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 the lesion through mechanisms of excitotoxicity, oxidative and nitrosative stress, calcium dysregulation, cortical spreading depolarization, edema, disruption of the blood-brain barrier (BBB), and inflammation^2,3. 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 injury^5,6,7,8. 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 tissue^8,9,10.

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 efficacy^11,12,13,14. 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 neurons^15,16. 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 field^15,17,18,19. 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/injury^{13,20,21,22,23,24}.

Herein, this protocol Neurod1, a transcription factor of interest, is ectopically expressed in transduced cells^13,22. 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 impairment^22,23,25. 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.

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 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.

1. Before starting the surgery, set up the recovery area by placing a clean cage on a heating pad set to 37 °C. Alternatively, if a heating lamp is being used, place only half of the cage under the heat.
   1. To anesthetize mice using isoflurane, follow the protocol approved by the Animal Care Committee. Place the mouse in the anesthetic induction chamber and turn on the oxygen to 1-1.5 L/min, then adjust the isoflurane vaporizer to 3%-5% to initiate anesthesia.
2. Remove the animal from the chamber once it lies on its side and can no longer stand, and place it onto a clean surface. Ensure the animal is adequately anesthetized by confirming the absence of a toe pinch reflex before and throughout the surgery.
   1. Secure the nose cone extended from the anesthetic machine on the animal and adjust isoflurane to 1%-2% for maintenance. Minimize the exposure to isoflurane (5 min at 3%-5% for induction and the remaining time at 1%-2%), as the procedure can take up to 40 min to complete.
3. Use animal hair trimmers to remove fur from the neck/behind ears to the nose of the animal to expose the skin on the skull for surgery.
4. Weigh the animal and administer the appropriate dose of pre-operative analgesic subcutaneously (Meloxicam (2.0 mg/kg)) based on the weight.
5. Apply eye lubricant ointment on each eye of the mouse to prevent corneal desiccation during anesthesia.
6. Sterilize the fur-clipped skin area (step 1.3) with 70% ethanol, followed by Chlorhexidine alcohol twice to form a surgical field.
7. Transfer the mouse onto a mouse stereotaxic frame by first placing its nose in the stereotaxic nose cone, then stabilize the skull with ear bars. Keep the mouse on a heating pad set to 37 °C for thermal support throughout the surgery procedure. Maintain isoflurane at 1%-2% so that the mouse displays a respiratory rate of 1 breath/s.
8. Repeat application of eye lubrication to prevent corneal desiccation during the procedure if needed. Apply a sterile surgical drape on the body of the mouse, below the ears, to extend the size of the sterile field.
9. Make a vertical incision parallel to the sagittal plane of the animal from behind the mid-point of the eyes to the back of the head (parietal bone) with a #10 scalpel blade. Expose the skull by gently retracting the skin towards the sides.
10. Use a Q-tip to disrupt the fascia on the skull and allow the skull to dry to expose bregma.
11. Set up manipulator arms with drill holder carrying high-speed stereotaxic drill with a .018" drill bit.
12. Drill 3 burr holes (.018" diameter) with a high-speed stereotaxic drill at the coordinates in the right sensorimotor cortex at 2.25 mm lateral to bregma and +0.0 mm rostral, +1.0 mm rostral and -1.0 mm caudal to bregma.
13. Replace the drill with a 26 G syringe with a 0.375″ long needle.
14. Load 1.5 µL of 400 µM ET-1 dissolved in sterile PBS into the syringe. To ensure good flow, release a small volume of ET-1 until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection.
15. Lower the tip of the needle past the skull in the middle burr hole (in the right sensorimotor cortex at + 0.0 anterior and 2.25 mm lateral to bregma) without puncturing the dura mater. Once aligned, lower the needle 1 mm into the brain.
16. Inject 1 µL of ET-1 by dispensing 0.1 µL at a time. Push the plunger of the Hamilton syringe forward to dispense 0.1 µL and wait for a minute before injecting the next 0.1 µL, inject until 1 µL has been dispensed.
    1. After the final 0.1 µL has been dispensed (0.1 µL x 10 injections = 1 µL total), keep the needle in place for 10 min to prevent backflow and then slowly withdraw upward to remove the syringe.
17. To ensure the needle does not become blocked during injection, release a small volume of the ET-1 until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection. Sterilize syringes before injecting the next animal with PREempt, followed by 70% alcohol, and then sterile PBS.
18. To close the wound, suture the overlying skin on the skull together using a 4-0 polysorb polyester sterile suture.
19. Administer antibiotic ointment using a cotton swab on the sutured area to prevent post-operative infection.
20. Remove the mouse from the stereotaxic frame and transfer it to a clean, pre-heated animal housing cage in the recovery area. Turn off the isoflurane vaporizer and oxygen.
21. Keep the cage on top of a heating pad for the duration of recovery from anesthesia. Alternatively, if using a heating lamp place the mouse on the side of the cage under the lamp so that the mouse can move to the cool side of the cage as it wakes up from anesthesia to prevent overheating. Monitor the mouse as it comes out of anesthesia in the next 5-10 min.
22. Monitor mouse over the next 3 days (weight monitoring), provide post-operative care (moist mashed chow to ensure hydration), and analgesics (2 mg/kg meloxicam, 2x daily subcutaneously, for 2-3 days) in accordance with regulations set out by local animal care committee.

