Introduction

Ischemic brain injury often results in complex pathophysiological events including excitotoxicity, peri-infarct depolarizations, inflammation, and programmed cell death, ultimately leading to the detrimental loss of neurons. In transient global ischemia the delayed neuronal death has been observed in selectively vulnerable brain regions such as the hippocampal CA1 region and caudate putamen ¹. During reduced cerebral blood flow, a mismatch between energy requirements and availability lead to excessive release of the amino acid neurotransmitter glutamate into the extracellular space. Excessive activation of glutamate receptors such as N-methyl-D-aspartate (NMDA) and AMPA/kainate receptors, which are expressed on CNS neurons, leads to massive influx of Na⁺ and Ca²⁺ and triggers cell death ^2,3. This sequelae of events that ultimately lead to neuronal loss in the vulnerable brain regions is termed excitotoxicity.

An increased understanding of the mechanisms underlying the pathophysiology of cerebral ischemia has enabled therapeutic interventions at certain points in the death cascade to prevent further cellular death, thereby ameliorating recovery after ischemia. One approach has been to use gene therapy to deliver therapeutic molecules that are neuroprotective to target brain areas. Gene therapy strategies have included the use of adenoviral vectors to express glial-derived neurotrophic factor (GDNF) ^4,5, neuronal apoptosis inhibitory protein ⁶, intercellular adhesion molecule 1 ⁷, or SAG (sensitive to apoptosis gene), an antioxidant protein ⁸. Other examples of viral vector approaches include the use of adenoviral-associated viral vectors to express Bcl-2 ⁹ or GDNF ¹⁰ or herpes simplex vectors overexpressing Bcl-2 ^11,12, heat shock protein 72 ¹³, or a rat brain glucose transporter, Glut-1 ¹⁴.

To date there have been no reports on the use of lentiviral vectors to deliver neuroprotective genes to the brain in an excitotoxicity model. Lentiviral vectors can efficiently transduce both dividing and nondividing cells, including postmitotic neurons, and mediate long-term and stable expression of the therapeutic protein in transduced cells with minimal inflammatory and immunological responses ¹⁵. In particular, minimal lentiviral vectors based on the equine infectious anemia virus (EIAV) have been used successfully in several neurodegenerative disease models such as functional recovery in rat models of Parkinson disease ¹⁶ and diabetes insipidus ¹⁷. The low immunogenicity of EIAV vectors makes them ideal candidates as gene delivery tools in ischemic models as inflammation often exacerbates neuronal damage in cerebral ischemia.

In this study we sought to utilize these EIAV-based lentiviral vectors to deliver neuroprotective molecules in an in vivo excitotoxicity model mimicking events that occur after cerebral ischemia. Activation of glutamate receptors, through the failure of ion homeostasis and an increase in Ca²⁺ concentration, is a major factor involved in initiating ischemic cell death. Therefore by direct injection of ionotropic glutamatergic agonists such as NMDA into the hippocampus we could induce neuronal degeneration that was reminiscent of that following cerebral ischemia. The CA1 region of the hippocampus is particularly rich in NMDA receptors ¹⁸, and consequently the CA1 is especially vulnerable to glutamate excitotoxicity after ischemic injury. Here we show that the delivery of Bcl-2- or GDNF-expressing EIAV vectors protected cultured hippocampal neurons from glutamate-induced cell death. Furthermore Bcl-2 or GDNF expression mediated by EIAV vectors in the hippocampus rescued neurons from NMDA-induced excitotoxicity and promoted cell survival. These data suggest that EIAV-based lentiviral vectors can be utilized for gene therapy in cerebral ischemia.

