Methods in molecular biology
-
The blood-brain barrier (BBB) proper is composed of endothelial cells (ECs) of the cerebral microvasculature, which are interconnected by tight junctions (TJs) that in turn form a physical barrier restricting paracellular flux. Tight control of vascular permeability is essential for the homeostasis and functionality of the central nervous system (CNS). In vitro BBB models have been in use for decades and have been of great benefit in the process of investigating and understanding the cellular and molecular mechanisms underlying BBB establishment. ⋯ Additionally, this chapter provides guidance through subsequent experiments such as permeability analysis (Pe, flux), expression analysis (qRT-PCR and Western blotting), and localization analysis of BBB junction proteins (immunocytochemistry) using the same inserts subjected earlier to impedance analysis. As numerous diseases are associated with BBB breakdown, researchers aim to continuously improve and refine in vitro BBB models to mimic in vivo conditions as closely as possible. This chapter summarizes protocols with the intention to provide a collection of BBB in vitro assays that generate reproducible results not only with primary brain ECs but also with EC lines to open up the field for a broader spectrum of researchers who intend to investigate the BBB in vitro particularly aiming at therapeutic aspects.
-
Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca(2+) indicators is widely used to record transient intracellular Ca(2+) increases associated with bioelectrical activity. The natural bioluminescent Ca(2+) sensor aequorin has been historically the first Ca(2+) indicator used to address biological questions. ⋯ Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca(2+) transients in single neurons or multiple neurons simultaneously.
-
Complex, interrelated systems exist to maintain the fluidity of the blood in the vascular system while allowing for the rapid formation of a solid blood clot to prevent hemorrhaging subsequent to blood vessel injury. These interrelated systems are collectively referred to as haemostasis. The components involved in the haemostatic mechanism consist of vessel walls, platelets, coagulation factors, inhibitors, and the fibrinolytic system. ⋯ Once the fibrin clot is formed, the fibrinolytic system ensures that the clot is lysed so that it does not become a pathological complication. Taken together, the systems exist to balance each other and maintain order. The balance of coagulation and fibrinolysis keeps the haemostatic system functioning efficiently.
-
Composite tissue transplantation is an emerging new era in transplant medicine and has become a viable reconstructive option for patients with large and devastating tissue defects. Advances in microsurgical techniques, transplant immunology and the development of potent immunosuppressive agents have enabled the realization of such types of transplants. ⋯ However, despite the fact that surgical, immunological and functional results are highly encouraging, the need for long-term and high-dose immunosuppression to enable graft survival and to treat/reverse acute skin rejection episodes remains a pace-limiting obstacle towards wide spread application. In this chapter we review the history and development of this novel field, the functional and immunological outcomes based on the world experience, unique biological features of such transplants, mechanisms and treatment protocols for acute skin rejection, as well as novel concepts for immune modulation and tolerance induction.
-
Fibrinogen is the final essential building block of the clotting process. Thus, all of the preliminary "cause and effect" events in the clotting cascade rely on the work of this molecule to measure their success. The most commonly used laboratory method for measuring fibrinogen is the Clauss fibrinogen assay. ⋯ The following chapter includes detailed information on the Clauss fibrinogen assay. Other fibrinogen assays used include fibrinogen levels derived from prothrombin time assays and antigenic methods. Fibrinogen measurements using the prothrombin time and antigenic based assays are described in brief.