Gene therapy targeting the blood-brain barrier improves neurological symptoms in a model of genetic MCT8 deficiency.

A genetic deficiency of the solute carrier monocarboxylate transporter 8 (MCT8), termed Allan-Herndon-Dudley syndrome, is an important cause of X-linked intellectual and motor disability. MCT8 transports thyroid hormones across cell membranes. While thyroid hormone analogues improve peripheral changes of MCT8 deficiency, no treatment of the neurological symptoms is available so far. Therefore, we tested a gene replacement therapy in Mct8- and Oatp1c1-deficient mice as a well-established model of the disease. Here, we report that targeting brain endothelial cells for Mct8 expression by intravenously injecting the vector AAV-BR1-Mct8 increased tri-iodothyronine (T3) levels in the brain and ameliorated morphological and functional parameters associated with the disease. Importantly, the therapy resulted in a long-lasting improvement in motor coordination. Thus, the data support the concept that MCT8 mediates the transport of thyroid hormones into the brain and indicate that a readily accessible vascular target can help overcome the consequences of the severe disability associated with MCT8 deficiency.

The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

The trophoblast clock controls transport across placenta in mice.

In mammals, 24-h rhythms of physiology and behavior are organized by a body-wide network of clock genes and proteins. Despite the well-known function of the adult circadian system, the roles of maternal, fetal and placental clocks during pregnancy are poorly defined. In the mature mouse placenta, the labyrinth zone (LZ) is of fetal origin and key for selective nutrient and waste exchange. Recently, clock gene expression has been detected in LZ and other fetal tissues; however, there is no evidence of a placental function controlled by the LZ clock. Here, we demonstrate that specifically the trophoblast layer of the LZ harbors an already functional clock by late gestation, able to regulate in a circadian manner the expression and activity of the xenobiotic efflux pump, ATP-binding cassette sub-family B member 1 (ABCB1), likely gating the fetal exposure to drugs from the maternal circulation to certain times of the day. As more than 300 endogenous and exogenous compounds are substrates of ABCB1, our results might have implications in choosing the maternal treatment time when aiming either maximal/minimal drug availability to the fetus/mother.

Telemedical emergency services: central or decentral coordination?

BACKGROUND AND OBJECTIVE: Teleemergency doctors support ambulance cars at the emergency site by means of telemedicine. Currently, each district has its own teleemergency doctor office (decentralized solution). This paper analyses the advantages and disadvantages of a centralized solution where several teleemergency doctors work in parallel in one office to support the ambulances in more districts. METHODS: The service of incoming calls from ambulances to the teleemergency doctor office can be modelled as a queuing system. Based on the data of the district of Vorpommern-Greifswald in the Northeast of Germany, we assume that arrivals and services are Markov chains. The model has parallel channels proportionate to the number of teleemergency doctors working simultaneously and the number of calls which one doctor can handle in parallel. We develop a cost function with variable, fixed and step-fixed costs. RESULTS: For the district of Greifswald, the likelihood that an incoming call has to be put on hold because the teleemergency doctor is already fully occupied is negligible. Centralization of several districts with a higher number of ambulances in one teleemergency doctor office will increase the likelihood of overburdening and require more doctors working simultaneously. The cost of the teleemergency doctor office per ambulance serviced strongly declines with the number of districts cooperating. DISCUSSION: The calculations indicate that centralization is feasible and cost-effective. Other advantages (e.g. improved quality, higher flexibility) and disadvantages (lack of knowledge of the location and infrastructure) of centralization are discussed. CONCLUSIONS: We recommend centralization of telemedical emergency services. However, the number of districts cooperating in one teleemergency doctor office should not be too high and the distance between the ambulance station and the telemedical station should not be too large.

Brain Distribution of Dual ABCB1/ABCG2 Substrates Is Unaltered in a Beta-Amyloidosis Mouse Model.

