Costanza Prosperi

β-Amyloid-Induced Inflammation and Cholinergic Hypofunction in the Rat Brain in Vivo: Involvement of the p38MAPK Pathway

Injection into the nucleus basalis of the rat of preaggregated Abeta(1-42) produced a congophylic deposit and microglial and astrocyte activation and infiltration and caused a strong inflammatory reaction characterized by IL-1beta production, increased inducible cyclooxygenase (COX-2), and inducible nitric oxide synthase (iNOS) expression. Many phospho-p38MAPK-positive cells were observed around the deposit at 7 days after Abeta injection. Phospho-p38MAPK colocalized with activated microglial cells, but not astrocytes. The inflammatory reaction was accompanied by cholinergic hypofunction. We investigated the protective effect of the selective COX-2 inhibitor rofecoxib in attenuating the inflammatory response and neurodegeneration evoked by Abeta(1-42). Rofecoxib (3 mg/kg/day, 7 days) reduced microglia and astrocyte activation, iNOS induction, and p38MAPK activation to control levels. Cholinergic hypofunction was also significantly attenuated by treatment with rofecoxib. We show here for the first time in vivo the pivotal role played by the p38MAPK microglial signal transduction pathway in the inflammatory response to the Abeta(1-42) deposit.

Effect of metrifonate on extracellular brain acetylcholine and object recognition in aged rats.

The effects of metrifonate were investigated in 4-6- and 22-24-month-old rats. Extracellular acetylcholine levels were measured by transversal microdialysis in vivo. Baseline extracellular acetylcholine levels in the cerebral cortex and hippocampus were 42% and 60% lower, respectively, in old than in young rats. Old rats did not discriminate between familiar and novel objects. In old rats, metrifonate (80 mg/kg p.o.) brought about 85% inhibition of cholinesterase activity in the cortex and hippocampus, a 4-fold increase in extracellular acetylcholine levels in the cortex only, and restored object recognition. In young rats, metrifonate caused 75% cholinesterase inhibition in the cerebral cortex and hippocampus, a 2-fold increase in cortical and hippocampal extracellular acetylcholine levels, and no effect on object recognition. The slight cholinesterase inhibition following metrifonate (30 mg/kg) in aged rats had no effect on cortical acetylcholine levels and object recognition. In conclusion, metrifonate may improve the age-associated cholinergic hypofunction and cognitive impairment.

The selective cyclooxygenase-2 inhibitor rofecoxib suppresses brain inflammation and protects cholinergic neurons from excitotoxic degeneration in vivo

Brain inflammatory processes underlie the pathogenesis of Alzheimers disease, and non-steroidal anti-inflammatory drugs have a protective effect in the disease. The aim of this work was to study in vivo whether attenuation of brain inflammatory response to excitotoxic insult by the selective cyclooxygenase-2 inhibitor, rofecoxib, may prevent neurodegeneration, as a contribution to a better understanding of the role inflammation plays in the pathology of Alzheimers disease. We investigated, by immunohistochemical methods, glia reaction, the activation of p38 mitogen-activated protein kinase (p38MAPK) pathway with an antibody selective for the phosphorylated form of the enzyme and the number of choline acetyltransferase-positive neurons and, by in vivo microdialysis, cortical extracellular levels of acetylcholine following the injection of quisqualic acid into the right nucleus basalis of adult rats. Seven days after injection, a marked reduction in the number of choline acetyltransferase-positive neurons was found, along with an intense glia reaction, selective activation of p38MAPK at the injection site and a significant decrease in the extracellular levels of acetylcholine in the cortex ipsilateral to the injection site. The loss of cholinergic neurons persisted for at least up to 28 days. Rofecoxib (3 mg/kg/day, starting 1 h prior to injection of quisqualic acid) treatment for 7 days significantly attenuated glia activation and prevented the loss of choline acetyltransferase-positive cells and a decrease in cortical acetylcholine release. The prevention of cholinergic cell loss by rofecoxib occurred concomitantly with the inhibition of p38MAPK phosphorylation. Our findings suggest an important role of brain inflammatory reaction in cholinergic degeneration and demonstrate a neuroprotective effect of rofecoxib, presumably mediated through the inhibition of p38MAPK phosphorylation.

Brain inflammatory reaction in an animal model of neuronal degeneration and its modulation by an anti-inflammatory drug: implication in Alzheimer's disease.

