Transient Middle Cerebral Artery Occlusion with an Intraluminal Suture Enables Reproducible Induction of Ischemic Stroke in Mice.

Ischemic stroke is a leading cause of mortality and chronic disability worldwide, underscoring the need for reliable and accurate animal models to study this disease's pathology, molecular mechanisms of injury, and treatment approaches. As most clinical strokes occur in regions supplied by the middle cerebral artery (MCA), several experimental models have been developed to simulate an MCA occlusion (MCAO), including transcranial MCAO, micro- or macro-sphere embolism, thromboembolisation, photothrombosis, Endothelin-1 injection, and - the most common method for ischemic stroke induction in murine models - intraluminal MCAO. In the intraluminal MCAO model, the external carotid artery (ECA) is permanently ligated, after which a partially-coated monofilament is inserted and advanced proximally to the common carotid artery (CCA) bifurcation, before being introduced into the internal carotid artery (ICA). The coated tip of the monofilament is then advanced to the origin of the MCA and secured for the duration of occlusion. With respect to other MCAO models, this model offers enhanced reproducibility regarding infarct volume and cognitive/functional deficits, and does not require a craniotomy. Here, we provide a detailed protocol for the surgical induction of unilateral transient ischemic stroke in mice, using the intraluminal MCAO model. Graphic abstract: Overview of the intraluminal monofilament method for transient middle cerebral artery occlusion (MCAO) in mouse.

Nanotransfection-based vasculogenic cell reprogramming drives functional recovery in a mouse model of ischemic stroke.

Ischemic stroke causes vascular and neuronal tissue deficiencies that could lead to substantial functional impairment and/or death. Although progenitor-based vasculogenic cell therapies have shown promise as a potential rescue strategy following ischemic stroke, current approaches face major hurdles. Here, we used fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis, intracranially, as a potential therapy for ischemic stroke. Perfusion analyses suggest that intracranial delivery of EFF-nanotransfected fibroblasts led to a dose-dependent increase in perfusion 14 days after injection. MRI and behavioral tests revealed ~70% infarct resolution and up to ~90% motor recovery for mice treated with EFF-nanotransfected fibroblasts. Immunohistological analysis confirmed increases in vascularity and neuronal cellularity, as well as reduced glial scar formation in response to treatment with EFF-nanotransfected fibroblasts. Together, our results suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming represent a promising strategy for the treatment of ischemic stroke.

Nanochannel-Based Poration Drives Benign and Effective Nonviral Gene Delivery to Peripheral Nerve Tissue.

While gene and cell therapies have emerged as promising treatment strategies for various neurological conditions, heavy reliance on viral vectors can hamper widespread clinical implementation. Here, the use of tissue nanotransfection as a platform nanotechnology to drive nonviral gene delivery to nerve tissue via nanochannels, in an effective, controlled, and benign manner is explored. TNT facilitates plasmid DNA delivery to the sciatic nerve of mice in a voltage-dependent manner. Compared to standard bulk electroporation (BEP), impairment in toe-spread and pinprick response is not caused by TNT, and has limited to no impact on electrophysiological parameters. BEP, however, induces significant nerve damage and increases macrophage immunoreactivity. TNT is subsequently used to deliver vasculogenic cell therapies to crushed nerves via delivery of reprogramming factor genes Etv2, Foxc2, and Fli1 (EFF). The results indicate the TNT-based delivery of EFF in a sciatic nerve crush model leads to increased vascularity, reduced macrophage infiltration, and improved recovery in electrophysiological parameters compared to crushed nerves that are TNT-treated with sham/empty plasmids. Altogether, the results indicate that TNT can be a powerful platform nanotechnology for localized nonviral gene delivery to nerve tissue, in vivo, and the deployment of reprogramming-based cell therapies for nerve repair/regeneration.