Effects of triptolide on hippocampal microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer's disease.

The principal pathology of Alzheimer's disease includes neuronal extracellular deposition of amyloid-beta peptides and formation of senile plaques, which in turn induce neuroinflammation in the brain. Triptolide, a natural extract from the vine-like herb Tripterygium wilfordii Hook F, has potent anti-inflammatory and immunosuppressive efficacy. Therefore, we determined if triptolide can inhibit activation and proliferation of microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer's disease. We used 1 or 5 µg/kg/d triptolide to treat APP/PS1 double transgenic mice (aged 4-4.5 months) for 45 days. Unbiased stereology analysis found that triptolide dose-dependently reduced the total number of microglial cells, and transformed microglial cells into the resting state. Further, triptolide (5 µg/kg/d) also reduced the total number of hippocampal astrocytes. Our in vivo test results indicate that triptolide suppresses activation and proliferation of microglial cells and astrocytes in the hippocampus of APP/PS1 double transgenic mice with Alzheimer's disease.

Age-related loss of noradrenergic neurons in the brains of triple transgenic mice.

Microscopic findings in Alzheimer's disease (AD) at autopsy include a wide cortical distribution of beta amyloid (Aß)-containing plaques and diminished numbers of pyramidal neurons in CA1 of hippocampus and tyrosine hydroxylase-positive (TH+) neurons in the locus coeruleus (LC). To better understand the neuropathology underlying cognitive decline in AD, we analyzed the AD-type neuropathology in brains of triple transgenic (3×Tg) mice harboring mutations for APP(swe), PS1(M146V), and tau(P301L). Histochemical and immunohistochemical staining and computerized stereology were carried out in age-matched young, early middle age, and late middle age 3×Tg mice. The 3×Tg mice showed an intracellular Aß deposition in subiculum and CA1 pyramidal neurons and an extracellular distribution of amyloid plaques specifically in the subiculum of hippocampal formation and in neocortical layer V. The 3×Tg mice also showed an age-related loss of TH+ neurons in LC, with a loss of 37% of these neurons at 15 months of age. There was no loss of CA1 neurons at any age examined. Reduced AD-type neuropathology in CA1 of 3×Tg mice suggests a possible neuroprotective role for high intracellular-to-extracellular ratios of insoluble Aß deposits. Understanding the neurobiology of this apparent neuroprotection could lead to an improved understanding of age-related cognitive function in general, and the development of novel strategies for the therapeutic management of AD patients.

Anti-depressant natural flavonols modulate BDNF and beta amyloid in neurons and hippocampus of double TgAD mice.

Increasing evidence suggests that depression may be both a cause and consequence of neurological disorders such as Alzheimer's disease (AD), and that anti-depressants could provide an alternative strategy to current AD therapies. Association of side effect and herbal-drug interaction with conventional anti-depressant and St. John's wort warrant investigating new anti-depressant drugs. Anti-depressant effects of ginkgo biloba extract (EGb 761) have been demonstrated in animal models of depression and in human volunteers. We report here that ginkgo flavonols quercetin and kaempferol stimulates depression-related signaling pathways involving brain-derived neurotrophic factor BDNF/phosphorylation of cyclic AMP response element binding protein CREB/postsynaptic density proteins PSD95, and reduces amyloid-beta peptide (Abeta) in neurons isolated from double transgenic AD mouse (TgAPPswe/PS1e9). In addition, enhanced BDNF expression and reduction of Abeta oligomers was confirmed in hippocampus of the double transgenic mice administered with flavonol, which correlates with cognitive improvement behaviors in these mice. The present results suggest that stimulating BDNF and reducing Abeta toxicity by natural flavonols provide a therapeutic implication for treatment of AD.

[Astrocyte activation and Bcl-2 protein expression induced by lipopolysaccharide in mouse brain].

