent increase of PGE(2) in CSF but not in plasma. NS-398 (5, 10, and 40 mg/kg) blocked the LPS-induced increase in CSF PGE(2), whereas resveratrol (10 mg/kg) did not. These results support a role of COX-2 in mediating the anorectic response to peripheral LPS and point at PGE(2) as a potential neuromodulator involved in this response.

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.

Abnormal patterns of microtubule-associated protein-2 (MAP-2) immunolabeling in neuronal nuclei and Lewy bodies in Parkinson's disease substantia nigra brain tissues.

Parkinson's disease (PD) is a neurodegenerative disorder associated with the appearance of cytoplasmic Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra and the progressive loss of these neurons. Cytoskeleton alterations and associated impairments of neuronal transport may contribute to neuronal death. Microtubule-associated protein-2 (MAP-2), a cytoskeleton protein is localized primarily in neuronal dendrites and is known to stabilize microtubule assembly and mediate their interactions with other neuronal cell components. To determine if alterations in MAP-2 morphology are present in PD neurons, we used single and double immunohistochemical and immunofluorescent techniques to characterize MAP-2 in PD neuronal tissues. We report abnormal MAP-2 immunolabeling in some neurons of the substantia nigra of PD brain tissues, which were not observed in the normal, age-matched, control brain tissues. Furthermore, MAP-2 was co-localized with alpha-synuclein and ubiquitin in cytoplasmic LBs of neurons. Surprisingly, MAP-2 was also found to form fibrous aggregates and crystal-like structures within neuronal nuclei. These PD-associated alterations in MAP-2 morphology and distribution suggest that impaired neuronal transport may contribute to the progression of neuronal loss in the brains of PD patients.

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.

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.

Cytokines and feeding.

Various categories of cytokines participate in the control of feeding, including interleukin-1 and -6 and other activators of gp 130, leptin (ob protein), interleukin-8 and other chemokines, tumor necrosis factor-alpha, and interferon-alpha. These feeding-inhibitory cytokines may play a role in the regulation of food intake during physiological (eg a role proposed for leptin) and pathophysiological (eg proinflammatory cytokines) conditions. Data show that various cytokines participate in acute and chronic disease-associated anorexia such as during infection, inflammation or malignancy. Food intake suppression (reported as anorexia) is also a common central manifestation observed during cytokine immunotherapy in humans. The concept of local production of various cytokines within specific brain regions in response to peripheral challenges and pathophysiological processes has broad implications for the interpretation of brain cytokines as mediators or participants in CNS modulation of feeding and anorexia.

Central nervous system mechanisms contributing to the cachexia-anorexia syndrome.

The cachexia-anorexia syndrome occurs in chronic pathophysiologic processes including cancer, infection with human immunodeficiency virus, bacterial and parasitic diseases, inflammatory bowel disease, liver disease, obstructive pulmonary disease, cardiovascular disease, and rheumatoid arthritis. Cachexia makes an organism susceptible to secondary pathologies and can result in death. Cachexia-anorexia may result from pain, depression or anxiety, hypogeusia and hyposmia, taste and food aversions, chronic nausea, vomiting, early satiety, malfunction of the gastrointestinal system (delayed digestion, malabsorption, gastric stasis and associated delayed emptying, and/or atrophic changes of the mucosa), metabolic shifts, cytokine action, production of substances by tumor cells, and/or iatrogenic causes such as chemotherapy and radiotherapy. The cachexia-anorexia syndrome also involves metabolic and immune changes (mediated by either the pathophysiologic process, i.e., tumor, or host-derived chemical factors, e.g., peptides, neurotransmitters, cytokines, and lipid-mobilizing factors) and is associated with hypertriacylglycerolemia, lipolysis, and acceleration of protein turnover. These changes result in the loss of fat mass and body protein. Increased resting energy expenditure in weight-losing cachectic patients can occur despite the reduced dietary intake, indicating a systemic dysregulation of host metabolism. During cachexia, the organism is maintained in a constant negative energy balance. This can rarely be explained by the actual energy and substrate demands by tumors in patients with cancer. Overall, the cachectic profile is significantly different than that observed during starvation. Cachexia may result not only from anorexia and a decreased caloric intake but also from malabsorption and losses from the body (ulcers, hemorrhage, effusions). In any case, the major deficit of a cachectic organism is a negative energy balance. Cytokines are proposed to participate in the development and/or progression of cachexia-anorexia; interleukin-1, interleukin-6 (and its subfamily members such as ciliary neurotrophic factor and leukemia inhibitory factor), interferon-gamma, tumor necrosis factor-alpha, and brain-derived neurotrophic factor have been associated with various cachectic conditions. Controversy has focused on the requirement of increased cytokine concentrations in the circulation or other body fluids (e.g., cerebrospinal fluid) to demonstrate cytokine involvement in cachexia-anorexia. Cytokines, however, also act in paracrine, autocrine, and intracrine manners, activities that cannot be detected in the circulation. In fact, paracrine interactions represent a predominant cytokine mode of action within organs, including the brain. Data show that cytokines may be involved in cachectic-anorectic processes by being produced and by acting locally in specific brain regions. Brain synthesis of cytokines has been shown in peripheral models of cancer, peripheral inflammation, and during peripheral cytokine administration; these data support a role for brain cytokines as mediators of neurologic and neuropsychiatric manifestations of disease and in the brain-to-peripheral communication (e.g., through the autonomic nervous system). Brain mechanisms that merit significant attention in the cachexia-anorexia syndrome are those that result from interactions among cytokines, peptides/neuropeptides, and neurotransmitters. These interactions could result in additive, synergistic, or antagonistic activities and can involve modifications of transducing molecules and intracellular mediators. Thus, the data show that the cachexia-anorexia syndrome is multifactorial, and understanding the interactions between peripheral and brain mechanisms is pivotal to characterizing the underlying integrative pathophysiology of this disorder.

