Key words

1 Introduction

1.1 Stroke and Gene Therapy

Stroke is the leading cause of death in China and the United States. Stroke is a multigenic complex disorder; it can be classified into two major categories, hemorrhagic stroke and ischemic stroke. Of the two types, ischemic stroke results from vascular occlusion and accounts for about 80% of all stroke (1). Stroke induces rapid neuronal death and loss of brain function due to the lack of oxygen and energy. Currently, apart from t-PA, the only medication approved by FDA, there are no other effective medications for the treatment of ischemic stroke.

Focal cerebral ischemia-injured brain tissue can be divided into three zones: ischemic core, ischemic penumbra, and extra penumbral cortical zone. Since neuronal injury in the ischemic penumbra may be reversible, inducing angiogenesis and neurogenesis in this zone may provide a unique approach in rescuing ischemic brain injury (2). The gene therapy technique in cerebral ischemic stroke was first applied by Betz’s group in 1995 and showed exciting effect (3). Since then, different genes have been delivered through gene therapy in experimental studies with positive results. Hence, further study to improve transduction efficiency is necessary. There are mainly two types of experimental animal models in ischemic stroke research including focal (4–7) and global ischemic stroke (8–10). They can further be divided into transient or permanent ischemia based on whether reperfusion is followed. Focal ischemia, especially middle cerebral artery occlusion (MCAO) in rodents is widely used as an experimental focal cerebral ischemia model because this model mimics acute focal ischemia in humans.

Several gene therapy strategies have been introduced to rescue the damaged brain after ischemic stroke such as providing neuroprotection, promoting neurogenesis and angiogenesis, anti-apoptosis, and anti-inflammation. Growth factors are the most selected genes to be transduced in the ischemic brain because they are capable of inducing neurogenesis and angiogenesis in the brain. Such growth factors include vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), fibroblast growth factor -2 (FGF-2), and epidermal growth factor (EGF). Among them, the role of BDNF and GDNF is of special interest because both exert potent neurotrophic effects.

Angiogenesis is a process involving the growth of neo-microvessels from pre-existing vessels. Several factors have been demonstrated to activate angiogenesis such as VEGF, PDGF, and TGF- β. It is noteworthy that some factors are not only simply angiogenic but also play roles in promoting neurogenesis. The shortage of oxygen and nutrients supply is followed by neuronal death. The increase of ROS and breakdown of cytoskeleton further induces apoptosis and necrosis. Gene delivery of B-cell lymphoma 2(Bcl-2) and heat shock protein (HSP) may attenuate neuronal death (11).

Inflammatory factors play important roles in ischemic-induced brain injury (12). When stroke occurs, various cells such as leukocytes, microglia/macrophages, and lymphocytes are able to migrate to the damaged site and release a variety of inflammatory mediators such as cytokines (Interleukin-1/IL1, IL-6, IL-8, and TNF-α), chemokines (monocyte chemotactic protein-1/MCP-1 and macrophage inflammatory protein-1/MIP-1), and adhesion molecules (intracellular adhesion molecule-1/ICAM-1 and platelet endothelial cell adhesion molecule/PECAM-1). Acute inflammatory response induces further brain tissue damage. Therefore, in vivo introduction of the antagonist of those factors through gene transfer will provide appropriated approaches to protect tissue from further injury. Recently, a number of experiments using anti-inflammatory factor such as IL-1 receptor antagonist (IL-1ra) or matrix metalloproteinase (MMP) inhibitors have been studied in ischemic animal models; the results are encouraging (13, 14).

Currently, several novel strategies have been developed in the stroke treatment using gene delivery. One of them is the delivery of microRNAs (miRNAs). miRNAs are small, endogenous, conserved, and non-coding RNAs that modulate gene expression by either promoting the degradation of mRNA or down regulating the protein production through translational repression. miRNAs are pervasive in human and animals. More than 400 miRNAs have been identified in human. Estimated 1000 miRNA genes have been predicted by computational programs (15). Discovery of miRNAs in brain tissue provides a glimpse of undiscovered regulatory mechanisms underlying cerebrovascular diseases. Current study demonstrates that miRNA can be delivered via gene transfer approach (16). Key technical aspects such as optimization of selectivity, stability, in vivo delivery, efficacy, and safety need to be extensively investigated before RNA silencing can become a successful therapeutic strategy (17, 18).