Brain inflammatory processes underlie the pathogenesis of Alzheimers disease, and nonsteroidal anti‐inflammatory drugs have a protective effect in the disease. The aim of this study was to characterize in vivo in the rat brain the inflammatory reaction in response to excitotoxic insult and to investigate the efficacy of nimesulide treatment. Quisqualic acid was injected into the right nucleus basalis of rats. The excitotoxin induced cholinergic degeneration, an intense glial reaction and the production of inflammatory mediators. Three hours after injection, a five‐fold elevation in the concentration of interleukin‐1β in the injected area was observed. This elevation was reduced by 50% by nimesulide (10 mg/kg, i.m.) pretreatment. Electron microscope examination and immunocytochemical staining revealed an intense activation of microglia and astrocytes at both 24 h and 7 days after injection. Cyclooxygenase‐2‐immunoreactivity was induced in the blood vessels of the injected hemisphere in perivascular microglial and endothelial cells 24 h after injection. Seven days postinjection, a cyclooxygenase‐2‐positive signal was induced in the parenchymal microglia and large amounts of prostaglandin‐E2 were measured in the injected area. Twenty‐four hours and 7 days after injection, many inducible nitric oxide synthase‐positive cells and a high level of nitrite were detected at the injection site. Seven days of nimesulide (10 mg/kg/day, i.m.) treatment strongly attenuated the microglial reaction, reduced the number of inducible nitric oxide synthase‐positive cells and completely abolished the increase in prostaglandin‐E2 formation. These data provide valuable support in vivo for the potential efficacy of cyclooxygenase‐2 inhibitors in Alzheimers disease therapy.

Comparison between flurbiprofen and its nitric oxide-releasing derivatives HCT-1026 and NCX-2216 on Abeta(1-42)-induced brain inflammation and neuronal damage in the rat.

Brain inflammation is an underlying factor in the pathogenesis of Alzheimers disease (AD) and epidemiological studies indicate that a sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) has a protective effect on the disease. We investigated, in vivo, whether differences exist in the antiinflammatory and neuroprotective actions of flurbiprofen and its two nitric oxide-donor derivatives, HCT-1026 and NCX-2216, and the ability of these two derivatives to release nitric oxide in the brain. In adult rats injected into the nucleus basalis with preaggregated Aβ(1-42) we investigated by immunohistochemical methods glia reaction, the induction of inducible nitric oxide synthase (iNOS), the activation of p38 mitogen-activated protein kinase (p38MAPK) pathway and the number of choline acetyltransferase (ChAT)-positive neurons and, in naïve rats we investigated, by microdialysis, cortical extracellular levels of nitrite. Injection of Aβ(1-42) induced iNOS at the injection site, activated p38MAPK 7 days after injection and brought about an intense microglia and astrocyte reaction along with a marked reduction in the number of ChAT-positive neurones, persisting up to at least up to 21 days. Flurbiprofen, HCT-1026 and NCX-2216 (15 mg/kg) significantly attenuated the Aβ(1-42)-induced glia reaction, iNOS induction and p38MAPK activation 7 days after treatment and astrocytes reaction 21 days after treatment. On an equimolar basis, HCT-1026 resulted the most active agent in reducing the Aβ(1-42)-induced microglia reaction. The cholinergic cell loss was also significantly reduced by 21 days of HCT-1026 treatment. No differences in the body weight were found between the animals treated for 21 days with 15 mg/kg of either HCT-1026 or NCX-2216 and the controls. An oral administration of HCT-1026 (15 mg/kg) or NCX-2216 (100 mg/kg) to naiïve rats was followed by significant and long long -lasting increases in cortical nitrite levels. These findings indicate that the addition of a nitric oxide donor potentiates the anti-inflammatory activity of flurbiprofen in a model of brain inflammation.

β(1–40) Amyloid peptide injection into the nucleus basalis of rats induces microglia reaction and enhances cortical γ-aminobutyric acid release in vivo

Abstract The nucleus basalis of adult rats was injected with β(1–40) amyloid peptide. A marked increase in basal and K+-evoked GABA release in the ipsilateral cortex and a significant decrease in GAD activity in the injected NB were found 30 days after injection. An intense activation of microglial cells that surrounded and infiltrated the deposit was observed. These data demonstrate that a local injection of β(1–40) peptide into the NB induces glia activation and affects GABAergic neurons.

Neutrophils CD11b and fibroblasts PGE2 are elevated in Alzheimer’s disease

To evaluate whether inflammation-like mechanisms present in the brain of Alzheimers disease (AD) patients are reflected in the periphery, the expression of CD11b in peripheral blood neutrophils and the expression and activity of inflammatory markers in cultured skin fibroblasts were examined. We found significantly higher levels of CD11b in neutrophils from sporadic AD patients than in controls and this elevation was positively correlated with disease severity and progression rate of mental decline. Cultured skin fibroblasts from familial (FAD) and sporadic AD patients and from controls were immunopositive for both isoforms of cyclooxygenase with no differences between groups. In unstimulated culture, the production of prostaglandin-E2 in the medium was significantly higher in fibroblasts from sporadic AD and FAD patients than in controls, and this elevation was reverted by the addition of 25 microM of ibuprofen. Our findings provide further evidence of the presence of inflammatory and immuno-related markers in the periphery of AD patients and support those studies indicating the beneficial effects of anti-inflammatory therapy in AD.

Morphological, biochemical and behaviouralchanges induced by neurotoxic and inflammatory insultsto the nucleus basalis

Interest in the basal forebrain cholinergic system has greatly increased sinceneuropathological studies in humans provided evidence that this system is severely affected in Alzheimers disease and other dementing disorders. In laboratory animals, disruption of the nucleusbasalis cholinergic neurones has been produced by several neurotoxic insults in order to obtain amodel reproducing the behavioural impairment related to the cholinergic deficits.