BACKGROUND: ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein) are co-localized at the blood-brain barrier (BBB), where they restrict the brain distribution of many different drugs. Moreover, ABCB1 and possibly ABCG2 play a role in Alzheimer's disease (AD) by mediating the brain clearance of beta-amyloid (Aß) across the BBB. This study aimed to compare the abundance and activity of ABCG2 in a commonly used ß-amyloidosis mouse model (APP/PS1-21) with age-matched wild-type mice. METHODS: The abundance of ABCG2 was assessed by semi-quantitative immunohistochemical analysis of brain slices of APP/PS1-21 and wild-type mice aged 6 months. Moreover, the brain distribution of two dual ABCB1/ABCG2 substrate radiotracers ([11C]tariquidar and [11C]erlotinib) was assessed in APP/PS1-21 and wild-type mice with positron emission tomography (PET). [11C]Tariquidar PET scans were performed without and with partial inhibition of ABCG2 with Ko143, while [11C]erlotinib PET scans were only performed under baseline conditions. RESULTS: Immunohistochemical analysis revealed a significant reduction (by 29-37%) in the number of ABCG2-stained microvessels in the brains of APP/PS1-21 mice. Partial ABCG2 inhibition significantly increased the brain distribution of [11C]tariquidar in APP/PS1-21 and wild-type mice, but the brain distribution of [11C]tariquidar did not differ under both conditions between the two mouse strains. Similar results were obtained with [11C]erlotinib. CONCLUSIONS: Despite a reduction in the abundance of cerebral ABCG2 and ABCB1 in APP/PS1-21 mice, the brain distribution of two dual ABCB1/ABCG2 substrates was unaltered. Our results suggest that the brain distribution of clinically used ABCB1/ABCG2 substrate drugs may not differ between AD patients and healthy people.

Costing of helicopter emergency services- a strategic simulation based on the example of a German rural region.

BACKGROUND: Helicopter emergency services (HEMS) are of increasing relevance for emergency medical services (EMS) of developed countries. Despite the known cost intensity of HEMS, there is only very limited knowledge of its cost dynamics and structures. This averts an efficient resource allocation of scarce EMS resources in an environment that is characterized by socio-political, medical and economic challenges. The objective of this study is the exemplary modeling of HEMS cost structures. METHODS: We defined three scenarios with each five variations to illustrate different models of HEMS provision. Into these, we included varying availability times, technical features for off-shore or alpine rescue and differing numbers of operations. Cost data is based on a broad literature review and primary data from a German HEMS organization resulting in a cost function. We calculated average costs per primary missions and total costs, whilst differentiating between fixed, jump-fixed, variable and maintenance costs for every scenario variation. The costs were further used to evaluate the profitability of operations by executing a break-even analysis. RESULTS: Average costs per HEMS operation decrease with increasing number of operations due to the digression of fixed costs. Depending on special equipment, availability times or other assumptions, total costs differ significantly with the different scenario variations. For the basic scenario (12 h of operations per day), the total costs per year of HEMS are 1,697,546.20 € and the unit costs are 763.41 € per primary mission at 1200 primary and 92 secondary operations. At an engine-runtime based revenue of 70 € per minute, global cost covering is possible after 728 missions (c.p.). CONCLUSIONS: Considering a revenue of 70 € per minute of engine run-time, HEMS can be operated at a profit for companies. However, the necessary remuneration represents a high financial effort for the societal cost bearers of helicopter emergency services. This leads to the question of the cost-benefit ratio of HEMS, which could be approached in further researches by using this model. The valuation of mission costs also opens a new view to the framework of HEMS disposition procedures and criteria. This cost analysis enhances the necessity of better planning of HEMS networks to use available resources efficiently in order to improve social welfare.

Detection and Prediction of Mild Cognitive Impairment in Alzheimer's Disease Mice.

