Estrogen replacement regimen and brain infusion of lipopolysaccharide differentially alter steroid receptor expression in the uterus and hypothalamus.

The regimen of estrogen replacement can alter the consequences of estrogen therapy and stressors. To determine the long-term effects and interaction of these systems on the brain and periphery, adult female rats were infused with lipopolysaccharide (LPS) into the fourth ventricle of the brain for 4 weeks, and ovariectomized rats were administered either constant or pulsed regimens of estrogen replacement (17beta-estradiol) until sacrifice at 8 weeks. Constant, but not pulsed, estrogen replacement reduced ERalpha and increased HSP90, HSP70, and PR(B) uterine protein levels. Both estrogen regimens increased ERbeta, HSP27, and PR(A) uterine proteins. Both regimens reduced hypothalamic levels of ERalpha, but not ERbeta, HSP, or PR. No changes were observed in the hippocampus. Long-term brain infusion of LPS activated microglia and reduced body weight, but did not alter corticosterone or nitrotyrosine levels. LPS infusion into intact rats suppressed uterine weight, increased ERalpha and decreased HSP90 in the uterus. LPS did not alter uterine weight in ovariectomized rats treated with constant or pulsed estrogen. Together, these data suggest the timing of estrogen replacement and neuroinflammatory stressors can profoundly affect uterine and hypothalamic steroid receptor expression and may be important parameters to consider in the post-menopausal intervention with estrogen.

Brain infusion of lipopolysaccharide increases uterine growth as a function of estrogen replacement regimen: suppression of uterine estrogen receptor-alpha by constant, but not pulsed, estrogen replacement.

The effects of estrogen therapy can differ depending on the regimen of estrogen administration. In addition, estrogen can modulate the effects of stressors. To examine the interaction between these systems, we infused adult female rats with lipopolysaccharide (LPS) into the fourth ventricle of the brain for 6 d and compared the effects of constant and pulsed estrogen replacement. Constant, but not pulsed, estrogen treatment reduced estrogen receptor-alpha (ERalpha) protein by 90% in the uterus and increased heat-shock proteins 70 and 90 by 74 and 48%, respectively, whereas progesterone receptor levels increased in all ovariectomized rats receiving estrogen replacement. In contrast to the uterine decline in ERalpha, no changes in ERalpha were observed in the hypothalamus or hippocampus, and ERbeta levels were unchanged in all regions tested. Brain infusion of LPS did not alter these proteins but increased the number of activated microglia in the thalamus and reduced body weight in all rats as well as activated the hypothalamic-pituitary-adrenal axis in ovariectomized rats, as determined by elevations in circulating corticosterone and progesterone. Estrogen treatments did not alter these markers, and no differences were observed in cortical choline acetyltransferase activity or nitrotyrosine for any of the treatment groups. The current study found an unexpected increase in uterine weight in lipopolysaccharide-infused rats treated with constant, but not pulsed, estrogen. This report suggests that constant and pulsed regimens of estrogen administration produce different effects and that stress may be an important factor in the postmenopausal intervention with estrogen.

The presence of the APP(swe) mutation in mice does not increase the vulnerability of cholinergic basal forebrain neurons to neuroinflammation.

Neuroinflammation, and elevated levels of inflammatory proteins, such as tumor necrosis factor-alpha, and the deposition of beta-amyloid may interact to contribute to the pathogenesis of Alzheimer's disease. We reproduced a component of the neuroinflammatory state within the basal forebrain cholinergic system, a region that is vulnerable to degeneration in Alzheimer's disease, of transgenic Tg2576 mice that express the Swedish double mutation of the human amyloid precursor protein (APPswe). We have previously shown that basal forebrain cholinergic neurons are selectively vulnerable to the consequences of neuroinflammation. In the current study, tumor necrosis factor-alpha was infused into the basal forebrain region of APPswe and nontransgenic control mice for 20 days with the expectation that the presence of the transgene would enhance the loss of cholinergic neurons. Chronic infusion of tumor necrosis factor-alpha significantly decreased cortical choline acetyltransferase activity, reduced the number of choline acetyltransferase-immunoreactive cells and increased the number of activated astrocytes and microglia within the basal forebrain. The presence of the APPswe gene did not enhance the vulnerability of forebrain cholinergic neurons to the chronic neuroinflammation. Furthermore, combined treatment of these mice with memantine demonstrated that the neurotoxic effects of tumor necrosis factor-alpha upon cholinergic cells did not require the activation of the N-methyl-d-aspartate receptors. In contrast, we have previously shown that memantine was able to provide neuroprotection to cholinergic forebrain neurons from the consequences of exposure to the inflammogen lipopolysaccharide. These results provide insight into the mechanism by which neuroinflammation may selectively target specific neural systems during the progression of Alzheimer's disease.