OBJECTIVE: To investigate the changes of anti-apoptotic protein Bcl-2 expression in neurons and activation of brain astroglial cells, and the relationship between astrocytes and neurons in mice after a single intracerebroventricular (ICV) stereotaxic injection of lipopolysaccharide (LPS). METHODS: C57BL/6J mice of different ages were divided into a control group and an experiment group. Immunohistochemistry to Bcl-2 and that to GFAP were conducted to observe the expression of Bcl-2 protein in neurons and GFAP in astrocytes in the brain at different time-points after the LPS injection. The glial cell type expressing Bcl-2 was characterized with immunofluorescence double labeling. RESULTS: GFAP-immunoreactive cells in the control mice were observed mainly within hippocampal formation, piriform, entorhinal cortex, septum, striatum, amygdaloid nucleus, subcortical white matter, as well as in the main fiber tracts. At 24 h after the LPS treatment there was no obvious difference in GFAP immunoreactivity compared with the controls. Astrocytes were markedly activated in periventricular brain regions such as hippocampus, the hypothalamic parenchyma surrounding the third ventricle, with larger cell body and hypertrophic processes 2 days after the endotoxin treatment. After the LPS injection, Bcl-2 positive cells were distributed widely in the brain, such as in the cortex (primary and secondary motor cortex, somatosensory cortex), hypothalamic parenchyma surrounding the third ventricle, diagonal band, hippocampus, septum and the red nucleus of the midbrain. At these sites, Bcl-2 induction increased significantly 2 days after the ICV LPS injection, with some subregional differences, peaking on 4th day. No immunofluorescent double labeling cells for GFAP and Bcl-2 were observed in the brain of the mice after the LPS administration, but merging GFAP positive astrocytes and Bcl-2 positive neurons were seen. Double staining for Bcl-2 and GFAP also showed that the projections of activated astrocytes were found in the sheath of Bcl-2 positive neurons 4 days after the ICV LPS administration. CONCLUSION: LPS can activate astroglial cells and upregulate of Bcl-2 expression in the neurons in the mouse brain, which may participate in the administration of central nervous system to central-immunity stimulated regulation and the protective response to the inflammatory stimulus. The projections of activated astrocytes are found in the sheath of Bcl-2 positive neurons, indicating that there is close relationship between astrocytes and neurons.

Catecholaminergic neuronal loss in locus coeruleus of aged female dtg APP/PS1 mice.

Alzheimer's disease (AD) is the most common type of dementia afflicting the elderly. In addition to the presence of cortical senile plaques and neurofibrillary tangles, AD is characterized at autopsy by extensive degeneration of brainstem locus coeruleus (LC) neurons that provide noradrenergic innervation to cortical neuropil, together with relative stability of dopaminergic neuron number in substantia nigra (SN) and ventral tegmental area (VTA). The present study used design-based stereological methods to assess catecholaminergic neuronal loss in brains of double transgenic female mice that co-express two human mutations associated with familial AD, amyloid precursor protein (APP(swe)) and presenilin-1 (PS1(DeltaE9)). Mice were analyzed at two age groups, 3-6 months and 16-23 months, when deposition of AD-type beta-amyloid (Abeta) plaques occurs in cortical brain regions. Blocks of brain tissue containing the noradrenergic LC nucleus and two nuclei of dopaminergic neurons, the SN and VTA, were sectioned and sampled in a systematic-random manner and immunostained for tyrosine hydroxylase (TH), a specific marker for catecholaminergic neurons. Using the optical fractionator method we found a 24% reduction in the total number of TH-positive neurons in LC with no changes in SN-VTA of aged dtg APP/PS1 mice compared with non-transgenic controls. No significant differences were observed in numbers of TH-positive neurons in LC or SN-VTA in brains of young female dtg APP/PS1 mice compared to their age-matched controls. The findings of selective neurodegeneration of LC neurons in the brains of aged female dtg APP/PS1 mice mimic the neuropathology in the brains of AD patients at autopsy. These findings support the use of murine models of Abeta deposition to develop novel strategies for the therapeutic management of patients afflicted with AD.

