New therapies beyond the hyperacute phase of stroke are needed to be able to treat much more patients in delayed phases of this devastating disease. The idea that neurovascular plasticity contributes to stroke recovery can be a powerful concept for stroke therapy. Obviously, the therapeutic time window for interventions based on promoting recovery would be much larger than those for targeting acute stroke.  In this context, long-term neuroreparative therapies will  have to target the two essential phenomena to achieve brain neurorecovery after stroke: to restore the cerebral blood flow and to promote Neuroregeneration.

To achieve these major goals, both angiogenesis and neurogenesis need to be enhanced in the ischemic brain.

Classically, the formation of new blood vessels was thought to be mediated exclusively by embryogenic vasculogenesis followed by the sprouting of endothelial cells from preexisting vessels during angiogenesis. In the last decade, this standard dogma was overturned with the identification of the existence of circulating bone marrow-derived endothelial progenitor cells (EPCs). These cells are capable of differentiating, ex vivo, into endothelial-phenotyped cells, and now comprise a new model for endothelial generation and vessel repair (Asahara et al., 1997). These cells comprise a potential cell-based and growth-factor source of an alternate approach to enhance angio-neurogenic responses. In fact, newborn neurons (neurogenesis) and new vascular components (angiogenesis) form a microenvironment that has been termed the neurovascular niche [Ohab et al., 2006] were angiogenesis and neurogenesis are linked thorough specific growth factors.

Angiogenesis and neurogenesis occur endogenously after stroke. Our goal is to study these two complex phenomena both in experimental and human studies to finally potentiate them correctly to improve brain function and neurorecovery after stroke.

Experimental Models and Techniques

In vivo stroke models:

Cerebral ischemia affecting the cortical territory of the Middle Cerebral Artery (MCA) is occluded at the level of the M1 portion (distal occlusion). This model has been chosen because presents very low mortality rates allowing long-term studies. Besides, the infarct is restricted to the cortex with clear boundary areas with normal cerebral blood flow and never affects neuroblast-rich areas such as the subventricular zone (then, both angiogenesis and neurogenesis can occur).

Functional outcome is assessed by the cylinder and corner tests which have been reported to be appropriate test for this type of cortical infarcts. Besides, histology and immunohistochemistry studies are conducted to evaluate brain repair and angio-neurogenic processes.

Endothelial Progenitor Cell Cultures:

EPCs are obtained from the Mononuclear cell fraction of human blood and from mouse spleen. MNCs are cultured in fibronectin-coated plates with complete cell culture medium EGM-2MV.

Both in human and murine cell cultures yield an early EPC population (also called Circulating Angiogenic Cells) obtained at day 4-7 after plating and late outgrowth EPC colonies (also called Endothelial Colony Forming Cells) appear from day 10 as colonies with high proliferation capacity and tubulogenic capacity.

In vitro Oxygen-Glucose Deprivation: endothelial cells and Endothelial progenitor cells are challenged to a transitory Oxygen and Glucose deprivation to study their angio-vasculogenic responses to ischemia and to test how potential treatments that could modify these responses.

Angiogenesis-related techniques: angio-vasculogenic mechanisms are studied in a variety of in vitro assays including Matrigel® tubulogenesis, cell migration using trans-well assays or cell adhesion to a mature monolayer of endothelial cells. Our studies focus on the angio-vasculogenic responses of both Endothelial Progenitor Cells and mature endothelial cells such as the human cell line of microvascular endothelial cells (hCMEC/D3).

NMR Imaging: Bruker-BIOSPEC 70/30 USR, 7 T Preclinical MRI System is used for the neuroimaging studies. Neuroimaging studies are conducted in vivo to follow-up the ischemic lesion. Specific sequences are performed to assess axonal degeneration/regeneration and changes in cerebral blood flow and angiogenesis.