Neuropil and neuronal changes in hippocampal NADPH‐diaphorase histochemistry in the ME7 model of murine prion disease

Abstract

Nitric oxide (NO) has been implicated in neurotoxicity and cerebral blood flow changes in chronic neurodegeneration, but its activity in the mammalian prion diseases has not been studied in detail. Nicotine adenine dinucleotide phosphate (NADPH)-diaphorase (NADPH-d) histochemistry is a simple and robust histochemical procedure that allows localization of the tissue distribution of NO synthases. The aim of the present study is to assess whether NADPH-d histochemical activity is altered in the hippocampus in the ME7 model of prion disease in C57BL/6J mice. At early and late stages after the initiation of the disease we assessed features of the NADPH-d positive cells and the neuropil histochemical activity in CA1 and dentate gyrus using densitometric analysis. In C57BL/6J mice 13 weeks postinjection of the prion agent ME7, when behavioural changes first become apparent, neuropil NADPH-d histochemical staining increases, whereas at late stages it decreases dramatically. Both type I and type II NADPH-d positive cells were found to survive throughout the hippocampal formation into the late stages of the disease, but diaphorase activity was reduced in dendritic branches and abnormal varicosities were present in both dendritic and axonal processes of NADPH-d positive type I cells. The pathophysiological implications of the results remain to be investigated but both blood flow alteration and NO neurotoxicity may be features of the disease.

Morphological and Functional Abnormalities in Mitochondria Associated with Synaptic Degeneration in Prion Disease

“…there is a significant increase in neuronal-nitric oxide synthase in the hippocampus. 47 The increase in neuronal-nitric oxide synthase occurs in the stratum radiatum and declines in late disease stage consistent with the changes in COX activity that we report here. Thus the idea that nitric oxide could damage mitochondria in ME7-animals is justified.…”

Neuropil reactivity, distribution and morphology of NADPH diaphorase type I neurons in the barrel cortex of the adult mouse

“…In early reports, however, the presence of type II neurons in rodents ranged from being neglected to altogether dismissed (Mitrovic and Schachner, 1996;Yan and Garey, 1997;Oermann et al, 1999). Later, we were able to demonstrate that NADPH-d type II cells are unequivocally present in the rodent's brain (Pereira Jr. et al, 2000;Picanço-Diniz et al, 2004;Freire et al, 2004).…”

“…Sections were washed three times in a medium containing 0.05% 0.1 M phosphate buffer saline-Tween (PBS-T) and incubated in 10% normal goat serum in PBS for 1 h. After that, sections were incubated in mouse anti-NOS primary antibody (dilution at 1:150 in PBS, Serotec, UK) for 48 h at 10 8C, washed three times in PBS-T and incubated with a biotinylated goat anti-mouse secondary antibody (dilution at 1:200 in PBS, Serotec) for 1 h, washed three times in PBS-T, and then incubated in avidin-biotin-peroxidase solution (Vectastain Standard ABC kit, Vector Laboratories, USA) for 1 h. In this study, we used the DAB/nickel method for revealing NOS antibody in NADPH-d labeled tissue, as described by Picanço-Diniz et al (2004), with the difference that the NADPH-d reaction was performed before the immunohistochemistry procedure, yielding to a lighter reaction product than if the immunohistochemistry is performed first. This protocol also ensures a lighter reaction product with better visualization of double labeling because a smaller amount of nickel is used, as compared to regular DAB/nickel protocol (Shu et al, 1988).…”

The Role of Microglia in Synaptic Stripping and Synaptic Degeneration: A Revised Perspective

“…It is at present not clear whether the mitochondrial changes are a cause or a consequence of the synaptic degeneration and the events leading to mitochondrial cytochrome c oxidase impairment are not known. However, during the period of early synaptic loss and mitochondrial abnormalities, there is an enhanced expression and activity of nNOS in the stratum radiatum (Picanco-Diniz et al, 2004). This could, as discussed earlier, modulate synaptic function and additionally impair mitochondrial function.…”