Direct Evidence for P2Y2 Receptor Involvement in Vascular Response to Injury

Objectives Extracellular nucleotide release at the site of arterial injury mediates proliferation and migration of vascular smooth muscle cells (SMC). Our aim was to investigate the role of the P2Y2 nucleotide receptor (P2Y2R) in neointimal hyperplasia. Approach and Results Vascular injury was induced by implantation of a polyethylene cuff around the femoral artery in wild-type and P2Y2 receptor-deficient mice (P2Y2R−/−). Electron microscopy was used to analyze monocyte and lymphocyte influx to the intima 36 hours post-injury. Compared to wild-type (WT) littermates, P2Y2R−/− mice exhibited a 3-fold decreased number of mononuclear leukocytes invading the intima (p<0.05). Concomitantly, migration of smooth muscle cells was decreased by more than 60% (p<0.05) a resulting in a sharp inhibition of intimal thickening formation in P2Y2R−/− mice (n=15) 14 days after cuff placement. In vitro, loss of P2Y2 receptor significantly impaired monocyte migration in response to nucleotide agonists. Furthermore, transgenic rats over-expressing the P2Y2R developed accelerated intimal lesions resulting in more than 95% luminal stenosis (P<0.05, n=10). Conclusions Loss-and gain-of-function approaches established a direct evidence for P2Y2 receptor involvement in neointimal hyperplasia. Specific anti-P2Y2 receptor therapies may be used against restenosis and bypass graft failure

APP21 transgenic rats develop age-dependent cognitive impairment and microglia accumulation within white matter tracts.

Background Most of the animal models commonly used for preclinical research into Alzheimer\u27s disease (AD) largely fail to address the pathophysiology, including the impact of known risk factors, of the widely diagnosed sporadic form of the disease. Here, we use a transgenic rat (APP21) that does not develop AD-like pathology spontaneously with age, but does develop pathology following vascular stress. To further the potential of this novel rat model as a much-needed pre-clinical animal model of sporadic AD, we characterize APP21 transgenic rats behaviorally and histologically up to 19 months of age. Methods The open field test was used as a measure of activity; and the Morris water maze was used to assess learning, memory, and strategy shift. Neuronal loss and microglia activation were also assessed throughout the brain. Results APP21 transgenic rats showed deficits in working memory from an early age, yet memory recall performance after 24 and 72 h was equal to that of wildtype rats and did not deteriorate with age. A deficit in strategy shift was observed at 19 months of age in APP21 transgenic rats compared to Fischer wildtype rats. Histologically, APP21 transgenic rats demonstrated accelerated white matter inflammation compared to wildtype rats, but interestingly no differences in neuron loss were observed. Conclusions The combined presence of white matter pathology and executive function deficits mirrored what is often found in patients with mild cognitive impairment or early dementia, and suggests that this rat model will be useful for translationally meaningful studies into the development and prevention of sporadic AD. The presence of widespread white matter inflammation as the only observed pathological correlate for cognitive deficits raises new questions as to the role of neuroinflammation in cognitive decline

Microglial Inflammation and Cognitive Dysfunction in Comorbid Rat Models of Striatal Ischemic Stroke and Alzheimer\u27s Disease: Effects of Antioxidant Catalase-SKL on Behavioral and Cellular Pathology

Ischemic stroke often co-occurs with Alzheimer\u27s disease (AD) leading to a worsened clinical outcome. Neuroinflammation is a critical process implicated in AD and ischemic pathology, associated with cognitive decline. We sought to investigate the combined effects of ischemic stroke induced by endothelin-1 injection in two AD rat models, using motor function, memory and microglial inflammation in the basal forebrain and striatum as readouts. In addition, we sought to determine the effectiveness of the antioxidant biologic CAT-SKL in one of the models. The early AD model employed the bilateral intracerebroventricular injections of the toxic β-amyloid peptide Aβ25–35, the prodromal AD model used the transgenic Fischer 344 rat overexpressing a pathological mutant human amyloid precursor protein. Motor function was assessed using a cylinder, modified sticky tape and beam-walk tasks; learning and memory were tested in the Morris water maze. Microglial activation was examined using immunohistochemistry. Aβ25–35 toxicity and stroke combination greatly increased microglial inflammation in the basal forebrain. Prodromal AD-pathology coupled with ischemia in the transgenic rat resulted in a greater microgliosis in the striatum. Combined transgenic rats showed balance alterations, comorbid Aβ25–35 rats showed a transient sensorimotor deficit, and both demonstrated spatial reference memory deficit. CAT-SKL treatment ameliorated memory impairment and basal forebrain microgliosis in Aβ25–35 rats with stroke. Our results suggest that neuroinflammation could be one of the early processes underlying the interaction of AD with stroke and contributing to the cognitive impairment, and that therapies such as antioxidant CAT-SKL could be a potential therapeutic strategy

Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD). Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP). Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated.</p> <p>Results</p> <p>Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ)₄₀were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ₄₂levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP) in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP.</p> <p>Conclusion</p> <p>This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of APP gene regulation. Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.</p

Cloning and characterization of bovine pyruvate carboxylase and phosphoenolpyruvate carboxykinase

Our objective was to clone the bovine pyruvate carboxylase (PC) and the cytosolic (C) and mitochondrial (M) forms of phosphoenolpyruvate carboxykinase (PEPCK) and determine their expression during the immediate periparturient interval in dairy cows. Bovine PEPCK-C cDNA contains 2592 nucleotides and contains 84% similarity to the coding sequence of human PEPCK-C cDNA. A 449 nt partial clone of the 3′ end of PEPCK-M is 76% similar to the corresponding sequence of human PEPCKM. Northern blot analysis revealed single transcripts of 2.85 and 2.35 kb for PEPCK-C and PEPCK-M, respectively. The transition to lactation did not alter PEPCK-M transcript expression but expression of PEPCK-C mRNA is increased during lactation indicating that enhanced hepatic gluconeogenesis during lactation is partly due to a change in PEPCK-C. The coding region plus 3′ untranslated region (UTR) of PC mRNA is 3926 bases. A 5′ rapid amplification of cDNA ends protocol was used to clone the 5′ end of the mRNA. Six 5′ UTR which contain 68 (bPC5′ A), 263 (bPC5′B), 363 (bPC5 ′C), 89 (bPC5′D), 275 (bPC5′E), and 178 by (bPC5′ F) were cloned. All PC 5′ UTR variants contain a common coding sequence. The abundance of PC mRNA, determined by Northern blot analysis, indicates that PC is more abundant in gluconeogenic and lipogenic tissues where all PC variants are expressed compared with tissues that do not possess the full spectrum of PC transcripts. One day after parturition, all 5′ UTR variants were increased as was total PC mRNA. Transcripts bPC5′A and bPC5′ B are the most abundant, bPC5′ D, and bPC5′F are intermediate and bPC5′E and bPC5′ C are the least abundant. Results demonstrate regulation of PEPCK at the level of mRNA abundance that is specific for PEPCK-C and regulation of PC that may be coupled to changes in the spectrum of 5′UTR variants expressed