The toxicity of tumor necrosis factor-alpha upon cholinergic neurons within the nucleus basalis and the role of norepinephrine in the regulation of inflammation: implications for Alzheimer's disease.

Inflammation and reduced forebrain norepinephrine are features of Alzheimer's disease that may interact to contribute to the degeneration of specific neural systems. We reproduced these conditions within the basal forebrain cholinergic system, a region that is vulnerable to degeneration in Alzheimer's disease. Tumor necrosis factor-alpha was infused into the basal forebrain of young mice pretreated with a norepinephrine neuronal toxin, N-(2-chloroethyl)-N-ethyl-2 bromobenzylamine (DSP4), with the expectation that the loss of noradrenergic input would enhance the loss of cholinergic neurons. The results indicate that chronic infusion of tumor necrosis factor-alpha alone significantly decreased cortical choline acetyltransferase activity and increased the number of activated microglia and astrocytes within the basal forebrain. The loss of forebrain norepinephrine following systemic treatment with DSP4 did not alter the level of cortical choline acetyltransferase activity or activate microglia but significantly activated astrocytes within the basal forebrain. Infusion of tumor necrosis factor-alpha into DSP4-pretreated mice also reduced cortical choline acetyltransferase activity on the side of the infusion; however, the decline was not significantly greater than that produced by the infusion of tumor necrosis factor-alpha alone. The neurodegeneration seen may be indirect since a double-immunofluorescence investigation did not find evidence for the co-existence of tumor necrosis factor-alpha type I receptors on choline acetyltransferase-positive cells in the basal forebrain. The results suggest that noradrenergic cell loss in Alzheimer's disease does not augment the consequences of the chronic neuroinflammation and does not enhance neurodegeneration of forebrain cholinergic neurons.

Long-term estrogen therapy worsens the behavioral and neuropathological consequences of chronic brain inflammation.

Alzheimer's disease (AD) is accompanied by chronic neuroinflammation and occurs with greater incidence in postmenopausal women. The increased incidence may be delayed by estrogen replacement therapy (ERT). The authors investigated the interaction of chronic ERT and lipopolysaccharide (LPS)-induced neuroinflammation in the female rat. Ovariectomy did not impair water maze performance; however, addition of chronic ERT or neuroinflammation resulted in an impairment that became exacerbated by the simultaneous occurrence of both conditions. Chronic LPS activated microglia, which was not reduced by ERT. Intact females receiving LPS infusion were not impaired in the water maze and had significantly fewer activated microglia. Results suggest that chronic ERT in postmenopausal women may exacerbate the memory impairment induced by the chronic neuroinflammation associated with AD.

Chronic brain inflammation results in cell loss in the entorhinal cortex and impaired LTP in perforant path-granule cell synapses.

Alzheimer's disease (AD) is characterized by chronic neuroinflammation, significant temporal lobe cell loss, and dementia. We investigated the influence of chronic neuroinflammation produced by chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle for 4 weeks upon the induction and maintenance of long-term potentiation (LTP) in the dentate gyrus of the hippocampus, a well-characterized model of cellular synaptic plasticity. We also examined for pyramidal cell loss within the entorhinal cortex an area of the brain that contains the cell bodies of the perforant path. The results demonstrate that chronic neuroinflammation results in the loss of pyramidal cells within layers II and III of the entorhinal cortex and a significant attenuation of LTP within the dentate gyrus. Similar changes may underlie the temporal lobe pathology and dementia associated with AD.

The cytotoxicity of chronic neuroinflammation upon basal forebrain cholinergic neurons of rats can be attenuated by glutamatergic antagonism or cyclooxygenase-2 inhibition.