1.2 Vector Types and Delivery

In general, vectors can be divided into three groups: viral, non-viral, and cellular forms. Viral vectors include adenovirus (Ad), adeno-associated virus (AAV), herpes simplex virus (HSV), Sendai virus, hemagglutinating virus of Japan (HVJ), lentivirus, etc. (8, 19–22). Ad, AAV and HSV are the most widely used vectors in brain tissue since they are more efficient, safer, and relatively easy to be delivered. Multiple brain cell types including neurons, glial cells, vascular smooth muscle cells, and endothelial cells can produce target genes via recombinant Ad, AAV, or HSV systems. However, viral vectors have immunogenic toxicities, which should be overcome before they can be clinically applied (23).

Employing cellular vectors is a novel approach to circumvent immune response to viral vectors. Potential cellular vehicles may be used include fibroblast cells, mesencyhmal stem cells (MSCs), endothelial progenitor cells (EPCs), and astrocytes. This approach presents the potential benefit because these cells may be acquired from the patient’s own body, which potentially reduces the incidence of immuno-rejection. For example, ex vivo gene delivery of NGF in fibroblasts has exhibited success in Alzheimer’s disease and animal ischemic stroke models (24, 25). Another benefit of cellular vectors is the potential use of stem cells. Circulating EPCs could be attracted by SDF-1, a chemokine which is released by injured brain cells. Neuro-progenitor cells (NPCs) could migrate from the subventricular zone (SVZ) to the ischemic brain region. EPCs and NPCs have been shown to play an important role in the remodeling, repairing, and regeneration of brain function following ischemic brain injury (26, 27).

Non-viral vectors have lower transfection efficiency but safer compared to their viral counterparts (28). Modes for non-viral gene delivery include ligand–DNA conjugate system, cationic conjugate, direct injection of naked DNA, and calcium phosphate precipitation. Many of these methods demonstrate promises for future gene therapy studies (29–32). The use of DNA-cationic liposome complexes in a gene therapy study for ischemic stroke has afforded successful results (31). Another possibility is to inject naked DNA directly into the tissue. Introducing naked DNA of hypoxia inducible factor-1 alpha (HIF-1) into cerebral ischemia model was reported to promote angiogenesis in the target tissue (29).

The available routes of administration to stroke animal model include injection into cerebrospinal fluid parenchyma, and cerebral vasculature. An alternative route is to inject a viral vector at a peripheral site that has the potential to retroactively transport into the central nervous system (33). Direct introduction of vectors into the cerebrospinal fluid (CSF), within either the ventricular or perivascular system, has been utilized in rodent stroke models (32, 34, 35). Intravascular injection is an optional approach to different neurological disease processes and cerebrovascular diseases (4, 36, 37). However, the blood-brain barrier (BBB) is an obstacle to the delivery of vectors through intravascular route. Hypertonic sugar solutions such as mannitol may induce the transient osmotic opening of the BBB, and allow vectors to travel through the BBB and reach the target regions (38). Injection of Ad or AAV vectors into the perivascular system is often used to deliver into the cortex and basal ganglia. This route is especially important in cerebral vasospasm model (39–41).

The viral vectors mostly infect epidermal cells, choroids plexus, and superficial cell layers of the ventricular wall after intraventricular injection (3, 34, 42, 43). Administration of vector containing fibroblast growth factor-2 (FGF-2) improved neurological function in a mouse transient MCAO model, despite the fact that infected cells were limited to the ependymal cell layer (34). Although the brain tissue that is around 2 mm away from the injecting point is believed to not been transfected, therapeutic gene products, especially cytokines and chemokines, could be released into the cerebral spinal fluid and may defuse appropriately to deeper locations in the brain (3).

