Clinical findings have linked multiple risk factors and associated pathologies to Alzheimer\''s disease (AD). Amongst them are vascular risk factors such as hypertension and pathologies such as stroke. Coexistence of AD and these associated pathologies worsenes dementia, the clinical hallmark of the disease, as compared to pure AD. One general common denominator of these associated pathologies is the presence of hypoxic tissue conditions. It was asked the question, whether there exists a mutual, causal interaction between hypoxia and AD pathology, that could explain the clinical observations. Alternatively, the worsened clinical state of multiple brain pathologies could \"simply\" be the consequence of multimorbidity, i.e. accumulated disease load, without any causal interaction between the constituents. To approach this question whether hypoxia influences AD progression, use was made of a murine animal model of AD (transgenic mice: APPswe, PSEN1dE). Animals of two ages (8 and 14 months, \"young\" and \"old\" respectively) and two genotypes (transgenic and wild- type) were either treated under hypoxia or normoxia, corresponding to 8% and 21% oxygen, for 20 consecutive days. The resulting changes in the brain were assessed with a variety of techniques, namely by histology, ELISA, dot and Western blotting. Additional experiments in primary cell cultures were performed.
Animals exposed to hypoxia showed an increased hematocrit (HCT), weight loss, reactive angiogenesis, but no infarctions. This illustrates that our hypoxic treatment put significant stress on the animals, without causing major pathologies. A large number of variables exists that could potentially be measured to assess the effect of hypoxia on AD. The focus was put on three of them: First, there is the Abeta1-42- protein, known to be the Abeta- isoform associated with the most detrimental disease progression. In AD, the self-combinatory Amyloid- beta peptide (Abeta) accumulates in the brain in so- called plaques, which is a main histologic finding of the disease. Its quantity was determined through histology and ELISA.
Secondly, it was attempted to estimate the structural quality of the Abeta- protein by assessing the amount of A!- oligomers present. Abeta- protein does self- accumulate in various grades of complexity, i.e. as monomer, oligomer or fibril. Since oligomers are known to be the most neurotoxic \"species\" of the Abeta- protein, it was hypothesized that under hypoxic treatment their quantity could increase.
And third, the organism\''s response to the Abeta- protein stimulus was investigated. Microglial cells have been described as the first cells to encounter the Abeta- protein \"threat\" in the shape of plaques, i.e. Abeta- protein aggregates. They then try to encapsulate and subsequently degrade them. Therefore, the attention was put on this cellular population.
It was asked whether hypoxia could change the Abeta- protein quantity in the brain. This was assessed in two ways: First histologically, by staining for Abeta- protein depositions and quantifying them. Second, an ELISA was performed. Our findings state that hypoxic treatment does not alter the Abeta1-42 protein load in the brain, neither in young nor old animals, as assessed by histology and by total ELISA quantification of Abeta1-42 protein.
Since hypoxia did not alter the quantity of the Abeta- protein, it was asked whether it influenced it qualitatively? If hypoxia increased oligomer formation, this change in the spectrum of the Abeta- species could, without any change in total Abeta- protein load, lead to increased neurotoxicity in animals under hypoxia. Initial experiments showed that oligomer formation in the brain seems to increase. However, this was not statistically significant and future experiments are necessary to evaluate this hypothesis further.
It was then asked, whether hypoxia alters the cellular response to the protein. The total number of microglia in the hippocampal dentate gyrus, our structure of interest for practical purposes, and, it can be argued, by extension the brain, changes dynamically with various factors. First, transgenic animals present an increase in microglia. Second, microglia increase with age. Third, microglia decrease under hypoxia, but only do so significantly in old animals. Next, a parameter called \"plaque occupancy\" was coined to assess the microglia function to confront Abeta- plaques. Plaque occupancy is defined as the number of microglia in spatial proximity to one square millimeter of Abeta- plaque. This means, that microglia restricting one plaque are counted, and then normalized to this plaque\''s area. It was hypothesized that hypoxia would decrease plaque occupancy. Indeed, plaque occupancy roughly halved under hypoxia.
Summarizing, our results demonstrate that long- term exposure to hypoxia significantly reduces the number of microglia. The reduced number results in significantly reduced plaque occupancy and compromizes the function of microglia to confront Abeta- plaques. The Abeta1-42 load, however, is not affected. On the other hand, Abeta shows an increased trend towards oligomer formation. A variety of possible explanations to these phenomena have been presented, that in our opinion deserve further investigation