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.

Neither acute nor chronic exposure to a naturalistic (predator) stressor influences the interleukin-1beta system, tumor necrosis factor-alpha, transforming growth factor-beta1, and neuropeptide mRNAs in specific brain regions.

Physical (neurogenic) stressors may influence immune functioning and interleukin-1beta (IL-1beta) 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-1beta, IL-1 receptor antagonist, IL-1 receptor type I, IL-1 receptor accessory proteins I and II, or tumor necrosis factor-alpha mRNA levels in the prefrontal cortex, amygdala, hippocampus, or hypothalamus. Likewise, there were no stressor effects on transforming growth factor-beta1, 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.

Cytokine-cytokine interactions and the brain.

Cytokine-cytokine interactions play a role in health and are crucial during immunological and inflammatory responses in disease. Cytokine interactions can result in additive, antagonist, or synergistic activities in maintaining physiological functions such as feeding, body temperature, and sleep, as well as in anorectic, pyrogenic, and somnogenic neurological manifestations of acute and chronic disease. These interactions involve signaling homology, convergence of signaling pathways, and/or positive or negative feedbacks within and among cytokine systems. The interplay of cytokines with neurotransmitters, peptides/neuropeptides, and hormones also influence cytokine action in the brain. Interactive chemical cascades involving cytokines are consistent with the homeostatic physiological mechanisms and with the multi-humoral, pleiotropic, and redundant processes that occur during acute and chronic disease.

Autoregulation of the interleukin-1 system and cytokine-cytokine interactions in primary human astrocytoma cells.

Cytokines are proposed to play important roles in brain tumor biology. Previous studies reported on interleukin-1beta (IL-1beta) production and IL-1 receptor type I (IL-1RI, signaling receptor) expression in human astrocytomas, and on IL-1beta action in astrocytoma cell lines. However, all studies that have tested the direct action of cytokines have used exclusively astrocytoma cell lines, which do not recapitulate the in situ astrocytoma. Here, we demonstrate that astrocytoma cells obtained shortly after tumor neurosurgical resection respond to the direct application of human IL-1beta with a significant upregulation of IL-1alpha, IL-1beta, IL-1RI, and tumor necrosis factor-alpha (TNF-alpha) mRNAs. IL-1 receptor antagonist (IL-1Ra, an endogenous inhibitor that blocks IL-1alpha and IL-1beta actions) mRNA was not upregulated. Application of heat-inactivated IL-1beta had no effect on any cytokine component examined, demonstrating specificity of action. On the other hand, IL-1beta application did not modulate any cytokine component in acutely resected and dissociated primitive neuroectodermal tumor cells. The data have implications for a positive autoregulatory IL-1beta feedback system and synergistic IL-1beta <=> TNF-alpha interactions, which can be involved in the growth of pilocytic astrocytomas. The results together with our previous studies also support the notion that IL-1Ra or a compound with similar cytokine inhibitory activity could be useful for brain immunotherapy of astrocytomas.

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.

Taste in the monkey cortex.