Neuropathological quantification of dtg APP/PS1: neuroimaging, stereology, and biochemistry.

Murine models that mimic the neuropathology of Alzheimer's disease (AD) have the potential to provide insight into the pathogenesis of the disease and lead to new strategies for the therapeutic management of afflicted patients. We used magnetic resonance imaging (MRI), design-based stereology, and high performance liquid chromatography (HPLC) to assess the age-related neuropathology in double transgenic mice that overexpress two AD-related proteins--amyloid precursor protein (APP) and presenilin 1 (PS1)--and age- and gender-matched wild-type (WT) controls. In mice ranging in age from 4-28 months, total volumes of the hippocampal formation (V (HF)) and whole brain (V (brain)) were quantified by the Cavalieri-point counting method on a systematic-random sample of coronal T2-weighted MRI images; the same stereological methods were used to quantify V (HF) and V (brain) after perfusion and histological processing. To assess changes in AD-type beta-amyloid (A beta) plaques, sections from the hippocampal formation and amylgdaloid complex of mice aged 5, 12, and 15 months were stained by Congo Red histochemistry. In aged mice with large numbers of amyloid plaques, systematic-random samples of sections were stained by GFAP immunocytochemistry to assess gender and genotype effects on total numbers of astrocytes. In addition, levels of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and 5-HT metabolites were assayed by HPLC in fresh-frozen samples from neocortex, striatum, hippocampus, and brainstem. We confirmed age-related increases in amyloid plaques, beginning with a few plaques at 5 months of age and increasing densities by 12 and 15 months. At 15 months of age, there were robust genotype effects, but no gender effects, on GFAP-immunopositive astrocytes in the amygdaloid complex and hippocampus. There were no effects on monoamine levels in all brain regions examined, and no volume changes in hippocampal formation or whole brain as quantified on either neuroimages or tissue sections. Strong correlations were present between volume estimates from MRI images and histological sections, with about 85% reduction in mean V (HF) or mean V (brain) between MRI and processed histological sections. In summary, these findings show that the double transgenic expression of AD-type mutations is associated with age-related increases in amyloid plaques and astrocytosis; however, this model does not recapitulate the cortical atrophy or neurochemical changes that are characteristic of AD.

[Effect of chronic stress on PKA and P-CREB expression in hippocampus of rats and the antagonism of antidepressors].

OBJECTIVE: To observe the effect of chronic unpredicted sequence of mild stress on the expression of cAMP-dependent protein kinase A(PKA) and phosphorylated cAMP-responsive element binding protein (P-CREB) in hippocampus of rats and the antagonism of antidepressors (fluoxetine). METHODS: Thirty-six male Sprague Dawley rats were randomly and equally allocated to 3 groups: A normal control group, a model group, and a fluoxetine group. All rats except the control group were singly housed and exposed to an unpredicted sequence of mild stressors. The different distribution and expression of PKA and P-CREB in the hippocampus of rats in different groups were investigated with immunohistochemistry and Westernblot technique. RESULTS: The positive PKA and P-CREB cells in the hippocampus of normal controls were the pyramidal cells and the granule cells. The PKA and P-CREB protein expression levels in the hippocampus of model rats were significantly lower than those of the normal controls (P<0.05). The PKA and P-CREB protein expression levels in the hippocampus of the fluoxetine group were significantly higher than those of the model group (P<0.05). CONCLUSION: Chronic unpredicted mild stress can affect the PKA and P-CREB expression in hippocampus of rats and fluoxetine has antagonism against it.

Triptolide inhibits COX-2 expression via NF-kappa B pathway in astrocytes.