BACKGROUND: The recent failure of clinical trials to treat Alzheimer's disease (AD) indicates that the current approach of modifying disease is either wrong or is too late to be efficient. Mild cognitive impairment (MCI) denotes the phase between the preclinical phase and clinical overt dementia. AD mouse models that overexpress human amyloid-ß (Aß) are used to study disease pathogenesis and to conduct drug development/testing. However, there is no direct correlation between the Aß deposition, the age of onset, and the severity of cognitive dysfunction. OBJECTIVE: To detect and predict MCI when Aß plaques start to appear in the hippocampus of an AD mouse. METHODS: We trained wild-type and AD mice in a Morris water maze (WM) task with different inter-trial intervals (ITI) at 3 months of age and assessed their WM performance. Additionally, we used a classification algorithm to predict the genotype (APPtg versus wild-type) of an individual mouse from their respective WM data. RESULTS: MCI can be empirically detected using a short-ITI protocol. We show that the ITI modulates the spatial learning of AD mice without affecting the formation of spatial memory. Finally, a simple classification algorithm such as logistic regression on WM data can give an accurate prediction of the cognitive dysfunction of a specific mouse. CONCLUSION: MCI can be detected as well as predicted simultaneously with the onset of Aß deposition in the hippocampus in AD mouse model. The mild cognitive impairment prediction can be used for assessing the efficacy of a treatment.

Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol.

INTRODUCTION: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are transmembrane proteins which actively transport a large variety of substrates across biological membranes. ABC transporter overexpression can be the underlying cause of multidrug resistance in oncology. Moreover, it has been revealed that increased ABCC1 transporter activity can ameliorate behavioural changes and Aß pathology in a rodent model of Alzheimer's disease and it is currently tested in AD patients. METHODS: Finding substances that modulate ABC transporter activity (inhibitors and activators) is of high relevance and thus, different methods have been developed to screen for potential modulators. For this purpose, we have developed a cell-based assay to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye using a common qPCR device (Agilent AriaMx). RESULTS: We validated the specificity of our method with vanadate and benzbromarone controls. Furthermore, we provide a step-by-step protocol including statistical analysis of the resulting data and suggestions how to modify the protocol specifically to screen for activators of ABCC1. DISCUSSION: Our approach is biologically more relevant than cell-free assays. The continuous detection of kinetics allows for a more precise quantification compared with assays with single end-point measurements.

Early Cognitive Training Rescues Remote Spatial Memory but Reduces Cognitive Flexibility in Alzheimer's Disease Mice.

BACKGROUND: Spatial memory dysfunction has been demonstrated in mouse models of Alzheimer's disease (AD) which is consistent with the clinical finding that the early signature of AD includes difficulties in the formation and/or storage of a memory. A stored memory-a long term memory-can be modulated via process called as memory retrieval that can either lead toward memory reconsolidation or even memory extinction. OBJECTIVE: We aim to shed light on the fate of the spatial memory during memory reactivation and memory extinction using a water maze task. METHODS: In Set-up I, we trained 3-month-old mice (wild-type mice and mice with cerebral ß-amyloidosis) and assessed the fate of remote memory after four months of retention interval (RI). In Set-up II, we performed an early-extensive training at 2 months of age, retrained the same mice at 3 months of age, introduced four months of RI, and finally assessed remote spatial memory at 7 months of age. RESULTS: We find in ß-amyloidosis mice that memory reactivation problems were detectable at 7 months of age and were alleviated by cognitive overtraining. Similarly, forgetting of remote spatial memory was also minimized by cognitive overtraining. Finally, we show that the cognitive training facilitates the recovery of the reactivated spatial memory while reducing the ability to form new spatial memory in AD mice. CONCLUSION: This result may explain the rationality behind the cognitive reserve observed in AD patients and elderly with severe ß-amyloidosis not corresponding to the actual low dementia symptoms.

Age dependency of cerebral P-glycoprotein function in wild-type and APPPS1 mice measured with PET.