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.

1. Before starting the surgery, set up the recovery area by placing a clean cage on a heating pad set to 37 °C. Alternatively, if a heating lamp is being used, place only half of the cage under the heat.
   1. To anesthetize mice using isoflurane, place the mouse in the anesthetic induction chamber and turn on the oxygen to 1-1.5 L/min, then adjust the isoflurane vaporizer to 3%-5% to initiate anesthesia (following the instutionally approved protocols).
2. Remove the animal from the chamber once it lies on its side and can no longer stand on a clean surface. Ensure the animal is adequately anesthetized by confirming the absence of a toe pinch reflex before and throughout the surgery.
   1. Secure the nose cone extended from the anesthetic machine on the animal and adjust isoflurane to 1%-2% for maintenance. Minimize the exposure to isoflurane (5 min at 3%-5% for induction and the remaining time at 1%-2%), as the procedure can take up to 40 min to complete.
3. Weigh the animal and administer the appropriate dose of pre-operative analgesic subcutaneously (Meloxicam (2.0 mg/kg)) based on the weight.
4. Apply eye lubricant ointment on each eye of the mouse to prevent corneal desiccation from being under anesthesia.
5. Sterilize the fur-clipped skin area with 70% ethanol, followed by Chlorhexidine alcohol twice to form a surgical field.
6. Transfer the mouse onto a mouse stereotaxic frame by first placing its nose in the stereotaxic nose cone, then stabilize the skull with ear bars. Keep the mouse on a heating pad set to 37 °C for thermal support throughout the surgery procedure. Maintain isoflurane at 1%-2% so that the mouse displays the respiratory rate of 1 breath/s.
7. Repeat application of eye lubrication to prevent corneal desiccation during the procedure if needed. Apply a sterile surgical drape on the body of the mouse, below the ears, to extend the size of sterile field.
8. If still present, cut sutures with a pair of surgical scissors and remove the scab with a pair of forceps. Retract the skin to expose the skull.
   NOTE: For the chronic model it may be necessary to excise the skin with #10 scalpel blade.
9. Use a sterile Q-tip to dry the skull and locate burr holes.
10. Set up manipulator arms of the stereotaxic apparatus with a needle holder carrying a 26 G Syringe with a 0.375″ long needle.
11. Load 3.4 µL of AAV (1 x 10⁹ Genome Copies(GC)/µL for acute study; 1 x 10¹⁰ GC/µL for chronic study) into the syringe. To ensure that the flow is good, release a small volume of AAV until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection.
12. Lower the tip of the needle past the skull in the first burr hole used for ET-1 (in the right sensorimotor cortex at + 0.0 anterior and 2.25 mm lateral to bregma) without puncturing the dura mater. Once aligned, lower the needle 1 mm into the brain. (0 AP, +2.25 ML, −1.0 DV from bregma).
13. Inject 1 µL of AAV (1 x 10⁹ GC/µL for acute study; 1 x 10¹⁰ GC/µL for chronic study) by dispensing 0.1 µL at a time. Push the plunger of the Hamilton syringe forward to dispense 0.1 µL and wait for a minute before injecting the next 0.1 µL; inject until 1 µL has been dispensed. After the final 0.1 µL has been dispensed (0.1 µL x 10 injections = 1 µL total), keep the needle in place for 10 min to prevent backflow and then slowly withdraw upward to remove the syringe.
14. To ensure the needle does not become blocked during injection, release a small volume of the AAV until a drop can be observed at the tip of the needle. Use a sterile Q-tip to wipe off the excess before performing an injection.
15. Repeat steps 2.11-2.14 for the AAV injections into the two remaining burr holes (in the right sensorimotor cortex at +1 AP, +2.25 ML, −1.0 DV; and −1 AP, +2.25 ML, −1.0 DV mm from bregma. Sterilize syringes before injecting the next animal with PREempt, followed by 70% alcohol, and finally sterile PBS.
16. To close the wound, suture the overlying skin on the skull together using a 4-0 polysorb polyester sterile suture.
17. Administer antibiotic ointment by using a cotton swab on the sutured area to prevent post-operative infection.
18. Remove the mouse from the stereotaxic frame and transfer it to a clean, pre-heated animal housing cage in the recovery area. Turn off the isoflurane vaporizer and oxygen.
19. Keep the mouse inside the cage and sterilize the outside of the cage with PREempt and 70% alcohol before placing it on top of a heating pad for recovery from anesthesia.
    1. Alternatively, if using a heating lamp, place the mouse on the side of the cage under the lamp so that the mouse can move to the cool side of the cage as it wakes up from anesthesia to prevent overheating. Monitor the mouse as it comes out of anesthesia in the next 5-10 min.
20. Monitor the mouse over the next 3 days (weight monitoring), provide post-operative care (moist mashed chow to ensure hydration), and analgesics (2 mg/kg meloxicam, 2x daily subcutaneously, for 2-3 days) in accordance with regulations set out by local animal care committee.