1.3 VEGF and AAV

VEGF, a 34- to 45-kDa dimeric glycosylated protein, is usually upregulated in ischemic tissues. VEGF is highly expressed in brain tissue and especially in the penumbra after cerebral ischemia (44). VEGF is expressed in different brain cells including endothelial cells, smooth muscle cells, astrocytes, and neurons (19). VEGF is explored as a therapeutic reagent in cerebral ischemia models because it not only induces cerebral angiogenesis but also enhances neuroprotection and promotes neurogenesis following cerebral ischemia. Pretreatment with VEGF through recombinant AAV-mediated gene transfer reduces infarct volume in mouse MCAO model (20). Intraventricular administration of VEGF activates angiogenesis in the ischemic penumbra and increases neuron survival, subsequently reducing infarct volume and improving neurological performance (20). Therefore, VEGF exerts its neuroprotective effect in the acute phase of cerebral ischemia with the longer latency effects on angiogenesis and on neo-neuron survival (45).

Adeno-associated virus (AAV) is a small single-stranded DNA virus, which infects human and primates. AAV based vector system is increasingly considered as a promising gene transfer vehicle because of its many advantages over adenovirus, retrovirus, or lentivirus. First, AAV is nonpathogenic and elicits no or less inflammatory response than other viral vectors (46). Second, recombinant AAV (rAAV) can infect both dividing and non-dividing cells and mediate lone-term gene expression up to months or several years in vivo (47–51). Third, rAAV probably maintains in vivo mostly as episomal genomes extrachromosomally and occasionally as integrated concatemers, which diminishes the insertional mutagenesis associated with random integration (52). Many kinds of serotypes of AAV have been characterized and different types show distinct tissue and cellular tropism transduction. AAV-2 and AAV-5 were always used in gene transduction into central nervous system (CNS), while current report demonstrates rAAV-1 also had much higher transduction efficiency in CNS (53). Distribution of rAAV1 transduced cells in the striatum is much wider than that of rAAV-2 (54).

Since uncontrolled VEGF expression can cause unwanted side effects such as hemangioma formation and synovium, it is important to control the expression of VEGF protein (55, 56). Examples of inducible promoters that have been used to control gene expression include the tetracycline operons, RU 486, edyasone, and other inducible systems (56–64). For the treatment of ischemic tissue, an ideal control would be for the expression of angiogenic factors to respond to hypoxia. Hypoxia inducible protein-1 (HIF-1) is a protein that accumulates in tissues under hypoxic condition. HIF-1α is greatly increased in ischemic myocardium from 48 h after left anterior descending coronary artery (LAD) occlusion (65). A method has been developed to achieve hypoxia-induced VEGF gene expression in vitro and in ischemic heart in vivo by controlling AAV-mediated VEGF expression using nine copies of hypoxia response elements (HRE) isolated from Epo gene enhancer and a minimum SV40 promoter (65). The AAVH9VEGF gene has been successfully transduced in the ischemic mouse brain and was shown to increase VEGF expression and consequently, reduce brain injury and promote local angiogenesis.

Although there is not yet experiment performed on human after stroke, AAV vectors has been used in treating Parkinson’s disease (66). AAV-GAD was injected subthalamically into 11 men and 1 woman with Parkinson’s disease. The results are encouraging; all patients improved their movement function. This is a promising start for using AAV vectors in gene therapy.

In the following paragraphs, we describe the delivery of VEGF through AAV-mediated pathway in detail.

2 Materials

2.1 AAV Construction, Production, and Purification

  1. 1.

    AAV Helper-Free Gene Delivery and Expression System (Strategene Inc., La Jolla, CA)

  2. 2.

    Restriction enzymes and T4 DNA ligase

  3. 3.

    Agarose gel equipment

  4. 4.

    Competent cells: E. coli strains XL1 blue, DH5γ

  5. 5.

    Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose and L-glutamine (Cellgro, Herndon, VA)

  6. 6.

    Fetal bovine serum (FBS) ( Hyclone, Logan, UT)

  7. 7.

    HEK 293 cells (American Type Culture Collection, Gaithersburg, MD)

  8. 8.

    2× HBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4·7H2O, 12 mM dextrose, and 50 mM HEPES)

  9. 9.

    Dialysis cassettes (Slide-A-Lyzer 7 K, Pierce, Rockford, IL)

  10. 10.

    High speed centrifuge and ultra speed centrifuge

  11. 11.

    Cell culture room equipped with incubators and biological hood

  12. 12.