P-glycoprotein (P-gp, ABCB1) is an efflux transporter at the blood-brain barrier (BBB), which mediates clearance of beta-amyloid (Aß) from brain into blood. We used (R)-[11C]verapamil PET in combination with partial P-gp inhibition with tariquidar to measure cerebral P-gp function in a beta-amyloidosis mouse model (APPtg) and in control mice at three different ages (50, 200 and 380 days). Following tariquidar pre-treatment (4 mg/kg), whole brain-to-plasma radioactivity concentration ratios (Kp,brain) were significantly higher in APPtg than in wild-type mice aged 50 days, pointing to decreased cerebral P-gp function. Moreover, we found an age-dependent decrease in cerebral P-gp function in both wild-type and APPtg mice of up to -50%. Alterations in P-gp function were more pronounced in Aß-rich brain regions (hippocampus, cortex) than in a control region with negligible Aß load (cerebellum). PET results were confirmed by immunohistochemical staining of P-gp in brain microvessels. Our results confirm previous findings of reduced P-gp function in Alzheimer's disease mouse models and show that our PET protocol possesses adequate sensitivity to measure these functional changes in vivo. Our PET protocol may find use in clinical studies to test the efficacy of drugs to induce P-gp function at the human BBB to enhance Aß clearance.

Measurement of cerebral ABCC1 transport activity in wild-type and APP/PS1-21 mice with positron emission tomography.

Previous data suggest a possible link between multidrug resistance-associated protein 1 (ABCC1) and brain clearance of beta-amyloid (Aß). We used PET with 6-bromo-7-[11C]methylpurine ([11C]BMP) to measure cerebral ABCC1 transport activity in a beta-amyloidosis mouse model (APP/PS1-21) and in wild-type mice aged 50 and 170 days, without and with pretreatment with the ABCC1 inhibitor MK571. One hundred seventy days-old-animals additionally underwent [11C]PiB PET scans to measure Aß load. While baseline [11C]BMP PET scans detected no differences in the elimination slope of radioactivity washout from the brain (kelim) between APP/PS1-21 and wild-type mice of both age groups, PET scans after MK571 pretreatment revealed significantly higher kelim values in APP/PS1-21 mice than in wild-type mice aged 170 days, suggesting increased ABCC1 activity. The observed increase in kelim occurred across all investigated brain regions and was independent of the presence of Aß plaques measured with [11C]PiB. Western blot analysis revealed a trend towards increased whole brain ABCC1 levels in 170 days-old-APP/PS1-21 mice versus wild-type mice and a significant positive correlation between ABCC1 levels and kelim. Our data point to an upregulation of ABCC1 in APP/PS1-21 mice, which may be related to an induction of ABCC1 in astrocytes as a protective mechanism against oxidative stress.

Imaging P-Glycoprotein Induction at the Blood-Brain Barrier of a ß-Amyloidosis Mouse Model with 11C-Metoclopramide PET.

P-glycoprotein (ABC subfamily B member 1, ABCB1) plays an important role at the blood-brain barrier (BBB) in promoting clearance of neurotoxic ß-amyloid (Aß) peptides from the brain into the blood. ABCB1 expression and activity were found to be decreased in the brains of Alzheimer disease patients. Treatment with drugs that induce cerebral ABCB1 activity may be a promising approach to delay the build-up of Aß deposits in the brain by enhancing clearance of Aß peptides from the brain. The aim of this study was to investigate whether PET with the weak ABCB1 substrate radiotracer 11C-metoclopramide can measure ABCB1 induction at the BBB in a ß-amyloidosis mouse model (APP/PS1-21 mice) and in wild-type mice. Methods: Groups of wild-type and APP/PS1-21 mice aged 50 or 170 d underwent 11C-metoclopramide baseline PET scans or scans after intraperitoneal treatment with the rodent pregnane X receptor activator 5-pregnen-3ß-ol-20-one-16α-carbonitrile (PCN, 25 mg/kg) or its vehicle over 7 d. At the end of the PET scans, brains were harvested for immunohistochemical analysis of ABCB1 and Aß levels. In separate groups of mice, radiolabeled metabolites of 11C-metoclopramide were determined in plasma and brain at 15 min after radiotracer injection. As an outcome parameter of cerebral ABCB1 activity, the elimination slope of radioactivity washout from the brain (kE,brain) was calculated. Results: PCN treatment resulted in an increased clearance of radioactivity from the brain as reflected by significant increases in kE,brain (from +26% to +54% relative to baseline). Immunohistochemical analysis confirmed ABCB1 induction in the brains of PCN-treated APP/PS1-21 mice with a concomitant decrease in Aß levels. There was a significant positive correlation between kE,brain and ABCB1 levels in the brain. In wild-type mice, a significant age-related decrease in kE,brain was found. Metabolite analysis showed that most radioactivity in the brain comprised unmetabolized 11C-metoclopramide in all animal groups. Conclusion:11C-metoclopramide can measure ABCB1 induction in the mouse brain without the need to consider an arterial input function and may find potential application in Alzheimer disease patients to noninvasively evaluate strategies to enhance the clearance properties of the BBB.