3. Tissue preparation and dissection

1. To euthanize mouse, intraperitoneally inject the mouse with Tribromoethanol (Avertin) (conc. 12.5 mg/mL, 250 mg/BW(kg)) and allow 2-3 min for complete induction. Ensure the animal is adequately anesthetized by removing the mouse from the cage and confirming the absence of a toe pinch reflex.
2. In a fume hood, perform transcardial perfusion²⁶ with saline followed by 4% paraformaldehyde. Spray the head of the euthanized animal with 70% ethanol until soaked.
3. Cut off the head at the base of the skull by using a pair of large scissors. Cut open the skin along the midline of the entire skull using straight 4-1/2" iris scissors. Retract the skin to expose the skull.
4. From the opening of the spinal cavity at the neck, insert the iris scissors and cut through the skull along the sagittal and inter-frontal suture.
5. Then insert scissors into the two eye sockets and cut through the frontal-nasal suture.
6. Take particular care not to damage the brain. Pry open the skull plates to the left and right until the brain is exposed.
7. Transfer the brain into a scintillation vial with 4% paraformaldehyde. Keep the brain in 4% paraformaldehyde at 4 °C for 4 h.
8. The brain is then cryoprotected by replacing 4% paraformaldehyde with 30% sucrose. Keep the brain in 30% sucrose at 4 °C until the brain sinks to the bottom of the vial (~12 h).

4. Sectioning frozen brains

1. Set the temperature of the cryostat chamber between -20 °C to -26 °C.
2. Make a coronal cut across the cerebellum and attach the flat, caudal part of the brain onto a cryostat chuck with optimal cutting temperature compound (OCT).
3. Place the chuck with the brain in the disk holder and align the olfactory bulb with the knife blade.
4. Section the brain into coronal slices with a thickness of 20 µm. To avoid streaks or scratch lines on sectioned brain slices, place a piece of tape on the sectioning platform to create a small gap between the anti-rolling plate and the platform. A paintbrush can be used to adjust the orientation of the section before capturing it on a positively charged glass slide.
5. Store slides containing brain sections at -20 °C.

