Vascular factors are critical in selective neuronal loss in an animal model of impaired oxidative metabolism

Thiamine deficiency (TD) models the cellular and molecular mechanisms by which chronic oxidative deficits lead to death of select neurons in brain. Region- and cell-specific oxidative stress and vascular changes accompany the TD-induced neurodegeneration. The current studies analyzed the role of oxidative stress in initiating these events by testing the role of intercellular adhesion molecule-1 (ICAM-1) and endothelial nitric oxide synthase (eNOS) in the selective neuronal loss that begins in the submedial thalamic nucleus of mice. Oxidative stress to microvessels is known to induce eNOS and ICAM-1. TD increased ICAM-1 immunoreactivity in microvessels within the submedial nucleus and adjacent regions 1 day prior to the onset of neuronal loss. On subsequent days, the pattern of ICAM-1 induction overlapped that of neuronal loss, and of induction of the oxidative stress marker heme oxygenase-1 (HO-1). The intensity and extent of ICAM-1 and HO-1 induction progressively spread in parallel with the neuronal death in the thalamus. Targeted disruption of ICAM-1 or eNOS gene, but not the neuronal NOS gene, attenuated the TD-induced neurodegeneration and HO-1 induction. TD induced ICAM-1 in eNOS knockout mice, but did not induce eNOS in mice lacking ICAM-1. These results demonstrate that in TD, an ICAM-1-dependent pathway of eNOS induction leads to oxidative stress-mediated death of metabolically compromised neurons. Thus, TD provides a useful model to help elucidate the role of ICAM-1 and eNOS in the selective neuronal death in diseases in which oxidative stress is implicated.     

Proinflammatory cytokines and apoptosis following glutamate-induced excitotoxicity mediated by p38 MAPK in the hippocampus of neonatal rats

The proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 rise during neuronal damage and activate the apoptotic mitogen-activated protein kinase p38. We studied apoptosis, the levels of TNF-alpha, IL-1beta, and IL-6, and the cell type producing TNF-alpha in rats at 8, 10, and 14 days of age after neonatal exposure to glutamate, which induces neuronal damage. TNF-alpha production was significantly increased by glutamate, but inhibited by SB203580 (a p38 inhibitor). TNF-alpha, IL-1beta, and IL-6 mRNA levels increased, but SB203580 did not modify their expression. Thus, the p38 signaling pathway influences the expression of inflammatory genes and its inhibition may offer anti-inflammatory therapy.     

Peripheral and central proinflammatory cytokine response to a severe acute stressor

The role of proinflammatory cytokines in the response to acute stressor exposure has received recent attention. Exposure to a single session of inescapable shock (IS) induces peripheral and central proinflammatory cytokines. Other stressors also increase expression of proinflammatory cytokine mRNA and/or protein in various tissues. However, the induction of central and peripheral proinflammatory cytokines by stressors remains controversial and the pattern of cytokine induction is not consistent across stressors. The present experiments sought to examine the pattern of the proinflammatory cytokine response to a stressor known to cause elevations of IL-1beta protein. mRNA expression for three proinflammatory cytokines, IL-1beta, TNF-alpha and IL-6, and IL-1beta protein was examined after IS. IS increases IL-1beta mRNA and/or protein in a variety of tissues, including hypothalamus, hippocampus, pituitary and spleen. Furthermore, IS concomitantly alters IL-1beta mRNA and protein in hypothalamus and spleen, while the IL-1beta mRNA increase in pituitary lags behind the increase of IL-1beta protein. Interestingly, IL-1beta mRNA is elevated in hippocampus 4 h after IS, but an increase of IL-1beta protein in hippocampus is not detected. Expression of TNF-alpha and IL-6 mRNA do not increase in response to IS. Indeed, TNF-alpha mRNA expression decreases in cortex, pituitary and liver immediately after IS. These findings suggest that alterations of proinflammatory cytokine expression by stressors, and IS in particular, are region- and cytokine-specific.     