Generation and Characterization of an Abcc1 Humanized Mouse Model (hABCC1flx/flx ) with Knockout Capability.

ATP-binding cassette (ABC) transporters such as ABCB1 (P-glycoprotein), ABCC1 (MRP1), and ABCG2 (BCRP) are well known for their role in rendering cancer cells resistant to chemotherapy. Additionally, recent research provided evidence that, along with other ABC transporters (ABCA1 and ABCA7), they might be cornerstones to tackle neurodegenerative diseases. Overcoming chemoresistance in cancer, understanding drug-drug interactions, and developing efficient and specific drugs that alter ABC transporter function are hindered by a lack of in vivo research models, which are fully predictive for humans. Hence, the humanization of ABC transporters in mice has become a major focus in pharmaceutical and neurodegenerative research. Here, we present a characterization of the first Abcc1 humanized mouse line. To preserve endogenous expression profiles, we chose to generate a knockin mouse model that leads to the expression of a chimeric protein that is fully human except for one amino acid. We found robust mRNA and protein expression within all major organs analyzed (brain, lung, spleen, and kidney). Furthermore, we demonstrate the functionality of the expressed human ABCC1 protein in brain and lungs using functional positron emission tomography imaging in vivo. Through the introduction of loxP sites, we additionally enabled this humanized mouse model for highly sophisticated studies involving cell type-specific transporter ablation. Based on our data, the presented mouse model appears to be a promising tool for the investigation of cell-specific ABCC1 function. It can provide a new basis for better translation of preclinical research.

Analysis of processes and costs of alternative packaging options of sterile goods in hospitals - a case study in two German hospitals.

BACKGROUND: Hospitals should monitor the costs of all direct and indirect processes in order to achieve efficiency and safeguard financial sustainability. One neglected process with significant costs is the processing of reusable medical devices and their packaging performed in the central sterilisation supply department and the operating room. The objective of this research is to analyse and compare processes and costs of four different packing alternatives, i.e. non-woven sterilisation wrap with two sheets, one-step wrap, sterilisation container with inner wrap and sterilisation container without inner wrap. METHODS: We defined sub-processes that are directly related to the packaging options and measured them through a comprehensive time study. For all sub-processes and the total processes a distribution fitting and a Monte-Carlo-Simulation were performed. We calculated the costs for all sub-processes, i.e., costs for personnel, variable costs and the respective share of fixed and jump-fixed costs (e.g. depreciation of containers) associated with each packaging option. All results are discussed through various scenarios to evaluate the advantageousness of all packaging options. RESULTS: The four packaging options are associated with different costs. "Sterile container without inner wrap" causes 2.05€ per use. The options "sterile container with inner wrap" (3.24€), "one-step sterilisation wrap" (3.44€) and "two sheets sterilisation wrap" (3.87€) cause higher costs. With regard to personnel costs the option "sterile container without inner wrap" clearly causes the lowest costs. In addition, variable costs are lower in case of sterile container. Sterile container only cause higher costs in the aspect of fixed and jump-fixed costs per packaging. CONCLUSIONS: The analysis shows that even under a broad set of scenarios the "sterile container without inner wrap" is the most cost-effective alternative. The evaluation of the options "sterile container with inner wrap" and "one-step sterilisation wrap" remains particularly interesting as they often yield comparable results. Both options cause approximately the same personnel costs, so the decision appears to be more dependent on the material prices for wrap or the frequency and duration of use for container. It turns out that the personnel time and consequently the personnel costs significantly influence the rational choice of the packaging options.