5. Immunohistochemistry and quantification

1. Process the sections for immunohistochemistry using a primary antibody against NeuN, a pan-neuronal marker, according to standard protocols^22,26.
2. Thaw cryopreserved brain sections for 5 min at room temperature. Wash the sections three times with 1x phosphate-buffered saline (PBS) for 5 min each.
3. Incubate the sections in 0.3% Triton X-100 in PBS for 20 min at room temperature to permeabilize.
4. Prepare the blocking solution (5% normal goat serum and 0.3% Triton X-100 in PBS) and incubate the sections in the blocking solution for 1 h at room temperature.
5. Dilute the primary antibody, rabbit anti-NeuN, at a 1:500 ratio in the blocking solution. Incubate the sections with the primary antibody solution overnight at 4 °C.
6. Wash the sections three times with 0.3% Triton X-100 in PBS, for 5 min each wash.
7. Dilute the secondary antibody, goat anti-rabbit 568 or 488, at a 1:1000 ratio in PBS. Incubate the sections with the secondary antibody solution for 1 h at room temperature, protected from light.
8. Perform three washes with 0.3% Triton X-100 in PBS, for 5 min each. Incubate the sections with DAPI (1:10,000 in PBS) for 5 min at room temperature.
9. Wash the sections three more times with PBS, for 5 min each. Mount the stained sections on a glass slide with a fluorescence mounting medium and a glass coverslip. Allow mounted slides to dry overnight.
10. Image stained slides using a 20x objective with a confocal microscope to obtain views of Reporter+ cells around the stroke lesion in 3 different coronal sections (between the anatomical regions of the crossing of the corpus callosum anteriorly and the crossing of the anterior commissure posteriorly) for each animal.
    NOTE: The distance of the AAV spread will vary within the hemisphere. However, based on our experience and post-tissue processing analysis of Reporter+ cells, the AAV spread generally spans approximately +3 mm to -3 mm from bregma. Reporter+ cells around the stroke lesion (as identified by the absence/sparse staining of NeuN) in the area between where the corpus callosum crosses the midline and the anterior commissure connects should be quantified.
11. For quantification of transduced neurons, perform colocalization analysis. Manually count the number of double-positive cells (Reporter+NeuN+) and the total number of transduced (Reporter+) cells in brain slices. For results herein, >3 fields of view were counted surrounding the lesion (medial, lateral, and inferior) from 3 coronal sections per mouse.

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 delivered to wild-type mice at day 7 post-stroke (Figure 1B). The second Flex vector contains the inverted sequence for GFP driven by the CAG promoter that is flanked by heterotypic, antiparallel loxP-type recombination sites (Figure 1B). Both viral constructs are injected together at the same time post-stroke. Once Cre is expressed in transduced cells, the GFP is inverted and excised at recombination sites, orienting the sequence in the sense direction, which allows for the expression of GFP. Hence, cells that express Cre will be GFP positive, indicating they have been transduced by the Cre-Flex vectors. In the subacute model, mice were sacrificed at 21 days post-transduction (i.e., 28 days post-stroke), and the number of transduced cells (GFP+), as well as the number of NeuN+GFP+ (transduced cells expressing NeuN) cells, were counted in the parenchyma of the stroke injured brains. As seen in Figure 2A, neurons are lost at the site of the ischemic injury. Quantification of transduced cells in the subacute paradigm using the Cre-Flex AAV system reveals significantly more labeled neurons (NeuN+GFP+/GFP+ cells) in the Neurod1 treated mice compared to Stroke-Cre control mice (43.05 +/- 5.50% vs. 18.43 +/- 3.03% NeuN+GFP+/GFP+ cells, in Stroke-Neurod1 vs. Stroke-Cre (control) treated mice, respectively) (p = 0.0172) (Figure 2B,C).

