AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke

Summary

This protocol describes the viral-mediated ectopic expression of Neurod1 following cortical ischemic stroke. Neurod1 is delivered (1) using the Cre-Flex AAV system in wild-type mice during the subacute phase post-stroke (7 days) and (2) using a single AAV vector in conditional reporter mice during the chronic phase post-stroke (21 days).

Abstract

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic helix-loop-helix (bHLH) transcription factors via non-propagating virus-like particle systems, including retrovirus, lentivirus, and adeno-associated virus (AAV), has been extensively reported. For in vivo experiments, AAVs are increasingly used due to their low pathogenicity and potential for translatability. This protocol describes two AAV systems for investigating the ectopic expression of transcription factors in transduced cells post-ischemic stroke. In both systems, Neurod1 expression is controlled by the short GFAP (gfaABC(1)D) promoter, which is upregulated in reactive astrocytes post-stroke as well as in endogenous neurons when combined with neurogenic factor expression. In the ischemic stroke model described, focal ischemia is induced by injecting endothelin-1 (ET-1) into the motor cortex of mice, creating a lesion surrounded by reactive GFAP-expressing astrocytes and surviving neurons. Intracerebral injections of AAV are performed to ectopically induce the expression of Neurod1 in the subacute (7 days) and chronic (21 days) phases post-stroke. Within weeks following AAV injection, a significantly higher number of neurons among transduced cells are identified in mice ectopically expressing Neurod1 compared to mice receiving AAV control viruses. The AAV-based strategies used replicated observed outcomes of increased numbers of neurons expressing the reporter gene in a model of mild-to-moderate cortical stroke. This protocol establishes a standard platform for exploring the effects of ectopic expression of transcription factors delivered with AAV-based systems, contributing to the understanding of neurogenic factor expression in the context of stroke.

Introduction

Stroke is a leading cause of disability worldwide¹. A stroke happens when blood flow to the brain is disrupted. This can occur either through a hemorrhagic stroke (~15% of cases), where a blood vessel in the brain bursts, or through an ischemic stroke, where blood flow to the brain is blocked¹. Ischemic strokes are most prevalent and account for ~85% of stroke cases¹. A stroke reduces the delivery of glucose and oxygen to the brain, leading to swift neuronal cell death and impaired neural function.

Ischemic stroke leads to the loss of cells within minutes in the core of....

Protocol

This protocol was approved by the Animal Care Committee at the University of Toronto and adheres to the Guide to the Care and Use of Experimental Animals (2^nd Edition, Canadian Council on Animal Care, 2017). For this study, wild-type (C57BL/6J) and transgenic tdTom-Cre reporter (B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J)²² mouse strains were used. Mice were 7-9 weeks old and included both males and females. Details of the reagents and equipment used are listed in the Table of Materials.

1. Endothelin-1 stroke surgery

NOTE: All....

Results

To examine the cellular outcomes of short GFAP (gfaABC(1)D) promoter driven, ectopic Neurod1 expression in wild-type (C57BL/6J) and in transgenic tdTom-Cre reporter strains (specifically, B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J)²² two AAV5-based systems were utilized in ET-1 stroke injured mice (Figure 1A-C). In a subacute model of stroke, Neurod1 was packaged in the Cre-Flex AAV system and deliver.......

Discussion

This protocol details two AAV systems and mouse models for investigating the ectopic expression of Neurod1 in the context of a mild-moderate ET-1 cortical stroke model. A number of critical steps relating to the ET-1 stroke are important to consider for the reproducibility and consistency of the injury. Burr holes must be carefully drilled without puncturing the dura mater to prevent unintentional surgical injury. It is important to use consistent landmarks, in this case bregma, to ensure a similar region of int.......

Materials

Name	Company	Catalog Number	Comments
#77 Drill Bit (.018”)	David Kopf Instruments	8177
AAV5-GFAP(0.7)-mNeurod1-2A-iCre	Vector Biolabs
Absorbable Suture with Needle Polysorb™ Polyester CV-15 3/8 Circle Taper Point Needle Size 4 - 0 Braided	Covidien	GL-881
Anti-NeuN Antibody (rabbit)	Millipore Sigma	ABN78
C57BL/6 Mice	Charles River	027
Chlorhexidine Solution	Partnar	PCH-020
Contec™ PREempt™ RTU Disinfectant Solution	Fisher Scientific	29-636-6212
Cryostat	Thermo Scientific	HM525 NX
Endothelin 1	Millipore Sigma	05-23-3800-0.5MG
Feather Safety Razor Microtome Blades	Feather	12-631P
Fisherbrand Cover Glasses: Rectangles	Fisher Scientific	12-545M
Fluorescence Mounting Medium	Agilent Technologies	S3023
Hamilton Syringe	Hamilton Company	7634-01
High Speed Stereotaxic Drill	David Kopf Instruments	1474
Metacam Solution (Meloxicam)	Boehringer Ingelheim
O.C.T Compound	Fisher Scientific	23-730-571
pAAV2/5-GFAP-iCre	Vector Builder	P190924-1001suq
Polyderm Ointment USP	TARO	2181908
SOMNI Scientific™ The Animal Temperature Heating Pad	Fisher Scientific	04-777-177
Stereotaxic Instruments	David Kopf Instruments	Model 902
Superfrost Plus Microscope Slides, white	Fisherbrand	12-550-15
tdTomato Reporter Mice	The Jackson Laboratory	007914
V-1 Tabletop Laboratory Animal Anesthesia System	VetEquip	901806