The sense of taste in humans differs substantially from that of rodents, from which a preponderance of gustatory electrophysiology derives. To establish a more appropriate neural model for human gustation, we recorded the activity of single neurons in the primary taste cortex in 11 alert cynomolgus macaques. Taste cells composed 6% of all neurons encountered. Another 24% responded during mouth and jaw movements, and 4% were sensitive to tactile stimulation of the mouth. Smaller numbers responded during olfactory or visual stimulation, or when the monkey extended his tongue. Taste cells could be divided into four statistically independent groups, corresponding to those most responsive to glucose (38%), NaCl (34%), quinine (22%), or HCI (5%). The location of a taste cell did not predict its response profile, i.e., there was no clear topographic organization of taste sensitivity. We established neural thresholds and intensity-response functions to the basic stimuli and determined that-with the exception of HCl, to which the macaque is relatively insensitive-they were similar to those reported by human subjects. We then turned to the coding of taste quality, as inferred in macaques from the patterns of neural activity elicited by each of greater than 100 stimuli. The results proved generally faithful to human reports of the perceived qualities of these same tastants. Finally, an investigation of taste mixtures revealed a degree of mixture suppression and interaction among basic qualities similar to those reported by humans. We conclude that the alert macaque offers a reliable neural model for human gustation.

Feeding status and bacterial LPS-induced cytokine and neuropeptide gene expression in hypothalamus.

This study determined the effects of feeding status on basal and lipopolysaccharide (LPS)-stimulated cytokine and neuropeptide gene expression in the hypothalamus. With the use of RNase protection assays, we measured mRNA levels of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1RA), IL-1 receptor type I (IL-1RI), IL-1R accessory proteins (AcP I and II), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), glycoprotein 130 (Gp 130), leptin receptor (OB-R), neuropeptide Y (NPY), preprodynorphin, and proopiomelanocortin (POMC). Analyses were done in ad libitum-fed, fasted, and fasted and refed rats treated with the intracerebroventricular administration of physiological saline or LPS. The data show that food deprivation increases the basal mRNA expression of IL-1beta, IL-1RA, TNF-alpha, IL-1RI, and IL-1R AcP I, whereas mRNA levels of POMC showed a decrease. Five hours of refeeding returned cytokine levels to those observed in the ad libitum-fed group. LPS administration induced a robust upregulation of IL-1beta, TNF-alpha, and IL-1RI during all three feeding conditions. Acute food deprivation did not modulate LPS-induced changes in hypothalamic cytokine mRNA profiles. These findings show that 1) cytokine modulation occurs as an adaptive response to the stress of acute fasting and 2) acute fasting does not affect LPS-induced cytokine mRNA modulation in the hypothalamus. The data have implications to gram-negative infections associated with acute anorexia.

Persistent Borna disease virus infection of neonatal rats causes brain regional changes of mRNAs for cytokines, cytokine receptor components and neuropeptides.

Borna disease virus (BDV) replicates in brain cells. The neonatally infected rat with BDV exhibits developmental-neuromorphological abnormalities, neuronal cytolysis, and multiple behavioral and physiological alterations. Here, we report on the levels of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), IL-1 receptor type I (IL-1RI), IL-1 receptor accessory protein (IL-1R AcP) I and II, glycoprotein 130, and various neuropeptide mRNAs in the cerebellum, parieto-frontal cortex, hippocampus and hypothalamus of BDV-infected rats at 7 and 28 days postintracerebral BDV inoculation. The data show that cytokine and neuropeptide mRNA components are abnormal and differentially modulated in brain regions. IL-1beta, TNF-alpha and TGF-beta1 mRNA levels were up-regulated in all brain regions following BDV inoculation. The same cerebellar samples from BDV-infected animals exhibited the highest levels of IL-1beta, IL-1Ra, TNF-alpha, IL-1RI, and IL-1R AcP II mRNA expression. The profiles of IL-1beta, IL-1Ra, TNF-alpha, and TGF-beta1 mRNA induction in the cerebellar samples were highly intercorrelated, indicating an association among cytokine ligand mRNAs. Cytokine mRNA induction was differentially up-regulated among brain regions, except for TGF-beta1. Specificity of transcriptional changes in response to BDV infection is also suggested by the up-regulation of cytokine and neuropeptide Y mRNAs associated with down-regulation of pro-opiomelanocortin, and with no change of IL-1R AcPI, dynorphin and leptin receptor mRNAs in the same brain region samples. Other data also show a differential mRNA component modulation in distinct brain regions obtained from the same rats depending on the stage of BDV infection. The conclusion of these studies is that cytokines may play a role in the neuropathophysiology of neonatally BDV-infected rats.