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* These authors contributed equally
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).
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.
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 the lesion through mechanisms of excitotoxicity, oxidative and nitrosative stress, calcium dysregulation, cortical spreading depolarization, edema, disruption of the blood-brain barrier (BBB), and inflammation2,3. Cells in the peri-infarct regions undergo apoptotic cell death within hours, and for days, post-stroke4. The peri-infarct environment is exposed to pro- and anti-inflammatory signals that lead to the activation of microglia and astrocytes ("reactive" astrocytes), that play diverse roles after injury5,6,7,8. Reactive astrocytes undergo a number of phenotypic changes that include the upregulation of glial fibrillary acidic protein (GFAP), and the formation of a border around the lesion within days post-stroke that helps to limit the extent of the lesion as well as impacts neuroplasticity within the injured tissue8,9,10.
Direct cellular reprogramming allows for the conversion of one mature cell type into a different mature cell type without going through a pluripotent state11. Turning resident brain cells into new neurons to substitute those lost to injury presents an appealing avenue for brain restoration. Stroke is an ideal target for cell-based treatments, given the necessity for therapeutic methods to replace lost neurons, broaden the therapeutic timeframe, and ultimately facilitate functional recovery. To this end, there have been many papers demonstrating astrocyte to neuron (AtN) reprogramming in vitro and in vivo, using a variety of basic- helix-loop-helix (bHLH) neurogenic transcription factors, including Ascl1, Neurog2, Neurod1 and more recently, mutated versions of Ascl1 (Ascl1-SA6) which demonstrated improved reprogramming efficacy11,12,13,14. Important to consider are the many experimental design factors that can impact outcomes, interpretation, and comparisons between studies. Ghazale et al. demonstrated that AtN reprogramming efficiency varied between different mouse strains, AAV serotypes, and promoters when using the same bHLH transcription factor14. Other considerations, such as the injury model, brain region, viral dosing, timing of gene delivery, the reprogramming factor, and the mode of delivery, will impact the outcomes. Recent reports have demonstrated that pre-existing transduced neurons have been misinterpreted as reprogrammed neurons15,16. While the reports do not necessarily negate that AtN conversion occurs, these studies have highlighted the importance of well-controlled experiments, and several papers have put forth recommendations for the next steps in the field15,17,18,19. What is apparent from the literature is that the ectopic expression of bHLH transcription factors can improve functional outcomes in a variety of animal models of neurodegenerative disease/injury13,20,21,22,23,24.
Herein, this protocol Neurod1, a transcription factor of interest, is ectopically expressed in transduced cells13,22. The well-established endothelin-1 (ET-1) model of ischemic stroke to the sensorimotor cortex in adult mice is used. Injection of ET-1 leads to local vasoconstriction mimicking the pathophysiology of ischemic stroke, including functional impairment22,23,25. The commonly employed short-GFAP promoter (gfaABC(1)D) is used to drive the ectopic expression of Neurod1 at the injury site and perilesional region using two viral strategies: (1) AAV5-CAG-Flex::GFP+AAV5-GFAP-Neurod1-Cre delivery in wild-type mice and (2) AAV5-GFAP-Neurod1-Cre delivery in tdTom-Cre reporter mice. To further understand the potential for ectopic expression of Neurod1 in the stroke-injured brain, AAVs were delivered in the subacute phase (7 days), or in the chronic phase (21 days), post-stroke. In both paradigms, significantly more labeled neurons were found within weeks following ectopic Neurod1 expression.
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 (2nd Edition, Canadian Council on Animal Care, 2017). For this study, wild-type (C57BL/6J) and transgenic tdTom-Cre reporter (B6.Cg-Gt(ROSA)26Sortm14(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 surgical instruments and materials are sterilized by autoclave prior to surgery. The stereotaxic frame is sterilized with PREempt. Syringes are sterilized with PREempt, 70% alcohol, and primed with sterile PBS prior to surgery. Endothelin-1 (ET-1) is stored on ice throughout the surgery.
2. Adeno-Associated Virus (AAV) delivery (7 days - subacute model or 21 days - chronic model) post-stroke
CAUTION: Refer to institutional biosafety guidelines for working with AAVs. As per the guidelines at the University of Toronto, this surgery is performed in a Containment Level 2 Biosafety Cabinet.
NOTE: All surgical instruments and materials are sterilized by autoclave prior to surgery. The stereotaxic frame is sterilized with PREempt. Syringes are sterilized with PREempt, 70% alcohol, and primed with sterile PBS. AAVs are stored on ice throughout the surgery.
3. Tissue preparation and dissection
4. Sectioning frozen brains
5. Immunohistochemistry and quantification
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)26Sortm14(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 wasΒ packaged in the Cre-Flex AAV system and deliver...
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...
The authors declare no competing interests.
This work was supported by the Heart and Stroke Foundation, Ontario Institute of Regenerative Medicine, Canada First Research Excellence Fund (Medicine by Design, MbD), and Connaught Innovation Award (University of Toronto).
Name | Company | Catalog Number | Comments |
#77 Drill Bit (.018β) | David Kopf Instruments | 8177 | |
AAV5-GFAP(0.7)-mNeurod1-2A-iCre | VectorΒ Biolabs | ||
Absorbable Suture with Needle Polysorbβ’ Polyester CV-15 3/8 Circle Taper Point Needle Size 4 - 0 Braided | Covidien | GL-881 | |
Anti-NeuN Antibody (rabbit)Β | Millipore Sigma | ABN78Β | |
C57BL/6 Mice | Charles River | 027 | |
Chlorhexidine Solution | Partnar | PCH-020 | |
Contecβ’Β PREemptβ’ RTU Disinfectant Solution | Fisher Scientific | 29-636-6212 | |
Cryostat | Thermo ScientificΒ | HM525 NXΒ | |
Endothelin 1 | Millipore Sigma | 05-23-3800-0.5MG | |
Feather Safety Razor Microtome Blades | Feather | 12-631P | |
FisherbrandΒ Cover Glasses: Rectangles | Fisher Scientific | 12-545MΒ | |
Fluorescence Mounting Medium | Agilent Technologies | S3023Β | |
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 Scientificβ’Β 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 MiceΒ | The Jackson Laboratory | 007914 | |
V-1 Tabletop Laboratory Animal Anesthesia System | VetEquip | 901806 |
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