Expression of connexin36 in the adult and developing rat brain

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neuronal marker (NeuN), we found that neuronal cells are responsible for the expression of Cx36 mRNA in inferior olive, cerebellum, striatum, hippocampus and cerebral cortex. Cx36 mRNA was also demonstrated in parvalbumin-containing GABAergic interneurons of cerebral cortex, striatum, hippocampus and cerebellar cortex. Analysis of developing brain further revealed that Cx36 reaches a peak of expression in the first two weeks of postnatal life, and decreases sharply during the third week. Moreover, in these early stages of postnatal development Cx36 is detectable in neuronal populations that are devoid of Cx36 mRNA at the adult stage. The developmental changes of Cx36 expression suggest a participation of this connexin in the extensive interneuronal coupling which takes place in several regions of the early postnatal brain.     

Changes in expressions of proinflammatory cytokines IL-1beta, TNF-alpha and IL-6 in the brain of senescence accelerated mouse (SAM) P8

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. The SAMP8 strain shows age-related deterioration of learning and memory at an earlier age than control mice (SAMR1). In the present study, we investigated the changes in expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the brain of SAMP8. In the hippocampus of 10 months old SAMP8, the expression of IL-1 mRNA was significantly elevated in comparison with that of SAMR1. In both strains of SAMs, increases in IL-1beta protein in the brain were observed at 10 months of age compared with 2 and 5 months. The only differences found between the strain in protein levels were at 10 months and were elevations in IL-1beta in the hippocampus and hypothalamus, and in TNF-alpha and IL-6 in the cerebral cortex and the hippocampus in SAMP8 as compared with SAMR1. However, lipopolysaccharide-induced increases in the expression of these cytokines in brain did not differ between SAMP8 and SAMR1. Increases in expression of proinflammatory cytokines in the brain may be involved in the age-related neural dysfunction and/or learning deficiency in SAMP8.     

Secretory phospholipase A2 IIA is up-regulated by TNF-alpha and IL-1alpha/beta after transient focal cerebral ischemia in rat

Cerebral ischemia initiates an inflammatory response in the brain that is associated with the induction of a variety of cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha/beta (IL-1alpha/beta) that contributes to stroke injury. Transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rat (SHR) resulted in significant increases in TNF-alpha and IL-1beta levels. We have previously demonstrated up-regulation of secretory phospholipase A2 IIA (sPLA2 IIA) mRNA and protein expression, increased PLA2 activity, and loss of phosphatidylcholine after 1-h tMCAO and 24-h reperfusion in SHR. Treatment with TNF-alpha antibody or IL-1 receptor antagonist significantly attenuated infarction volume, sPLA2 IIA protein expression, PLA2 activity and significantly restored phosphatidylcholine levels after tMCAO. This suggests that cytokine induction up-regulates sPLA2 IIA protein expression, resulting in altered lipid metabolism that contributes to stroke injury.     

Increased PKA and PKC activities accompany neuronal differentiation of NT2/D1 cells

After retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (C alpha and C beta) subunits of PKA increased after differentiation. Total PKA activity levels increased 7-fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12-fold induction of Type 2 (beta) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (beta) and Type 3 (alpha) PKC protein levels also accompanied differentiation. These changes in PKA- and PKC-specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state in these cells.     

Endogenous brain cytokine mRNA and inflammatory responses to lipopolysaccharide are elevated in the Tg2576 transgenic mouse model of Alzheimer's disease.

Beta-amyloid (Abeta) plaques have been shown to induce inflammatory changes in Alzheimer's disease brains. Cortical, but not cerebellar tissue from 16-month-old Tg2576 (Tg+) mice showed significant increases in interleukin (IL)-1alpha (2.2-fold), IL-1beta (3.4-fold), tumor necrosis factor-alpha (3.9-fold), and monocyte chemoattractant protein-1 (2.5-fold) mRNA levels compared to controls (Tg-). These changes were not apparent in 6-month-old Tg+ mice except for TNF-alpha. mRNA levels of glial fibrillary acidic protein and complement components, C1qA and C3 were also elevated in aged mice. Lipopolysaccharide (LPS) (25 microg/mouse, i.v.) induced a significantly greater production of IL-1beta protein in the cortices and hippocampi of Tg+ vs. Tg- mice at 1, 2, 4, and 6 h. Experiments in 6-month-old mice showed that not only was there less cytokine produced compared to 16-month-old mice, but the exacerbated cytokine response to LPS in Tg+ mice was not apparent. Higher levels of Abeta1-40 were measured in the cortices of 6- and 16-month-old Tg+ mice at 4-6 h after LPS, which returned to baseline after 18 h. We demonstrate that Abeta plaques elicit inflammatory responses in Tg2576 mice that are further exacerbated when challenged by an exogenous inflammatory insult, which may serve to amplify degenerative processes.     