The proinflammagen lipopolysaccharide (LPS) was infused chronically (37 days) into the basal forebrain of rats. The current study determined whether the chronic administration of either a non-competitive N-methyl-D-aspartate- (NMDA-) sensitive receptor antagonist, memantine, or a selective cyclooxygenase-2 (COX2)/lipoxygenase inhibitor, CI987, could provide significant neuroprotection from the cytotoxic effects of LPS-induced neuroinflammation. Chronic LPS infusions decreased cortical choline acetyltransferase activity, which paralleled a decline in the number of choline-acetyltransferase-immunoreactive-cells within the basal forebrain as well as the number of activated resident microglia. The infusions appeared to be selective for cholinergic neurons. Peripheral administration of memantine (i.p.) or CI987 (s.c.) significantly attenuated the cytotoxic effects of the chronic inflammatory processes upon cholinergic cells within the basal forebrain. However, only CI987 attenuated the neuroinflammation produced by LPS and the subsequent changes in microglial activation. These results indicate that the cytotoxic effects of chronic neuroinflammation may involve prostanoid synthesis and may operate through NMDA receptors, and that the effects of prostaglandins occur upstream to NMDA-receptor activation.

Quantitative volumetric analyses of brain magnetic resonance imaging from rat with chronic neuroinflammation.

Brain inflammation may have a pathogenic role in many neurodegenerative diseases, including Alzheimer's disease. In the present study, we investigated the effects of chronic neuroinflammation upon anatomical changes in two regions of interest in the temporal lobe using high-resolution magnetic resonance imaging techniques. We show that chronic infusion of lipopolysaccharide into the fourth ventricle for 4 consecutive weeks enlarged the lateral ventricles and significantly decreased the size of the hippocampal formation and the temporal lobe region. These changes are comparable to those observed in humans during the early stages of Alzheimer's disease.

Mechanisms to prevent the toxicity of chronic neuroinflammation on forebrain cholinergic neurons.

Inflammatory processes may play an important role in the degeneration of basal forebrain cholinergic cells Alzheimer's disease. We infused the proinflammagen lipopolysaccharide into the basal forebrain of young rats and determined whether the chronic administration of two novel non-steroidal anti-inflammatory drugs or a pan-caspase synthesis inhibitor, z-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD), could provide neuroprotection from the cytotoxic effects of the neuroinflammation. Chronic lipopolysaccharide infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain region. The level of caspases 3, 8 and 9 was increased in ventral caudate/putamen. Non-steroidal anti-inflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases 3, 8 and 9 activity in the caudate/putamen. zVAD treatment significantly decreased the levels of caspases 3, 8 and 9 but did not provide neuroprotection for the cholinergic neurons. These results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in Alzheimer's disease.

LPS-induced neuroinflammatory effects do not recover with time.

Inflammatory processes develop in the vicinity of the neuropathological hallmarks associated with Alzheimer's disease (AD) and may play a role in the progression of the disease and its clinical expression. We have previously reported that chronic infusion of LPS into the fourth ventricle of rat brains reproduced many of the inflammatory and pathological changes seen in the brain of AD patients. In the current study, we used the same animal model to investigate the effects of longer infusion of LPS and whether these effects could recover over time. The results show that doubling the time of LPS infusion did not increase the inflammatory reaction and did not produce a significantly greater behavioral impairment. Waiting for 37 days after the cessation of the LPS infusion did not decrease the density of activated microglia and did not improve performances in the Morris water maze task. The results suggest that inflammation may contribute to the pathogenic mechanisms that underlie the clinical expression of AD.

Behavioral and ultrastructural changes induced by chronic neuroinflammation in young rats.

We investigated the ultrastructural, immunohistochemical, biochemical and behavioral effects of chronic neuroinflammation in young rats produced by injection of lipopolysaccharide (LPS) into the 4th ventricle. The 37-day infusion of LPS impaired spatial memory but not object recognition ability. Electron microscopic studies of neurons within the hippocampus identified numerous paired cisternae of the rough endoplasmic reticulum (RER) and other ultrastructural changes that suggested impaired or reduced synthesis of cellular proteins within the cytoplasm. Immunohistochemical staining found numerous highly activated microglia distributed throughout the cingulate gyrus, entorhinal cortex, hippocampus and dentate gyrus. This animal model may be useful to test potential pharmacotherapies that are directed at the prevention of the cytotoxic consequences of chronic neuroinflammation associated with normal aging or Alzheimer's disease.

The effects of a novel NSAID on chronic neuroinflammation are age dependent.