Results

Neuroprotection by Bcl-2 or GDNF in Hippocampal Cultures

We transduced E18 rat hippocampal cultures with lentiviral vectors expressing -galactosidase (EIAV-LacZ), Bcl-2 (EIAV-Bcl-2), or GDNF (EIAV-GDNF) at a multiplicity of infection (m.o.i.) of 10 at 1 day in vitro. Incubation of the respective vectors with the rat hippocampal neurons for 5 days led to transgene expression in 50% of the total population of cells (Figs. 1A–1C). Most of the transduced cells were colocalized with the neuronal marker MAP-2 (Figs. 1A–1C). Counting of the doubly labeled cells indicated that 70–80% of cells that were transduced by EIAV-LacZ, EIAV-Bcl-2, or EIAV-GDNF were neurons, while 15–20% of transduced cells colocalized with glial fibrillary acidic protein (GFAP), a glial marker (Fig. 1D). Five days following transduction, we exposed the cultures to 100 M or 1 mM glutamate for 1 h, after which we washed them with conditioned medium. Twenty-four hours following glutamate exposure, we measured the mitochondrial dehydrogenase activity of the neuronal cultures to determine cell viability ¹⁹. Exposure to 100 M glutamate led to a significant 48.3 0.02% decrease in cell viability in EIAV-LacZ-transduced cultures (Fig. 1E; P < 0.001, Student's t test). By contrast in EIAV-Bcl-2-transduced cultures we observed no significant decrease in cell viability, indicating that hippocampal neurons were protected from glutamate-induced cell death (Fig. 1E). Similarly in hippocampal neurons that were transduced with EIAV-GDNF we observed no significant decrease in cell viability, suggesting protection from glutamate-induced neuronal degeneration by the EIAV-GDNF vector. Exposure to a higher concentration of glutamate (1 mM) in control (EIAV-LacZ) cultures led to a 41.8 0.05% decrease in cell viability (Fig. 1E; P < 0.01, Student's t test). Under similar conditions there was a 23.9 0.07% decrease in cell viability (Fig. 1E) in EIAV-Bcl-2-transduced cultures and 26.8 0.05% cell death in EIAV-GDNF-transduced cultures that were exposed to 1 mM glutamate (Fig. 1E). These data indicate that at high glutamate concentrations Bcl-2 or GDNF expression from the EIAV vectors gave significant protection of hippocampal neurons from glutamate-induced toxicity. However, in both cases there was some cell death, suggesting that there may be a threshold for neuroprotection by these vectors.

Figure 1.

Transduction of cultured hippocampal cells by EIAV-Bcl-2 and EIAV-GDNF and their neuroprotective effects. E18 hippocampal cultures were transduced with (A) EIAV-LacZ, (B) EIAV-Bcl-2, or (C) EIAV-GDNF at an m.o.i. of 10 and the expression of the appropriate transgenes was determined using antibodies against -galactosidase, Bcl-2, or GDNF, respectively (all indicated in green in A–C). Colocalization with a neuronal marker was determined using antibody against MAP-2 (red), while all cells were stained with DAPI (blue). Approximately 50% of total cells were transduced. (D) The percentage of cells transduced by EIAV-LacZ, EIAV-Bcl-2, or EIAV-GDNF that colocalized with MAP2 or a glial marker, GFAP, was assessed by counting the number of transduced cells that express MAP2 (MAP2⁺) or GFAP (GFAP⁺) and expressing it as a percentage of the total transduced cells. (E) Protection against glutamate excitotoxicity by EIAV-Bcl-2 and EIAV-GDNF in hippocampal cultures. Control hippocampal cultures transduced by EIAV-LacZ underwent cell death after exposure to 100 M or 1 mM glutamate. In contrast, EIAV-Bcl-2- and EIAV-GDNF-transduced hippocampal cells were protected against glutamate toxicity. Scale bars (A–C) represent 50 m.

Full figure and legend (161K)

Transduction of Rat Hippocampus by Lentiviral Vectors

To utilize the EIAV vectors in the excitotoxicity model, we investigated the transduction characteristics of EIAV-LacZ. We injected 1.5 10⁶ transduction units in a volume of 3 l of EIAV-LacZ pseudotyped with the rabies-G envelope bilaterally into the adult rat dorsal hippocampus. We observed strong expression of the LacZ marker gene in the CA1 layer of the hippocampus with vector (Fig. 2A); therefore in the subsequent experiments we chose to use EIAV vectors that were pseudotyped with the rabies-G envelope. The observation of stronger expression of EIAV vectors pseudotyped with the rabies-G envelope compared to those pseudotyped with the vesicular stomatitis virus glycoprotein in the CA1 and CA3 pyramidal neurons in the hippocampus has also been demonstrated in Mazarakis et al. ¹⁵. We injected EIAV-Bcl-2 or EIAV-GDNF vectors pseudotyped with the rabies-G envelope stereotaxically into the adult rat dorsal hippocampus. Bilateral injection of the respective EIAV vectors resulted in good transduction of the vector and high transgene expression in the CA1 layer of the hippocampus after 3 weeks (Figs. 2B and 2C). In hippocampus that received EIAV-LacZ injections, colocalization with a neuronal nuclei marker, NeuN, demonstrated that 47% of neurons in the CA1 layer of the hippocampus were transduced at the site of injection (Fig. 2D).