Central monoamine and plasma corticosterone changes induced by a bacterial endotoxin: sensitization and cross-sensitization effects

Low doses of lipopolysaccharide, tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or exposure to a stressor (restraint) increased plasma corticosterone levels. In animals pretreated with lipopolysaccharide, a marked sensitization of the corticosterone response was evident upon subsequent exposure to lipopolysaccharide, TNF-alpha, or restraint, 1 day later. As well, the sickness-inducing effects of lipopolysaccharide, TNF-alpha and IL-1 beta were markedly increased in mice pretreated with lipopolysaccharide. The sensitization effects were marked when the second treatment was administered 1 day after lipopolysaccharide administration, but not when a 28-day interval elapsed. In a second experiment, TNF-alpha influenced monoamine functioning in the paraventricular nucleus of the hypothalamus and within extrahypothalamic regions, including the central amygdala, locus coeruleus, prefrontal cortex. Moreover, serotonin activity within the central amygdala, as well as dopamine activity within the prefrontal cortex, were subject to a sensitization effect in animals pretreated with lipopolysaccharide 1 day earlier. Macrophage depletion by a suspension of clodronate liposomes attenuated the plasma corticosterone changes induced by TNF-alpha, but did not affect the sensitization. In contrast, the acute effects of TNF-alpha on central neurotransmitters were unaffected by the liposome suspension, but this treatment prevented the sensitization. These data may be relevant to clinical situations in which individuals exposed to bacterial infections may be rendered more susceptible to the behavioural and neurochemical effects of subsequently encountered stressors and immunological challenges.     

The novel beta-blocker, carvedilol, provides neuroprotection in transient focal stroke.

Increasing evidence supports a role for oxidative stress, proinflammatory cytokines, and apoptosis in the pathophysiology of focal ischemic stroke. Previous studies have found that the multi-action drug, carvedilol, is a mixed adrenergic antagonist, and that it behaves as an antioxidant and inhibits apoptosis. In the current study, the authors investigated whether carvedilol provides protection in focal cerebral ischemia and whether this protection is associated with reduced apoptosis and the downregulation of the inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin- 1beta (IL-1beta). Male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) by an intraluminal filament technique. Carvedilol (1, 3, and 10 mg/kg) was injected daily subcutaneously 2 or 4 days before the induction of ischemia. Neurologic scores, infarct volumes, TUNEL staining, and mRNA levels of TNF-alpha and IL-1beta were assessed at 24 hours reperfusion. The effect of carvedilol on microvascular cortical perfusion was studied with continuous laser-Doppler flowmetry. Twenty-four hours after MCAO, carvedilol at all three doses reduced infarct volumes by at least 40% and reduced neurologic deficits on average by 40% compared with vehicle-treated controls when given 2 or 4 days before the induction of ischemia. This protection was not mediated by changes in temperature or blood flow. Treatment with all three dose regimens resulted in fewer TUNEL positive cells compared with controls. At 24 hours reperfusion, carvedilol decreased TNF-alpha and IL-1beta expression by 40% to 50% in the ipsilateral ischemic cortex compared with the contralateral controls. The results of the current study indicate that carvedilol is neuroprotective in focal cerebral ischemia and may protect the ischemic brain by inhibiting apoptosis and attenuating the expression of TNF-alpha and IL-1beta.     