    Quantikine human VEGF ELISA kit (R&D Systems, Minneapolis, MN)

  13. 13.

    2× SSC (300 mM NaCl, 30 mM sodium citrate, pH 7)

2.2 Middle Cerebral Artery Occlusion (MCAO) Model and Viral Vector Injection

The optimal small animal surgery room needs to be kept quiet and pathogen-free with the room temperature maintained at 25°C. The following items are recommended for small animal surgery.

  1. 1.

    Operation microscope (M651, Leica, Germany)

  2. 2.

    Bipolar coagulator (Malis™ Precision-Control bipolar coagulator, CMC™-II-PC, Codman and Shurtleff Inc., Randolph, MA)

  3. 3.

    Exercise Physiological System (AD Instruments, PowerLab/4SP, Castle Hill, Australia)

  4. 4.

    Temperature controller (Homeothermic Blanket Control Unit, Harvard Apparatus, Cambridge, MA)

  5. 5.

    Laser Doppler Flowmeter (VMS-LDF2, Moor Instrument Ltd., Millwey,UK)

  6. 6.

    Dry sterilizer (Simon Keller AG, Fine Science Tools, CH 3400, Foster City, CA)

  7. 7.

    Vaporizer (Isoflurane Matrx, Midmark International, Orchard Park, NY)

  8. 8.

    Stereotaxic frame (World Precision Instruments Inc., Sarasota, FL)

  9. 9.

    pH/blood gas analyzer (Bayer, Radiolab 248, Tarrytown, NY)

  10. 10.

    High-speed micro drill (Fine Science Tools, Foster City, CA)

  11. 11.

    Surgical equipment kit (nylon suture, bipolar forceps, microscissors for small vessels, surgical scissors for animal skin and tissue use, microforceps, needle holder, skin hook for the exposure, ruler)

  12. 12.

    Hamilton syringe with replaceable beveled needle (WPI, Sarasota, FL)

2.3 Histology and Immunohistochemistry

2.3.1 X-Gal Staining

  1. 1.

    2% paraformaldehyde in 0.1 M PIPES, pH 6.9

  2. 2.

    0.5% glutaraldehyde

  3. 3.

    X-gal staining solution 1 (5 mM K3[Fe (CN)6], 5 mM K4[Fe (CN)6], 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40)

  4. 4.

    X-gal staining solution 2 (1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactoside in PBS)

2.3.2 Cresyl Violet Staining

  1. 1.

    0.1% cresyl violet acetate (Sigma, St. Louis, MO)

  2. 2.

    Distilled water

  3. 3.

    10% acetic acid (Sigma, St. Louis, MO)

2.3.3 Lectin Staining for Capillary Density

  1. 1.

    Luorescein-lycopersicin esculentum lectin (Vector Lab, Burlingame, CA)

2.3.4 Fluoro-Jade B Staining

  1. 1.

    0.06% potassium permanganate (KMnO4) (Sigma, St. Louis, MO).

  2. 2.

    A 0.001% Fluoro-Jade staining solution (Chemicon, Temecula, CA).

  3. 3.

    0.1% acetic acid (Sigma, St. Louis, MO).

3 Methods

3.1 AAV Vector Construction

Plasmid AAV-LacZ is included in the AAV Helper-Free Gene Delivery and Expression System (Strategen Inc.). The pCMV-VEGF vector can be generated by inserting the human VEGF165 cDNA to multiple cloning sites in pCMV-MCS vector. The expression cassette, including CMV promoter, VEGF cDNA and HGH polyadenylation(poly A) signal, can be isolated from pCMV-VEGF vector by Not I digestion. pAAV-VEGF vector can be generated by replacing the LacZ expression cassette with VEGF expression cassette. VEGF expression mediated by pAAV-VEGF can be confirmed by infecting 293 cells and analyzing VEGF concentration in supernatant using a Quantikine human VEGF ELISA kit (R&D Systems).

3.2 AAV Virus Production

3.2.1 Transfection

  1. 1.

    First, 4.5 × 106 HEK 293 cells are seeded in 15-cm tissue culture dishes 2 days before transfection. Cells in one 90% confluent dish can be passed to eight dishes. The cells are maintained in 25 ml DMEM with 10% fetal bovine serum (FBS), and 25 mM HEPES.