In a separate set of experiments, AtN conversion was performed in a chronic model of stroke (Figure 1B,C). The transcription factor Neurod1 was delivered to ET-1 stroke-injured tdTom-Cre reporter mice on day 21 post-stroke (Figure 1C). Identical to the Cre-Flex system used in wild-type mice, Neurod1 expression was driven by the short GFAP promoter. However, in this system, a single AAV construct was used with Cre recombinase following Neurod1 (Figure 1B). Hence, GFAP-expressing cells will produce Cre recombinase, inducing the tdTom reporter, indicating cells that have been AAV transduced. Mice were sacrificed at 77 days post-stroke (8 weeks post-AAV) (Figure 1C). As seen in Figure 3A, a cortical lesion is observed at day 77 post-stroke in tdTom-Cre mice. The number of transduced cells expressing the neuronal marker, NeuN+, was quantified (relative percentage of (NeuN+tdTom+/tdTom+ cells) in the chronic stage post-stroke and found that mice that received Stroke-Neurod1 had significantly more labeled neurons as compared to the Stroke-Cre control mice (74.81 +/- 4.835% vs. 53.05 +/- 5.028% NeuN+tdTom+/tdTom+ cells in Stroke-Neurod1vs. Stroke-Cre (control) treated mice, respectively) (p = 0.0355) (Figure 3B,C).

Overall, the results demonstrate baseline transduction of neurons and a higher number of transduced cells expressing NeuN+ with Neurod1 delivery in both the subacute and chronic post-stroke phases in wild-type and tdTom-Cre reporter mice, with two different AAV-based systems.

Figure 1: Ectopic expression of Neurod1, vectors, and timelines. (A) Graphical representation of stroke induction by Endothelin-1 (ET-1) injection into the mouse motor cortex. Adeno-Associated Virus (AAV) delivered at 3 sites, at the ET-1 injection site as well as at sites surrounding the lesion, 1mm anterior and posterior. Zoomed-in panel showing transduced cells with AAV-Neurod1 in the motor cortex. (B) Schematic of the two AAV vectors in the Cre-Flex system delivered to wild-type mice (top panel). AAV5 vector 1 contains the short GFAP promoter, the Neurod1 transcription factor, and the Cre recombinase sequence flanked by a loxP site (black triangle) and by a lox2272 site (white triangle). AAV5 vector 2 carries the inverted GFP gene reporter sequence flanked by a pair of loxP and lox2272 sites. Transduction of both vectors in cells results in Cre-mediated excision and inversion, enabling GFP expression to label transduced cells. Neurod1 was also delivered to tdTom-Cre mice using one AAV5 vector (bottom panel). In AAV5 vector 1 the short GFAP promoter, the Neurod1 transcription factor, and the Cre recombinase sequence are packaged. Cre expression in transduced cells drives excision of the Stop codon at loxP sites (black triangle), enabling tdTomato expression. (C) The Cre-Flex system was tested in wild-type mice with AAVs injected in the subacute period post-stroke (Post-Stroke Day 7). Wild-type mice were sacrificed 21 days post-AAV transduction (28 days post-stroke). AAV was delivered to tdTom-Cre mice in the chronic period post-stroke (Post-Stroke Day 21), and mice were sacrificed 56 days post-AAV transduction (77 days post-stroke). Please click here to view a larger version of this figure.

Figure 2: Ectopic Neurod1 delivery using Cre-Flex AAVs in the subacute phase post-stroke leads to increased numbers of transduced cells expressing NeuN. (A) Immunostaining of coronal brain sections through the motor cortex at day 28 post-stroke from Stroke-Cre control (left) and Stroke-Neurod1 (right) mice. Neuronal loss is observed at the site of the lesion (white dashed lines). Transduced cells (GFP+, green), neurons (NeuN+, red), and colocalized NeuN+GFP+ (orange) cells are seen in the cortex. Scale bars: 250 µm. (B) Representative images of Stroke-Cre control (top) and Stroke-Neurod1 (bottom) brain tissue showing neurons (NeuN+, red) and AAV transduced GFP+ (green). Insets outlined in white show a close-up of cells for each individual channel. White arrowheads indicate AAV-transduced cells (GFP+). White arrows indicate colocalized NeuN+GFP+ cells. Scale bars: 125 µm. (C) Quantification of NeuN+GFP+/GFP+ cells in Stroke-Cre and Stroke-Neurod1 mice. N = 3 mice per group. Data are represented as average ± SEM. Shapiro-Wilk Test (p = 0.3223). Unpaired t-test, *p < 0.05. Please click here to view a larger version of this figure.