Influence of Multidrug Resistance-Associated Proteins on the Excretion of the ABCC1 Imaging Probe 6-Bromo-7-[11C]Methylpurine in Mice.

PURPOSE: Multidrug resistance-associated proteins (MRPs) mediate the hepatobiliary and renal excretion of many drugs and drug conjugates. The positron emission tomography (PET) tracer 6-bromo-7-[11C]methylpurine is rapidly converted in tissues by glutathione-S-transferases into its glutathione conjugate, and has been used to measure the activity of Abcc1 in the brain and the lungs of mice. Aim of this work was to investigate if the activity of MRPs in excretory organs can be measured with 6-bromo-7-[11C]methylpurine. PROCEDURES: We performed PET scans with 6-bromo-7-[11C]methylpurine in groups of wild-type, Abcc4(-/-) and Abcc1(-/-) mice, with and without pre-treatment with the prototypical MRP inhibitor MK571. RESULTS: 6-Bromo-7-[11C]methylpurine-derived radioactivity predominantly underwent renal excretion. In blood, MK571 treatment led to a significant increase in the AUC and a decrease in the elimination rate constant of radioactivity (kelimination,blood). In the kidneys, there were significant decreases in the rate constant for radioactivity uptake from the blood (kuptake,kidney), kelimination,kidney, and the rate constant for tubular secretion of radioactivity (kurine). Experiments in Abcc4(-/-) mice indicated that Abcc4 contributed to renal excretion of 6-bromo-7-[11C]methylpurine-derived radioactivity. CONCLUSIONS: Our data suggest that 6-bromo-7-[11C]methylpurine may be useful to assess the activity of MRPs in the kidneys as well as in other organs (brain, lungs), although further work is needed to identify the MRP subtypes involved in the disposition of 6-bromo-7-[11C]methylpurine-derived radioactivity.

Humanization of the Blood-Brain Barrier Transporter ABCB1 in Mice Disrupts Genomic Locus - Lessons from Three Unsuccessful Approaches.

ATP-binding cassette (ABC) transporters are of major importance for the restricted access of toxins and drugs to the human body. At the body's barrier tissues like the blood-brain barrier, these transporters are highly represented. Especially, ABCB1 (P-glycoprotein) has been a priority target of pharmaceutical research, for instance, to aid chemotherapy of cancers, therapy resistant epilepsy, and lately even neurodegenerative diseases. To improve translational research, the humanization of mouse genes has become a popular tool although, like recently seen for Abcb1, not all approaches were successful. Here, we report the characterization of another unsuccessful commercially available ABCB1 humanized mouse strain. In vivo assessment of transporter activity using positron emission tomography imaging revealed a severe reduction of ABCB1 function in the brain of these mice. Analyses of brain mRNA and protein expression showed that the murine Abcb1a gene is still expressed in homozygous humanized animals while expression of the human gene is minimal. Promoter region analyses underpinned that the introduced human gene might dysregulate normal expression and provided insights into the regulation of both transcription and translation of Abcb1a. We conclude that insertion of the human coding DNA sequence (CDS) into exon 3 instead of exon 2 most probably represents a more promising strategy for Abcb1a humanization.

ABCA7 Downregulation Modifies Cellular Cholesterol Homeostasis and Decreases Amyloid-ß Peptide Efflux in an in vitro Model of the Blood-Brain Barrier.