  2. 2.

    The AAV vector containing the gene of interest is co-transfected with pAAV-RC and pHelper vectors into 293 cells by using the calcium phosphate precipitation method. pAAV-RC contains AAV rep and cap genes (see Note 1). pHelper has the adenoviral VA, E2A, and E4 regions that mediate AAV vector replication. To transfect one 15-cm dish, a total of 50 μg DNA (17 μg DNA of each plasmid) is mixed with 1 ml of 300 mM CaCl2. A mixture for transfection of four dishes can be prepared in one 50-ml Corning tube.

  3. 3.

    A sterile pipet is placed into the tube. Air is gently bubbled while 1 ml (4 ml for four plates) of 2× HBS is added to the tube drop by drop.

  4. 4.

    The transfection mix is distributed into each plate.

  5. 5.

    The plates are incubated in 37°C for 6 h.

  6. 6.

    The medium is changed to DMEM containing 2% FBS and 25 mM HEPES. Cells are cultured at 37°C for 54 h.

3.2.2 Purification ( see Note 2)

  1. 1.

    Cells are dislodged from the dishes by gentle pipeting and transferred into 50-ml Corning tubes.

  2. 2.

    Media is removed by centrifugation (1000×g for 5 min at 4°C).

  3. 3.

    Cells are resuspended in 100 mM Tris–HCl, 150 mM NaCl, pH 8.0 (1 × 107 cells/ml) and lysed by three freeze and thaw cycles (alternating between dry ice-ethanol and 37°C water baths).

  4. 4.

    The cell lysate is centrifuged at 10,000×g for 15 min to remove the cell debris.

  5. 5.

    The cleared supernatant is precipitated with 25 mM CaCl2 at 0°C for 1 h.

  6. 6.

    Precipitate is removed by centrifugation (10,000×g, for 15 min at 4°C).

  7. 7.

    NaCl and PEG (8000) are added into the supernatant to make a final concentration of 620 mM NaCl and 8% PEG.

  8. 8.

    The supernatant is incubated on ice for 3 h.

  9. 9.

    AAV vector containing precipitate is collected by centrifugation (300×g, for 30 min at 4°C) and resuspended in 5 ml of 50 mM HEPES, 150 mM NaCl, 25 mM EDTA, pH 8.0 (Adjust pH with NaOH).

  10. 10.

    Insoluble material is removed by centrifugation (10,000×g for 15 min at 4°C).

  11. 11.

    The AAV vector is purified by CsCl2 gradient centrifugation. Solid CsCl2 is added to the supernatant to produce a density of 1.4 g/ml. Samples are centrifuged at 15°C for 16 h at 22,300×g.

  12. 12.

    The gradient is fractionated (0.5–1 ml/fraction) and assayed by dot blot to detect the viral particles (see Section 3.2.3).

  13. 13.

    The AAV vector-containing fractions are pooled, put in dialysis cassettes (Slide-A-Lyzer 7 K, Pierce) and dialyzed against 1 l buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, 0.1% Tween-80, 5% Sorbital three times at 4°C. The dialysis buffer is changed every 2 h.

3.2.3 Titer Determination

Viral titers are determined by dot blot analysis of the DNA content (see Note 3).

  1. 1.

    First, 2 μl of each fraction collected after CsCl2 centrifugation or dialyzed viral stock are added to 20 μl of 500 mM NaOH and dotted on Hybond H+ membrane (Strategene).

  2. 2.

    Then 1 ng of gene fragment (about 1 kb) is serially diluted and dotted on the same membrane as standards.

  3. 3.

    The membrane is air-dried and neutralized in 500 mM Sodium phosphate buffer, pH 7.4, for 10 min, washed in 2× SSC briefly, and air-dried.

  4. 4.

    The membrane is hybridized with isotope or digoxin-labeled (Roche Diagnostics, Indianapolis, IN) probes for genes in the AAV vectors using a standard protocol.

  5. 5.

    The density of each fraction or viral stock is compared with that of the standards. The relative copy number can be calculated. Copies of a 1-ng gene fragment can be calculated as follows (copies/ml): (1×10–9/length of the fragment (bp)×660)×6×1023×2×103.