Figure 3: Ectopic Neurod1 delivery in tdTom-Cre reporter mice in the chronic phase post-stroke leads to increased numbers of transduced cells expressing NeuN. (A) Immunostaining of coronal brain sections through the motor cortex at day 77 post-stroke from Stroke-Cre control and Stroke-Neurod1 mice. Neuronal loss is seen at the ischemic injury site (white dashed lines). Transduced cells are labeled with tdTom (red) in the peri-infarct. NeuN+tdTom+ (orange) are transduced cells that express neuronal markers. Scale bars: 250 µm. (B) Representative images of Stroke-Cre control (top) and Stroke-Neurod1 (bottom) brain tissue showing neurons (NeuN+, green) and AAV transduced cells (tdTom+, red). Insets outlined in white show a close-up of cells for each individual channel. White arrowheads indicate AAV transduced cells (tdTom+). White arrows indicate colocalized NeuN+tdTom+ cells. Scale bars: 125 µm. (C) Quantification of NeuN+tdTom+/tdTom+ cells in the chronic paradigm shows significantly more transduced cells expressing NeuN+ (NeuN+tdTom+/tdTom+ cells) in the Neurod1 treated mice. N = 3 mice per group. Data are represented as average ± SEM. Shapiro-Wilk Test (p = 0.2044). Unpaired t-test, *p < 0.05. Please click here to view a larger version of this figure.

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 interest (sensorimotor cortex in this study). This is particularly important for studies using behavioral assessments since the lesion location and size will impact functional outcomes^13,22,25,27. Most importantly, the injection rate of ET-1 and the pressure applied by the surgeon on the syringe need to be consistent for reproducible results. Of note, the needle of the syringe can get clogged and needs to be cleaned between animals to ensure accurate ET-1 delivery. During cellular analysis of tissue, if a stroke lesion is not observed, the needle was likely clogged during surgery. If the issue persists, obtain a new lot of endothelin-1 from the manufacturer, as lot-dependent potency issues have been observed in the past.

The steps for the surgical procedure to deliver AAV are similar to that of the ET-1 stroke induction protocol. Hence, the aforementioned considerations are critical. Additionally, the AAV viral titer and volume must be consistent to ensure the transduction efficiency can be compared across groups. In this protocol, 1 x 10⁹ GC/µL of AAV for the acute study and 1 x 10¹⁰ GC/µL of AAV for the chronic study was administered. It is crucial to select a viral dose that is low enough to minimize artefactual misexpression while still being high enough to ensure sufficient cell transduction, enabling the detection of differences across experimental groups. The optimal viral dose range for such studies has been determined to be 10⁷-10¹⁰ GC/µL^13,24. Higher titers of AAV not only result in extensive labeling of neurons in the control group but have also been shown to induce astrocytosis, disrupt the blood-brain barrier, and upregulate inflammatory responses^28,29. Studies investigating viral-mediated transcription factor delivery post-stroke have used different delivery sites for AAV injections. One study described the successful labeling of transduced cells using a single AAV injection into the stroke lesion site¹³. Other studies target one to three areas around the infarct area³⁰. Herein, AAV was delivered in three locations, with one injection at the coordinates for the site of the ET-1 injection and 2 surrounding the lesion site (one injection at 1.0 mm anterior and one injection at 1.0 mm posterior) with the goal of transducing reactive astrocytes in the perilesional glial border⁹. During cellular analysis of tissue, if none or very few (1-10 cells/section) fluorescent cells are observed, the needle may have been clogged during injection, or the AAV has been freeze-thawed too many times, leading to the inactivation of the virus.