Cytokine regulation of E-selectin in rat CNS microvascular endothelial cells: differential response of CNS and non-CNS vessels

We have compared the induced expression of E-selectin in primary cultures of rat brain microvascular endothelial cells (EC), pericytes and in non-CNS microvascular endothelium stimulated with the cytokines, IL-1beta (20 ng/ml), and tumor necrosis factor (TNF)-alpha (75 ng/ml). Expression was studied at both the protein and mRNA levels. Fluorescence in-situ hybridization (FISH) was used to examine de novo synthesis of E-selectin mRNA. Laser cytometric analysis was used as a novel approach to the quantitaion of FISH. In-situ hybridization was performed using two PCR-generated probes. The first probe (517 bp) spanned the lectin and epidermal growth factor (EGF)-like domain. The second probe (562 bp) spanned the CR3, 4, and 6 domains. E-selectin-specific mRNA was localized to the perinuclear regions of the EC. Both cytokines, IL-1beta and TNF-alpha significantly increased E-selectin gene expression in CNS EC but not pericytes. IL-1beta induced higher E-selectin mRNA levels than TNF-alpha. The maximum number of mRNA-positive cells was observed after stimulation for 4--6 h. Surface protein expression was sustained for up to 48 h following addition of cytokine. This was in contrast to the transient expression in non-CNS EC indicating that pure primary CNS EC display slightly different kinetics of E-selectin expression than non-CNS EC.     

Modulation of IL-18 production in the adrenal cortex following acute ACTH or chronic corticosterone treatment

Interleukin (IL)-18 is a proinflammatory cytokine and a stimulator of cell-mediated immune responses. We have previously reported that acute stress stimulates the production of IL-18 mRNA in the glucocorticoid (GC)-producing cells of the adrenal cortex. In order to investigate the mechanisms governing the expression of IL-18 in the adrenal cortex, the effects of acute ACTH or chronic corticosterone treatment on the levels of IL-18 mRNA and protein were examined by in situ hybridization and Northern and Western blot assays. Adult male Sprague-Dawley rats received a subcutaneous injection of ACTH or subcutaneous implantation of slow-release corticosterone pellets followed by an injection of saline or ACTH. After 4 h, ACTH induced a 4-fold increase in IL-18 mRNA levels and elevated the content of pro-IL-18 peptide. Six days of chronic corticosterone treatment did not alter the basal levels of IL-18 mRNA and reduced those of pro-IL-18. Finally, ACTH treatment of animals under the corticosterone regimen induced a 2-fold increase in IL-18 mRNA and elevated the levels of the pro-IL-18 protein. The levels of the precursor, p45, and the active subunit p10 peptides of the IL-18-processing enzyme, IL-1beta-converting enzyme (ICE), showed no substantial differences in all the conditions tested. IL-1beta was not detected under these experimental conditions. These data demonstrate that the production of IL-18 in the adrenal cortex is stimulated by ACTH treatment and is not inhibited by the direct action of corticosterone. In contrast to the anti-inflammatory action of GCs, IL-18 may have an immunostimulatory role during acute stress.     

Induction and activation of protein kinase C delta in hippocampus and cortex after kainic acid treatment

Various isoforms of protein kinase C (PKC), especially the novel PKC subtypes delta, epsilon, and the atypical subtype PKC zeta, are involved in delayed cell death. We studied the expression and late activation of the latter PKC isoforms in comparison with classic PKC alpha, beta, and gamma in the brains of rats exposed to systemic kainate injection. The expression of PKC delta mRNA was strikingly upregulated (13-fold) in the cortex and the CA1 and CA3 hippocampal regions on 1 day after kainate administration, whereas PKC zeta mRNA was only moderately increased (about 100%) in these three brain regions on day 2 following the drug. PKC epsilon mRNA was slightly increased only in the cortex on days 2 and 6, while the mRNA levels of the classic PKC subtypes (alpha, beta, and gamma) remained unchanged or decreased after the treatment. Immunoblotting analyses revealed that the level of PKC delta protein started to increase on day 1 after kainate and was significantly elevated on day 2 in both the membrane and cytosol fractions of cortex and hippocampus. PKC epsilon protein only showed a marginal increase and the level of PKC zeta protein remained unaltered in response to the treatment. Cortical and CA1-3 pyramidal neurons displayed strong immunoreactivity for PKC delta on days 1 and 2, and microglia on days 1, 2, and 4 after the drug. The results indicate that the expression of apoptosis-associated isoforms of PKC, most notably that of delta, but to lesser extent also that of epsilon and zeta, is increased during kainate-induced neuronal death. The predominant induction of PKC delta in neurons and microglia suggests that PKC delta could be the major mediator or modulator of apoptotic and inflammatory responses to excitotoxic insults.