Chronic inflammation may play an important role in the pathogenesis of Alzheimer's disease (AD). The present study compared the effects of chronic neuroinflammation, produced by infusion of lipopolysaccharide (LPS) into the fourth ventricle, upon memory in young, adult, and old rats. Nonsteroidal anti-inflammatory drug (NSAID) therapy may delay the onset of AD. We show that NO-Flurbiprofen (NFP), a novel NSAID that lacks gastrointestinal side effects, attenuated the neuroinflammatory reaction and reduced the inflammation-induced memory deficit. Chronic LPS infusions impaired performance of young rats but not adult or old rats. Treatment with NFP improved the performance of LPS-infused young rats, but not LPS-infused adult or old rats. LPS infusions increased the number of activated microglia in young and adult rats but not old rats. NFP treatment attenuated the effects of LPS upon microglia activation in young and adult rats, but not old rats. The results suggest that NSAID therapies designed to influence the onset of AD should be initiated in adults before age-associated inflammatory processes within the brain have a chance to develop.

Altered cholinergic function in the basal forebrain of girls with Rett syndrome.

Rett syndrome (RS) is a neurodevelopmental disorder that is predominant in females and is associated with cortical atrophy, stereotyped hand movements and severe mental deficiency. Previous studies have demonstrated a significant decline in number of choline acetyltransferase (ChAT)-containing neurons throughout the forebrain of RS girls. The loss of these ChAT-positive cells may be caused by a lack of nerve growth factor (NGF). In the current study, cortical levels of NGF were normal in RS girls as compared to age-and sex-matched controls. The number of neurons within the basal forebrain that express the 75 kDa (p75) low-affinity receptor for NGF was unchanged. In contrast, the number of ChAT-positive neurons was significantly decreased. The results suggest that normal amounts of NGF are available for binding to the p75 receptor and for retrograde transport to forebrain cholinergic cells, however, these neurons do not respond by producing the ChAT protein that is necessary for the production of the neurotransmitter acetylcholine.

Pathological and biochemical consequences of acute and chronic neuroinflammation within the basal forebrain cholinergic system of rats.

Inflammatory processes may play a critical role in the degeneration of basal forebrain cholinergic cells that underlies some of the cognitive impairments associated with Alzheimer's disease. In the present study, the proinflammagen lipopolysaccharide, from the cell wall of Gram-negative bacteria, was used to produce inflammation within the basal forebrain of rats. The effects of acute, high-dose injections of lipopolysaccharide (2, 20 or 40 microg) upon basal forebrain chemistry and neuronal integrity were compared with the effects of chronic, low-dose lipopolysaccharide infusions (0.18, 0.25, 1.8 or 5.0 microg/h) for either 14, 37, 74 or 112 days. Acute exposure to lipopolysaccharide decreased cortical choline acetyltransferase activity and the number of immunoreactive choline acetyltransferase-positive cells within a small region of the basal forebrain. Regional levels of five different neuropeptides were unchanged by acute, high-dose lipopolysaccharide injections. Chronic lipopolysaccharide infusions produced (i) a time-dependent, but not dose-dependent, decrease in cortical choline acetyltransferase activity that paralleled a decline in the number of choline acetyltransferase- and p75-immunoreactive cells within the basal forebrain, and (ii) a dense distribution of reactive astrocytes and microglia within the basal forebrain. Chronic neuroinflammation might underlie the genesis of some neuropathological changes associated with normal ageing or Alzheimer's disease.

Peripheral administration of novel anti-inflammatories can attenuate the effects of chronic inflammation within the CNS.

In the present study we investigated whether nitroflurbiprofen (NFP) or nitro-aspirin can reduce the inflammatory response induced by continuous infusion of lipopolysaccharide (LPS) into the fourth ventricular space of the rat's brain for 30 days. The chronic LPS infusion produced an extensive inflammation that was particularly evident in the hippocampus, subiculum and entorhinal and piriform cortices. Daily peripheral administration of NFP dose-dependently, and significantly, attenuated the brain inflammation as indicated by the decreased density and reactive state of microglial cells. Daily peripheral administration of nitro-aspirin also attenuated the brain inflammation, but to a much lesser degree than NFP. The results demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs) could reduce brain inflammation and that NFP is an effective anti-inflammatory agent.

Neuroprotection by novel antagonists at the NMDA receptor channel and glycineB sites.