The efficiency of transduction was examined at different times in the mild-moderate stroke model, and the results demonstrate that significantly more neurons are labeled in Neurod1-treated brains at both the subacute and chronic times post-stroke. The work shown here is the first to demonstrate Neurod1 transduction in the chronic phase post-stroke and reveals neurons ectopically expressing Neurod1 as long as 8 weeks post-stroke. While the findings that increased numbers of transduced neurons are seen in stroke-injured brains that received Neurod1 versus control vectors are consistent with Neurod1 playing a role in AtN conversion, interpreting the cellular outcomes requires a sound knowledge of the limitations of the experimental paradigm. The vast majority of published studies investigating AtN conversion utilize GFAP as the promoter as GFAP is upregulated in reactive astrocytes in neurodegenerative diseases, including stroke^4,31. However, it has been shown that low GFAP expression also occurs in neurons³². This is consistent with the findings reported here wherein a percentage of transduced cells in the control group (Stroke-Cre) are pre-existing neurons³². Specifically, the Stroke-Cre control groups in this protocol showed 18.43 +/- 3.03% and 53.05 +/- 5.028% labeling of NeuN+Reporter+ cells, which represents the labeling of pre-existing neurons, indicating GFAP promoter-driven expression in neurons. Indeed, targeted expression of Neurod1 depends on a number of factors, including viral tropism. AAV5 has been shown to transduce neurons with different efficiency in different brain regions, including the sensorimotor cortex, indicating that AAV serotypes have regional cell tropism, which needs to be considered in studies inducing ectopic expression of transcription factors^31,33. Mouse strains can also affect AAV transduction^14,34. A study comparing AAV transduction in the striatum of wild-type (C57BL/6J) and FVB/N mice found variable cell tropism and transduction efficiency in the two strains³⁴.

An advantage of the Cre-Flex system is that it can be used for long-term labeling in wild-type mice, which may reduce the issue of non-specific Cre expression present in Cre-reporter mouse lines^15,35. A disadvantage of the Cre-Flex system is that Cre-independent transgene expression may occur due to 0.1%-0.8% of viral particles having recombined LoxP sites during viral production³⁶. These findings highlight the importance of lineage tracing experiments, for example, pre-labeling astrocytes before AAV administration using the Aldh1l1-CreERT2 mouse line, to ensure that AtN conversion is not overestimated when reporting conclusions¹⁵. Studies have suggested that activation of the human GFAP promoter (as used in these studies) through cis-regulation by downstream genes (such as Neurod1) may result in GFAP expression in endogenous neurons, leading to a higher number of labeled neurons in the treatment group^15,32. Together, these studies and the findings herein highlight the importance of lineage tracing experiments, for example, pre-labeling astrocytes or neurons before AAV administration, to differentiate between pre-existing and endogenous neurons during analysis¹⁵.

In considering the next steps that could reduce neuronal transduction, different AAV serotypes and/or the use of a less promiscuous astrocyte-specific promoter would be useful¹⁹. Increasing numbers of reports describing astrocyte heterogeneity may further impact gene expression specificity if different subpopulations of astrocytes are more susceptible to AtN conversion in general^37,38,39,40.

The age of animals used in pre-clinical models of injury or disease is also an important consideration that may introduce variable outcomes. For example, in the case of neurotrauma, age can affect astrocyte reactivity, which would impact study outcomes for AtN conversion⁴¹. In addition, Cre-inducible reporter lines can lose expression specificity over time, which can impact outcome measures⁴². To date, reprogramming in older animals has not been comprehensively investigated⁴³.

The protocol describes AAV delivery in 2 phases post-stroke: (1) the subacute phase (at 7 days) and (2) the chronic phase (at 21 days) post-ischemic stroke. Ectopic expression of Neurod1 in a chronic stroke model has not been examined; hence, the observation that significantly more neurons are labeled at this late stage has important implications for future studies aimed at promoting stroke recovery in chronic stroke. Studies examining the potential for ectopic expression of Neurod1 to enhance stroke outcomes will need to incorporate behavioral assessments, as functional outcomes following stroke are the most clinically relevant measures of success for a therapeutic intervention^13,22,25,27.

There are currently no treatments available for patients suffering from stroke-induced disabilities in the subacute and chronic phases post-stroke with the exception of rehabilitation, which has limited benefit. Forced expression of neurogenic bHLH genes in glia has been shown to improve functional outcomes post-stroke^22,30. The findings are consistent with AtN reprogramming studies demonstrating that the ectopic expression of Neurod1 increases neuronal transduction at 3 and 7 weeks post-AAV delivery compared to mice receiving the control vector. Most importantly, several articles have outlined recommendations for study controls, design, and interpretations in the AtN reprogramming field^17,18,19,22. To this end, this protocol has described AAV systems and mouse models for ectopic expression of transcription factors following stroke, which can be used to inform and advance further investigations into the cellular mechanisms underlying the observations.

Materials

List of materials used in this article

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