3.2.4 Toxicity

The viral stocks’ toxicity to cells is checked by infection of 293 cells.

  1. 1.

    293 cells are seeded in 48-well tissue culture dishes, 1 × 105 cells/well. Cells are cultured in 0.5 ml DMEM with 10% FBS for 24 h at 37°C.

  2. 2.

    Then 50 μl of viral stock is added to each well and cultured with the cells for 24 h.

  3. 3.

    The medium is changed after 24 h of incubation.

  4. 4.

    The status of the infected cells is monitored every day for 3–5 days and compared with uninfected control cells. The viral stock will be considered toxic if cell death or slower growth is observed. More dialysis steps can be used to reduce toxicity, if a viral stock is found to be toxic to cells.

3.3 Animal Procedure

3.3.1 Rat Middle Cerebral Artery Occlusion (see Note 4 for Mouse Model)

  1. 1.

    Adult rats are anesthetized using 1.5–2.0% isoflurane inhalation.

  2. 2.

    A PE-50 catheter is inserted into the femoral artery for continuous monitoring of arterial blood pressure, and sampling for analysis of blood gases and blood pH.

  3. 3.

    The temperature of both the temporal muscle and the rectum is measured by thermocouples (E type, OMEGA Engineering, Inc.). The body temperature of the animal is kept in normal physiological range with a feedback-enabled heating unit.

  4. 4.

    Drill a burr hole in the pericranium 3.5 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture (keep the inner layer intact).

  5. 5.

    Measure baseline of cerebral blood flow using a Laser Doppler Flowmeter; monitor and record surface blood flow before, during, and after occlusion.

  6. 6.

    Make a midline incision on the front of the neck, and expose left common carotid artery (CCA).

  7. 7.

    Isolate left internal carotid artery (ICA) and coagulate other small branches.

  8. 8.

    Ligate the pterygopalatine artery from the bifurcation of ICA and the pterygopalatine artery.

  9. 9.

    A 3-cm length of 3-0 nylon suture with a slightly enlarged and rounded tip is inserted into the transected lumen of the external carotid artery (ECA) and gently advanced from the ICA across to the opening of the MCA. The distance from the tip of the suture to the bifurcation of CCA is 17–19 mm.

  10. 10.

    Measure surface blood flow to make sure the blood flow drop to below 20% of baseline.

  11. 11.

    For reperfusion, the inserted suture is withdrawn into the ECA to restore ICA-MCA blood flow.

  12. 12.

    The skin is sutured and the animal is returned to the cage when it recovers from the anesthesia.

3.3.2 AAV-Mediated Gene Transfer (see Note 5 for Mouse Model)

  1. 1.

    Rat is anesthetized with 50 mg/kg ketamine and 10 mg/kg xylazine (Sigma, San Louis, MO) body weight intraperitoneally.

  2. 2.

    Rat is placed in a stereotactic frame with a mouth holder (David Kopf Instruments, Tujunga, CA).

  3. 3.

    Cut a skin incision in the midline of skull, drill a burr hole in the pericranium 3.5 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture.

  4. 4.

    A 50-μl Hamilton syringe is stereotactically inserted 5.5 mm into the lateral caudate.

  5. 5.

    Ten μl AAV viral suspension containing 2 × 1010 genome copies of virus is injected into the right caudate putamen at a rate of 0.2 μl/min (13). The volume and concentration can be adjusted based on different purpose. The needle is withdrawn after 15 min of injection.

  6. 6.

    Seal the bone hole and the suture the wound.

  7. 7.

    Rat is returned to their home cages after it recovers from anesthesia.

3.4 Histology and Immunohistochemistry

3.4.1 X-Gal Staining

  1. 1.

    The brains are cut into 20-μm coronal sections and fixed in 0.5% glutaraldehyde for 10 min.

  2. 2.

    The sections are incubated in X-gal staining solution for 2 h and photographed.

  3. 3.

    The transduction volumes are calculated by multiplying the transduction areas by the thickness of the sections using NIH Image 1.63 software.

3.4.2 Cresyl Violet Staining for Infarct Volume

  1. 1.