Figure 2.

Transduction of rat hippocampus by lentiviral vectors. (A) EIAV-LacZ pseudotyped with rabies-G was injected into the rat hippocampus and expression of the marker gene was assessed by X-gal staining. EIAV-LacZ pseudotyped with rabies-G demonstrated good expression in the CA1 pyramidal neurons. Similarly, good transduction was observed with (B) EIAV-Bcl-2 and (C) EIAV-GDNF. (D) In the EIAV-LacZ animals, the majority of -galactosidase expression (green) occurred in neurons, which express NeuN (red). Scale bars represent 100 m (A) and 50 m (B–D).

Full figure and legend (162K)

Neuroprotection by Bcl-2 in excitotoxicity model

We tested the neuroprotective effects of Bcl-2 in an adult rat model of excitotoxicity. Three weeks following introduction of the viral vectors, we injected 0.5 l of 5 mM NMDA unilaterally into the CA1 region of the hippocampus, while on the control side, we injected 0.5 l of PBS. Injection of 5 mM NMDA into the control EIAV-LacZ hippocampus resulted in neuronal degeneration and cell death, as exemplified by the loss of NeuN staining in the CA1 region of the hippocampus (Fig. 3A). In comparison we observed no visible toxicity in the control hippocampus, which received an injection of PBS. This was further illustrated when consecutive sections were stained with Fluorojade-B (FJB), which stains for areas of neurodegeneration ^20,21. We detected FJB-positive cells in the hippocampus in the presence of 5 mM NMDA (Fig. 3C). By contrast in the EIAV-Bcl-2 animals, injection of 5 mM NMDA into the hippocampus did not induce a large area of neuronal cell death in the CA1, as indicated by the presence of NeuN staining (Fig. 3B). Furthermore few if any FJB-positive neurons were detected in the Bcl-2-transduced hippocampus (Fig. 3D).

Figure 3.

Neuroprotection by EIAV-Bcl-2 against NMDA excitotoxicity in the rat hippocampus. (A) Injection of NMDA induced an excitotoxic lesion (indicated by arrowhead) in the CA1 layer of the hippocampus in control EIAV-LacZ-transduced rats. Lesion results in the loss of NeuN staining and widespread cellular damage to the CA1 layer of the hippocampus. (B) In contrast an insignificant lesion was induced in EIAV-Bcl-2-transduced rats. (C) Using Fluorojade-B staining a large number of fluorescent neurons in the CA1 were observed in the NMDA-induced lesion in EIAV-LacZ animals, whereas (D) only a few fluorescent cells could be detected in EIAV-Bcl-2 animals. (E) Quantitative assessment of excitotoxic lesion in both groups indicated that EIAV-Bcl-2 significantly protected hippocampal neurons from NMDA-induced toxicity. ^*P < 0.05 (Student's unpaired t test). Scale bar represents 1 mm (A and B) and 50 m (C and D).

Full figure and legend (274K)

We assessed and quantified the area of damage in the CA1 of the hippocampus. In hippocampus that was injected with PBS, we observed a small lesion in the CA1 region in the EIAV-LacZ-transduced animals (4.7 1.81% of total CA1 layer) and in the EIAV-Bcl-2-transduced animals (9.7 4.08%) (Fig. 3E). These lesions were not significant in size and may be due to surgical procedures. In the excitotoxic hippocampus, 5 mM NMDA induced a large lesion in the hippocampus of the EIAV-LacZ animals, destroying 70.1 6.55% of the CA1 hippocampus (Fig. 3E). In contrast Bcl-2 significantly protected the hippocampus from excitotoxicity; injection of 5 mM NMDA resulted in only a 27.3 7.79% loss of the CA1 hippocampus, leaving the majority of the CA1 hippocampus intact (Fig. 3E; P < 0.05, Student's t test). This suggested that Bcl-2 expression protected the hippocampus from cell death that was induced by the excitotoxic effects of NMDA.