The role of ABCA7 in brain homeostasis and Alzheimer's disease (AD) is currently under intense scrutiny, since it has been reported that polymorphisms in the Abca7 gene and a loss of function of the protein are closely linked to excessive accumulation of amyloid peptides and disturbed cholesterol homeostasis. The blood-brain barrier (BBB), which isolates the brain from the blood compartment, is involved in both of these processes. We therefore hypothesized that ABCA7 downregulation might affect cholesterol and amyloid exchanges at the BBB. Using siRNA and primary cultures of mouse endothelial cells purified from brain microvessels and seeded on Transwell ® inserts, we investigated the role of ABCA7 in cholesterol and amyloid exchanges across the BBB. Our results showed that a decrease in ABCA7 expression at the BBB provokes in vitro a reduction in ABCA1 expression and a decrease in APOE secretion. In vitro, these decreases reduce cholesterol exchange across the BBB, particularly for high-density lipoproteins and ApoA-I particles. When ABCA7 was absent, we observed a reduction in Aß peptide basolateral-to-apical transport in the presence of ApoA-I, with non-significant changes in the expression levels of Rage, Lrp1, Abcb1, Abcc1, and Abcg2. Our study in murine BBB model highlighted a putative new role for ABCA7 in AD via the protein's involvement in cholesterol metabolism and amyloid clearance at the BBB.

Improved method for cannula fixation for long-term intracerebral brain infusion.

BACKGROUND: Implanted osmotic minipumps are commonly used for long-term, brain-targeted delivery of a wide range of experimental agents by being connected to a catheter and a cannula. During the stereotactical surgery procedure, the cannula has to be placed correctly in the x-y directions and also with respect to the injection point in the z-direction (deepness). However, the flat fixation base of available cannula holders doesn't allow an easy, secure fixation onto the curve-shaped skull. NEW METHOD: We have developed a modified method for a better fixation of the cannula holder by using an easy-to-produce, skull-shaped silicone spacer as fixation adapter. RESULTS: We describe the application and its fast and reliable production in the lab. COMPARISON WITH EXISTING METHOD(S): Superglue or cement is currently being used as the method of choice. However, the curve-shaped skull surface does not fit well with the flat and rigid cannula adapter which leads to fixation problems over time causing wide infusion channels and often also to leakage problems from intracerebrally applied agents towards the surface meninges. As another consequence of the inappropriate fixation, the cannula may loosen from the skull before the end of the experiment or it causes damage to the brain tissue, harming the animals with leading to a failure of the whole experiment. CONCLUSIONS: The easy-to-produce spacer facilitates the crucial step of long-term, stereotactic brain infusion experiments with intracerebral catheters in a highly secure and reproducible way.

Expression of endogenous mouse APP modulates ß-amyloid deposition in hAPP-transgenic mice.

Amyloid-ß (Aß) deposition is one of the hallmarks of the amyloid hypothesis in Alzheimer's disease (AD). Mouse models using APP-transgene overexpression to generate amyloid plaques have shown to model only certain parts of the disease. The extent to which the data from mice can be transferred to man remains controversial. Several studies have shown convincing treatment results in reducing Aß and enhancing cognition in mice but failed totally in human. One model-dependent factor has so far been almost completely neglected: the endogenous expression of mouse APP and its effects on the transgenic models and the readout for therapeutic approaches.Here, we report that hAPP-transgenic models of amyloidosis devoid of endogenous mouse APP expression (mAPP-knockout / mAPPko) show increased amounts and higher speed of Aß deposition than controls with mAPP. The number of senile plaques and the level of aggregated hAß were elevated in mAPPko mice, while the deposition in cortical blood vessels was delayed, indicating an alteration in the general aggregation propensity of hAß together with endogenous mAß. Furthermore, the cellular response to Aß deposition was modulated: mAPPko mice developed a pronounced and age-dependent astrogliosis, while microglial association to amyloid plaques was diminished. The expression of human and murine aggregation-prone proteins with differing amino acid sequences within the same mouse model might not only alter the extent of deposition but also modulate the route of pathogenesis, and thus, decisively influence the study outcome, especially in translational research.