    Brain sections are incubated in xylene for 3 min.

  2. 2.

    The sections are incubated in 100, 95, 90, and 70% ethanol for 3 min each and washed in distilled water for 3 min.

  3. 3.

    Sections are incubated in cresyl violet solution for 5–10 min depending on the freshness of the staining solution.

  4. 4.

    Sections are differentiated by incubation in 70, 90, and 100% ethanol sequentially, 2 × 3 min each. Incubation time can vary slightly according to degree of staining and thickness of the sections.

  5. 5.

    Sections are then incubated in xylene for 2 × 5 min.

  6. 6.

    Sections are mounted with Permount.

  7. 7.

    Color assignment: nissl granules: violet; nuclei: pale violet; and background: colorless.

3.4.3 Fluoro-Jade B Staining

  1. 1.

    The frozen sections are fixed with 2% paraformaldehyde in 0.1 M PIPES, pH 6.9, for 20 min.

  2. 2.

    After being washed, the sections are stained with 0.06% potassium permanganate (KMnO4) for 30 min at room temperature.

  3. 3.

    The sections are immersed a 0.001% fluoro-Jade B staining solution (Chemicon, Temecula, CA) in 0.1% acetic acid for 20 min.

  4. 4.

    The staining sections are evaluated using a fluorescence microscope.

3.4.4 Determination of Capillary Density

  1. 1.

    Five minutes before sacrificing the animal, 100 μl of fluorescein-lycopersicon esculentum lectin (100 μg, Vector Labs, CA) is injected intravenously (19, 67).

  2. 2.

    After the animals are sacrificed, their brains are removed, and 20-μm-thick frozen sections are fixed with 100% ethanol at 20°C for 20 min.

  3. 3.

    Sections are incubated with 2 g/ml fluorescein-lycopersicin esculentum lectin (Vector Lab, Burlingame, CA) at 4°C overnight.

  4. 4.

    Two coronal sections from the lectin staining brain, 1 mm anterior and 1 mm posterior from the needle track, are chosen for capillary density analysis. The microvessel density is quantified by counting the total number of three areas of microscopic fields per tissue section immediately adjacent (left, right, and bottom) to the needle track.

  5. 5.

    As a surrogate of vessel counting, vessel density was determined by lectin optical density measurements using NIH Image 1.63 software.

  6. 6.

    The procedure needs to be analyzed blindly.

4 Notes

  1. 1.

    The AAV Helper-Free Gene Delivery and Expression System was generated based on AAV serotype 2 virus. Many new AAV serotypes have been cloned in recent years (68–73). Recombinant cross-packaging of the AAV genome of one serotype into capsids of other AAV serotypes to achieve optimal tissue-specific gene transduction is now possible. Studies by our group and others have shown that AAV serotype 1 results in more efficient transduction of genes in the murine and human adult heart compared with serotypes 2, 3, 4, and 5. AAV serotype 1 also mediated more efficient gene transduction in the brain the AAV serotype 2 (74, 75). The AAV packaged in serotypes other than 2 can be made by replacing the serotype 2 CAP sequence in pAAV-RC with the CAP sequence of the corresponding serotype.

  2. 2.

    AAV titer can also be determined by quantitative PCR assay (76). Infectious particles can be determined by using serial diluted viral stocks to infect 293 or other cells and counting the infected cells or transgenic expressions. DNase I digestion can be used to eliminate unpackaged DNA in viral stocks (77).

  3. 3.

    AAV can be purified by using other methods, such as nonionic iodixanol gradients, ion exchange, or heparin affinity chromatography by either conventional or high-performance liquid chromatography columns (77, 78). Ion exchange or heparin affinity chromatography can also be used in combination with CsCl2 gradients or nonionic iodixanol gradients.

  4. 4.

    The method for mouse MCAO is similar to the rat model described above. The only difference is the suture size (2-cm length of 5-0 nylon). The distance from the tip of the suture to the bifurcation of CCA is 10–11 mm.

  5. 5.

    The method for mouse AAV-mediated gene transfer is similar to the rat model described above. The differences are (1) the bone hole location is pericranium 2 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture, and (2) the total inject volume of AAV vector is 2 µl.