Previous investigations have showed that triptolide possessed potent anti-inflammatory and immunosuppressive properties. In the present study, we examined the protective effects of triptolide on the inflammatory response induced by bacterial lipopolysaccharide (LPS) both in vivo and in vitro. Intrahippocampal injection of LPS (4 microg) in rats significantly increased the immunoreactivity of glial fibrillary acid protein (GFAP) and cyclooxygenase-2 (COX-2) in the injected region, which was reduced by pretreatment with triptolide (10-50 microg/kg) for 5d. In the cultured human differentiated A172 astroglial cells, LPS (1mg/L) increased the expression of COX-2 mRNA and protein, the production of prostaglandin E(2) (PGE(2)) and the DNA binding activity of NF-kappa B, which were markedly attenuated by pretreatment with triptolide (0.2-5 microg/L) for 1h. These results suggested that the protective effect of triptolide on neuroinflammation is mediated by decreasing COX-2 expression, at least partly, via the inhibition of NF-kappa B signaling pathway.

Stereological analysis of microvascular parameters in a double transgenic model of Alzheimer's disease.

Morphological alterations in microvasculature occur as a common finding in the brains of non-demented aged persons and patients with Alzheimer's disease. Quantifying the extent of this vascular pathology, however, has been complicated by systematic error (bias) associated with the applications of assumption- and model-based morphometric techniques to human and animal tissues. The current study used novel assumption- and model-free stereological approaches to quantify capillary parameters in the corpus callosum of a double amyloid precursor protein/presenilin-1 transgenic murine model of Alzheimer's disease. The results revealed significant reductions in the total number of capillary segments in white matter of transgenic mice compared to non-transgenic littermates, with no differences in total capillary length. These findings support the view that the expression of mutant human genes for beta-amyloid peptides alters the normal architecture of cerebral capillary vessels in the white matter of mouse brain, which may model microvasculature changes reported in Alzheimer's disease.

[Experimantal study on the anti-freeze buffer preserving frozen sections].

OBJECTIVE: To investigate whether anti-freeze buffer preserves frozen sections for immunocytochemistry (ICC) and histochemistry. METHODS: C57BL/6NIA female mice (n = 4) aged 12 - 15 months were used in this study. It was frozen sectioned at 50 micron and stored in anti-freeze buffer at -20 degrees C. At the time of sectioning (Day 0) and after 3 intervals (3, 6, and 12 months) sections through the dentate gyrus were stained with beta-NADPH histochemistry for nitric oxide synthase (NOS) -containing neurons and capillaries, in combination with GFAP ICC for astrocytes. RESULTS: Analysis of the sections stained with beta-NADPH histochemistry revealed an equivalent level of staining for NOS-positive neurons and capillaries at both Day 0 and 6 months. Similarly, sections preserved in anti-freeze buffer and stained with GFAP ICC showed equivalent staining of astrocytes at Day 0, 6 months, and 12 months. CONCLUSION: These findings confirm that anti-freeze buffer provides long-term preservation of antigens associated with neurons, glial cells, and blood vessels for common histological and ICC staining procedures.

Age and gender effects on microglia and astrocyte numbers in brains of mice.

The morphological changes that occur during normal brain aging are not well understood. This study used modern stereology to assess the effects of age and gender on total numbers of astrocytes and microglia in the hippocampal formation in C57Bl/6NNIA (B6) mice. Astrocytes and microglia were visualized using immunocytochemistry for glial fibrillary acidic protein (GFAP) and complement receptor 3 (Mac-1), respectively, and numbers of each cell type in dentate gyrus (DG) and CA1 regions were estimated using the optical fractionator method. The results reveal significantly greater ( approximately 20%) numbers of microglia and astrocytes in aged females compared to young female B6 mice. We also report that on average female B6 mice have 25-40% more astrocytes and microglia in DG and CA1 regions than age-matched male C57Bl/6J mice. Since astrocytes and microglia are thought to be targets of gonadal hormones, the effects of sex hormones and reproductive aging may be responsible for these findings.