Microglial activation and beta -amyloid deposit reduction caused by a nitric oxide-releasing nonsteroidal anti-inflammatory drug in amyloid precursor protein plus presenilin-1 transgenic mice

Abstract

3-4-(2-Fluoro-alpha-methyl-[1,1'-biphenyl]-4-acetyloxy)-3-methoxyphenyl]-2-propenoic acid 4-nitrooxy butyl ester (NCX-2216), a nitric oxide (NO)-releasing derivative of the cyclooxygenase-1-preferring nonsteroidal anti-inflammatory drug (NSAID) flurbiprofen, dramatically reduced both beta-amyloid (Abeta) loads and Congo red staining in doubly transgenic (Tg) amyloid precursor protein plus presenilin-1 mice when administered at 375 ppm in diet between 7 and 12 months of age. This reduction was associated with a dramatic increase in the number of microglia expressing major histocompatibility complex-II antigen, a marker for microglial activation. In contrast, ibuprofen at 375 ppm in diet caused modest reductions in Abeta load but not Congo red staining, suggesting that the effects of this nonselective NSAID were restricted primarily to nonfibrillar deposits. We detected no effects of the cyclooxygenase-2-selective NSAID celecoxib at 175 ppm on amyloid deposition. In short-term studies of 12-month-old Tg mice, we found that the microglia-activating properties of NCX-2216 (7.5 mg small middle dot kg(-1) small middle dot d(-1), s.c.) were present after 2 weeks of treatment. Microglia were not activated by NCX-2216 in non-Tg mice lacking Abeta deposits, nor were microglia activated in Tg animals by flurbiprofen (5 mg small middle dot kg(-1) small middle dot d(-1)) alone. These data are consistent with the argument that activated microglia can clear Abeta deposits. We conclude that the NO-generating component of NCX-2216 confers biological actions that go beyond those of typical NSAIDs. In conclusion, NCX-2216 is more efficacious than ibuprofen or celecoxib in clearing Abeta deposits from the brains of Tg mice, implying potential benefit in the treatment of Alzheimer's dementia.

Fig. 1.

Aβ immunohistochemistry in control and NCX-2216-treated transgenic mice. Aβ immunohistochemistry of the frontal cortex (A) and hippocampus (C) of an APP+PS1 transgenic mouse fed the control diet from 7 to 12 months of age is shown. Aβ immunohistochemistry of the frontal cortex (B) and hippocampus (D) of an APP+PS1 transgenic mouse fed 375 ppm NCX-2216 in diet from 7 to 12 months of age is also shown. Scale bar, 1 mm.

Fig. 2.

Aβ load in the anterior cortex and hippocampus of NCX-2216-treated mice. The Aβ load is the percentage of the cross-sectional area occupied by the immunohistochemical reaction product. NCX-2216-treated APP+PS1 mice have significantly less Aβ in both the cortex (A) and hippocampus (B), as demonstrated by immunohistochemistry, compared with mice treated with ibuprofen (hippocampus,p < 0.05), celecoxib (cortex,p < 0.05; hippocampus, p < 0.005), or fed the control diet (cortex, p < 0.001; hippocampus, p < 0.001). In the cortex, ibuprofen significantly reduced Aβ load as well (p < 0.025) compared with mice fed the control diet.

Fig. 3.

Congo red staining in NCX-2216-treated transgenic mice. Shown is Congo red staining of the frontal cortex (A) and hippocampus (C) of an APP+PS1 transgenic mouse fed the control diet from 7 to 12 months of age. Congo red staining of the frontal cortex (B) and hippocampus (D) of an APP+PS1 transgenic mouse fed 375 ppm NCX-2216 in diet from 7 to 12 months of age is also shown. Scale bar, 1 mm.

Fig. 4.

Amyloid load in the anterior cortex and hippocampus of NCX-2216-treated mice. Amyloid load is the percentage of the cross-sectional area occupied by Congo red stain. NCX-2216-treated APP+PS1 mice have significantly less amyloid in both the cortex (A) and hippocampus (B), as demonstrated by Congo red staining, compared with mice treated with ibuprofen (cortex, p < 0.002; hippocampus,p < 0.005), celecoxib (cortex,p < 0.02; hippocampus, p < 0.02), or fed the control diet (cortex, p < 0.001; hippocampus, p < 0.01).

Fig. 5.

MHC-II immunohistochemistry with Congo red counterstaining of NCX-2216-treated transgenic mice is shown. MHC-II immunohistochemistry (black reaction product) of the frontal cortex (A) and hippocampus (C) of an APP+PS1 transgenic mouse fed the control diet from 7 to 12 months of age is shown. MHC-II immunohistochemistry of the frontal cortex (B) and hippocampus (D) of an APP+PS1 transgenic mouse fed 375 ppm NCX-2216 in diet from 7 to 12 months of age is also shown. Scale bar, 100 μm.

Fig. 6.

NCX-2216-treated APP+PS1 mice have larger numbers of MHC-II-positive microglia in the frontal cortex (A) and hippocampus (B) compared with mice fed a control diet (frontal cortex,p < 0.05; hippocampus, p < 0.02). Ibuprofen-treated mice have a small but not significant increase in the numbers of MHC-II-positive microglia in the anterior cortex compared with control-treated mice.

Fig. 7.

Effects of short-term NCX-2216 treatment on microglial markers in APP+PS1 mice. Animals were treated with 7.5 mg · kg⁻¹ · d⁻¹NCX-2216 or vehicle for 14 d before they were killed. MHC-II immunohistochemistry of the frontal cortex of an APP+PS1 transgenic mouse treated with vehicle (A) and NCX-2216 (B) is shown. CD-11 immunohistochemistry of the frontal cortex of an APP+PS1 transgenic mouse treated with vehicle (C) and NCX-2216 (D) is also shown. Scale bar, 100 μm.

Fig. 8.

Measurement of the effects of short-term NCX-2216 treatment on microglial markers in APP+PS1 mice. Animals were treated with 7.5 mg · kg⁻¹ · d⁻¹NCX-2216 or vehicle for 14 d before they were killed. Sections were stained with anti-MHC-II antibodies and Congo red, and positive microglial profiles were counted on systematic uniform random sections through the frontal cortex and hippocampus. Results are expressed as average number of MHC-II-positive microglial profiles per section in each region (A). NCX-2216 treatment resulted in a >10-fold increase in the average number of MHC-II-positive profiles in both the frontal cortex and hippocampus when compared with vehicle-treated mice (p < 0.025). Sections were stained with anti-CR3 antibody and Congo red, and total immunoreactivity measurements of ICC-RP were calculated (B). There is significantly greater total immunoreactivity of Congo red-associated ICC-RP in the hippocampus (p < 0.05); however, there is only a trend toward greater total immunoreactivity in the frontal cortex (p < 0.2).

Fig. 9.

Measurement of MHC-II immunohistochemistry after acute treatment of APP transgenic and nontransgenic mice with NCX-2216, flurbiprofen, or vehicle. NCX-2216 (7.5 mg · kg⁻¹ · d⁻¹) treatment of 12-month-old APP transgenic mice results in a significantly increased number of MHC-II-positive microglia compared with APP mice treated with either flurbiprofen (5 mg · kg⁻¹ · d⁻¹) or vehicle (p < 0.001). Nontransgenic mice treated daily with either NCX-2216 or vehicle for 2 weeks demonstrate no detectable MHC-II-positive microglia.