An important limitation of recombinant adeno-associated virus (rAAV) vector efficiency is the requirement of hostcell-mediated synthesis of double-stranded DNA from the single-stranded genome. We have bypassed this step in a specialized self-complementary rAAV (scAAV) vector, by utilizing the tendency of AAV to package DNA dimers when the replicating genome is half the length of the wild type (wt). To produce these vectors efficiently, we have deleted the terminal resolution site (trs) from one rAAV TR, preventing the initiation of replication at the mutated end. These constructs generate single-stranded, inverted repeat genomes, with a wt TR at each end, and a mutated TR in the middle. After uncoating, the viral DNA folds through intramolecular base pairing within the mutant TR, which then proceeds through the genome to form a double-stranded molecule. We have used the scAAV to investigate barriers to rAAV transduction in the mouse liver, muscle and brain. In each tissue, scAAV was characterized by faster onset of gene expression and higher transduction efficiency. This study confirms earlier predictions that complementary-strand DNA synthesis is the primary barrier to rAAV-2 transduction. The scAAV is unaffected by this barrier, and provides an extremely efficient vector for gene transfer into many types of cells in vivo.

Even in the 21st century, studies aimed at characterizing the pathological paradigms associated with the development and progression of central nervous system diseases are primarily performed in laboratory animals. However, limited translational significance, high cost, and labor to develop the appropriate model (e.g., transgenic or inbred strains) have favored parallel in vitro approaches. In vitro models are of particular interest for cerebrovascular studies of the blood–brain barrier (BBB), which plays a critical role in maintaining the brain homeostasis and neuronal functions. Because the BBB dynamically responds to many events associated with rheological and systemic impairments (e.g., hypoperfusion), including the exposure of potentially harmful xenobiotics, the development of more sophisticated artificial systems capable of replicating the vascular properties of the brain microcapillaries are becoming a major focus in basic, translational, and pharmaceutical research. In vitro BBB models are valuable and easy to use supporting tools that can precede and complement animal and human studies. In this article, we provide a detailed review and analysis of currently available in vitro BBB models ranging from static culture systems to the most advanced flow-based and three-dimensional coculture apparatus. We also discuss recent and perspective developments in this ever expanding research field.

A host of diabetes-related insults to the central nervous system (CNS) have been clearly documented in type-1 and -2 diabetic patients as well as experimental animal models. These host of neurological disorders encompass hemodynamic impairments (e.g., stroke), vascular dementia, cognitive deficits (mild to moderate), as well as a number of neurochemical, electrophysiological and behavioral alterations. The underlying causes of diabetes-induced CNS complications are multifactorial and are relatively little understood although it is now evident that blood-brain barrier (BBB) damage plays a significant role in diabetes-dependent CNS disorders. Changes in plasma glucose levels (hyper- or hypoglycemia) have been associated with altered BBB transport functions (e.g., glucose, insulin, choline, amino acids, etc.), integrity (tight junction disruption), and oxidative stress in the CNS microcapillaries. Last two implicating a potential causal role for upregulation and activation of the receptor for advanced glycation end products (RAGE). This type I membrane-protein also transports amyloid-beta (Aβ) from the blood into the brain across the BBB thus, establishing a link between type 2 diabetes mellitus (T2DM) and Alzheimer’s disease (AD, also referred to as “type 3 diabetes”). Hyperglycemia has been associated with progression of cerebral ischemia and the consequent enhancement of secondary brain injury. Difficulty in detecting vascular impairments in the large, heterogeneous brain microvascular bed and dissecting out the impact of hyper- and hypoglycemia in vivo has led to controversial results especially with regard to the effects of diabetes on BBB. In this article, we review the major findings and current knowledge with regard to the impact of diabetes on BBB integrity and function as well as specific brain microvascular effects of hyper- and hypoglycemia.