Glutamate may act via an N-methyl-D-Aspartate (NMDA)-sensitive receptor site to destroy cholinergic neurons within the nucleus basalis magnocellularis in age-associated neurodegenerative diseases. Multiple interesting properties of the NMDA receptor are relevant to its excitotoxic actions, e.g., glutamate is ineffective unless a glycine (gly) modulatory site is also occupied. Thus, the antagonism of glutamate receptor-related toxicity by blockade of either the NMDA-sensitive recognition site or the gly binding site may therefore have therapeutic applications. The current study investigated the ability of four novel noncompetitive antagonists at these two sites: one NMDA open channel antagonist (MRZ 2/579: 1-amino-1,3,3,5,5-pentamethyl-cyclohexane hydrochloride), and three glyB receptor antagonists (MRZ 2/570: 8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline salt; MRZ 2/57: 8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline; MRZ 2/576: 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline) administered acutely, to provide neuroprotection from a NMDA receptor agonist within the nucleus basalis magnocellularis of young rats. Injection of NMDA into the nucleus basalis magnocellularis significantly decreased cortical choline acetyltransferase activity. Acute administration (i.p.) of MRZ 2/579, 2/570, 2/571 and 2/576 provided significant neuroprotection from NMDA.

Brain inflammatory response induced by intracerebroventricular infusion of lipopolysaccharide: an immunohistochemical study.

Inflammatory processes may play a critical role in the pathogenesis of the degenerative changes associated with Alzheimer's disease (AD). In the present study, we used an animal model of brain inflammation in order to study a possible mechanism involved in AD. Lipopolysaccharide (LPS) was used to produce global microglial reactivity within the brain of young rats. Time-dependent changes in the inflammatory reaction and the participation of glial cells after acute injection of LPS (50 or 100 microg) into the lateral ventricle or the fourth ventricle were compared with the chronic infusion of LPS (0.15, 0.5, 1.5 or 5.0 microg/h) into the fourth ventricle (14 days). Several immunohistochemical markers were used to characterize the microglial response. Acute and chronic exposure to LPS induced major histocompatibility complex class II (MHC II) antigen expression, detected with OX-6 antibody, in a sub-population of microglial cells in defined brain areas. The morphological features and distribution of OX-6 positive cells observed in the proximity of the cannula track after LPS injection into the lateral ventricle suggested the recruitment of monocytes/macrophages from the periphery. The activation of the resident microglial cells was delayed and mainly concentrated within the temporal lobe regions and the limbic system. Chronic infusion to LPS into the fourth ventricle induced a comparable activation of microglial cells. Quantitative analysis of OX-6 positive cells showed a dose-dependent response to LPS exposure.

Chronic neuroinflammation in rats reproduces components of the neurobiology of Alzheimer's disease.

Inflammatory processes may play a critical role in the pathogenesis of the degenerative changes and cognitive impairments associated with Alzheimer's disease (AD). In the present study, lipopolysaccharide (LPS) from the cell wall of gram-negative bacteria was used to produce chronic, global inflammation within the brain of young rats. Chronic infusion of LPS (0.25 microgram/h) into the 4th ventricle for four weeks produced (1) an increase in the number of glial fibrillary acidic protein-positive activated astrocytes and OX-6-positive reactive microglia distributed throughout the brain, with the greatest increase occurring within the temporal lobe, particularly the hippocampus, (2) an induction in interleukin-1 beta, tumor necrosis factor-alpha and beta-amyloid precursor protein mRNA levels within the basal forebrain region and hippocampus, (3) the degeneration of hippocampal CA3 pyramidal neurons, and (4) a significant impairment in spatial memory as determined by decreased spontaneous alternation behavior on a T-maze.

Novel glycineB antagonists show neuroprotective activity in vivo.

The degeneration or dysfunction of cholinergic neurons within the basal forebrain of patients with Alzheimer's disease (AD) may be related to the vulnerability of these cells to endogenous glutamate (Beal, 1995; Greenamyre and Young, 1989). The administration of drugs that attenuate the toxic actions of glutamate in the early stages of the disease might significantly delay its rate of progression. Two approaches to neuroprotection from endogenous glutamatergic function were investigated and found to be effective: blockade of voltage-dependent, NMDA-type glutamate receptor channels and antagonism of an NMDA-receptor related glycineB modulatory site.