Cortical organotypic slice cultures as a tool to analyze the neurovascular unit in hypoxia/ischemia and hypothermia-induced neuroprotection

Neurons and glial cells of the central nervous system (CNS) communicate and work together to function and execute an array of complex tasks. In addition to them a third cell type which also works to keep the brain alive are the cerebral endothelial cells that create the vascular system which supply and deliver oxygen and nutrients. The cerebral endothelium is also specialized with a blood-brain barrier (BBB) that is important for protecting the CNS from harmful substances and for regulating access only to certain ions and nutrients for optimal maintenance and support of CNS activities. A complex of tight junction proteins which include occludin, Claudin-1/3, Claudin-5, and ZO1-3 are thought to keep the endothelium impermeable, while a system of transporters, such as GLUT1 and P-gp are involved in regulating the molecular trafficking across the BBB. The endothelium is also characterized by a low pinocytotic activity compared to vessels in peripheral organs. A lot is known on the formation and composition of the BBB, but less is understood on the maintenance. This is probably due to the heterogeneity of the cerebral endothelium, which makes it difficult to study. However, the cerebrovascular function is supported by a combination of interactions with glia, pericytes, and nerve cells, known together as the neurovascular unit (NVU). A number of in vitro studies show that co-culturing endothelial cells with astrocytes and pericytes, as well as nerve tissue is capable of expressing tight junction proteins and a tight endothelial barrier, suggesting that cell-cell interactions and production of essential factors such as FGF and TGF-beta are involved in BBB maintenance.
Disruption of the BBB is often associated with brain injury and considered to be detrimental to the recovery process. Although the mechanisms are poorly understood, breakdown of the BBB has been suggested to be due to production of matrix-metalloproteinases, growth factors and cytokines. Inflammatory mediators have been well known to modulate BBB permeability. Since inflammation typically follows excitotoxicity in the ischemic cascade to cell death, BBB disruption may be secondary in the injury process. Whether the vascular damage is precipitated by neurodegeneration is unclear. Understanding how the neurovasculature might be affected is important for prevention and treatment of neurovascular diseases, such as stroke or neonatal asphyxia since neuroprotectants, except for therapeutic mild-hypothermia, have failed in the clinics.
Cooling of the head or body down to about 33°C for up to 3 days has so far been the most effective neuroprotective strategy for treating cerebral ischemia observed in stroke or perinatal asphyxia. Along with reducing energy demand and energy consumption, mild hypothermia seems to affect many aspects of cellular injury but overall is very efficient at stopping cell death. A small group of proteins are expressed during mild-hypothermia. One of them is the RNA binding motif protein 3 (RBM3) and based on in vitro studies it is involved in cell proliferation and survival. Whether RBM3 is involved in hypothermia-induced neuroprotection deserves investigation as it may provide some hints into the mechanisms of how hypothermia prevents cell death.
A useful in vitro system to study aspects of neurodegeneration that is close to the in vivo situation is the organotypic slice culture system, which has been very well established for cortical, hippocampal, and cerebellar tissue. Previously our lab has established an in vitro BBB model utilizing the cortical organotypic slice culture system, which preserves tight junction proteins for more than a week under the presence of FGF2. The main objective of my PhD thesis was to analyze the expression of the neuronal and vascular elements of the blood-brain barrier (BBB) under normal and stressed conditions and to study the cell-cell interaction at the biochemical and cellular level utilizing the organotypic slice culture system established in our laboratory. In the first project we have studied the expression of transporter proteins in cortical organotypic slice cultures (COSCs). We could show that transporters such as glucose transporter 1 (GLUT-1) and the ATP-binding cassette (ABC) transporter, P-glycoprotein (P-gp) were present and functional in the blood vessels of COSCs. In the second project we used entorhino-hippocampal organotypic slice cultures (EHOSCs) derived from newborn mice to study the effect of oxygen-glucose deprivation (OGD) as well as excitotoxicity on neurodegeneration and accompanying neurovascular changes. We could show that changes in BBB integrity and vascular remodeling were linked to neurodegeneration. Selective loss of the neurovasculature in CA1 of the hippocampus was preceded by neuronal death indicating that not OGD by itself, but the OGD-induced neurodegeneration was responsible for the loss of local blood vessels. Finally, in the third project we have explored a potential function of the cold-inducible RNA binding motif protein 3 as a neuroprotectant in response to mild hypothermia that is employed after neonatal asphyxia. We could show that RBM3 expression was strongly induced in COSCs upon hypothermia and was required and sufficient for neuroprotection of dissociated PC12 cells from induced apoptosis.