EIAV-GDNF Protects against Excitotoxic Lesion in the Hippocampus

Expression of GDNF protected hippocampal neurons from NMDA-induced excitotoxicity as evident from the increased amount of NeuN staining in the lesioned hippocampus (Fig. 4B) compared to EIAV-LacZ animals (Fig. 4A). There was also a higher number of CA1 pyramidal neurons that were stained with FJB in EIAV-LacZ animals (Fig. 4C) compared to EIAV-GDNF animals (Fig. 4D). This suggests that the amount of neuronal degeneration induced by NMDA in the hippocampus in EIAV-GDNF animals was lower. Quantification revealed that NMDA induced the loss of 54.0 16.4% of the CA1 region of the hippocampus in the EIAV-LacZ-transduced animals. In contrast NMDA produced a lesion size of only 13.2 5.3% in EIAV-GDNF animals, suggesting significant protection (Fig. 4E; P < 0.05, Student's t test).

Figure 4.

EIAV-GDNF protects rat hippocampal neurons against NMDA excitotoxicity. (A) An excitotoxic lesion (indicated by arrowhead) in the CA1 layer of the hippocampus was produced by NMDA injection in control EIAV-LacZ-transduced r ats. In this lesion, there was significantly reduced NeuN staining compared to the contralateral control PBS-injected side. (B) In EIAV-GDNF-transduced rats, only a small area of lesion was induced by NMDA as indicated by the presence of NeuN staining. (C) A large number of CA1 neurons in the lesion were stained with Fluorojade-B in EIAV-LacZ animals, whereas (D) only a few fluorescent cells could be detected in EIAV-GDNF animals. (E) Quantitative assessment of excitotoxic lesion in both groups showed that EIAV-GDNF significantly reduced the lesion size compared to EIAV-LacZ rats. ^*P < 0.05 (Student's unpaired t test). Scale bar represents 1 mm (A and B) and 50 m (C and D).

Full figure and legend (250K)

Discussion

The present study describes the use of lentiviral vectors to provide neuroprotection in in vitro and in vivo excitotoxicity models and indicates that EIAV vectors have the capacity as gene delivery tools for distributing putative neuroprotective targets to the central nervous system. Both EIAV vectors expressing Bcl-2, an antiapoptotic molecule, or GDNF, a secreted neurotrophic factor, demonstrated protective effects against excitotoxic cell death induced by glutamate application in cultured hippocampal neurons. Furthermore, both vectors prevented the CA1 region of the hippocampus from undergoing neurodegeneration and cell death after NMDA injection. This suggests that EIAV vectors can be exploited in therapeutic strategies for the treatment of neurological diseases such as cerebral ischemia.

The use of other viral vectors to express Bcl-2 in ischemia models has been demonstrated in some studies ^11,12,22. Lawrence et al. ¹¹ demonstrated that overexpression of Bcl-2 from a herpes simplex virus vector enhanced neuronal survival in animals subjected to focal ischemia or adriamycin-induced oxidative damage; however, they suggested that the low efficiency of infection and titers of the viral inocula used may limit protection. In such circumstances, the good expression levels and relatively high titers of EIAV vectors offer an alternative strategy, and this study demonstrates the utility of these vectors for expressing a neuroprotective gene in a relevant disease model. The exact mechanism of Bcl-2-mediated neuroprotection remains unclear, though it has been observed that induction of tolerance to ischemia in the hippocampal CA1 is associated with increased endogenous Bcl-2 immunoreactivity ²². Studies determining the mechanism of estrogen-mediated neuroprotection against glutamate excitotoxicity in rat hippocampal neurons have implicated Bcl-2 in promoting mitochondrial tolerance of an increased [Ca²⁺] load ²³. Bcl-2 enhances the ability of mitochondria to sequester large quantities of cytosolic Ca²⁺ thus diverting the excitotoxic effects of Ca²⁺ from the cytoplasm. This provides a plausible mechanism by which Bcl-2 may inhibit neuronal death associated with ischemia and excitotoxicity in our model, as NMDA receptor activation is known to cause a rise in intracellular Ca²⁺ concentrations ²⁴. We cannot, however, exclude other possible mechanisms of neuroprotection by Bcl-2, for example blockage of lipid peroxidation ²⁵ or prevention of intracellular accumulation of reactive oxygen species (ROS) and lipid peroxides ²⁶. Indeed, glutamatergic excitation through NMDA receptors promotes the release of arachidonic acid and generation of oxygen radicals ²⁷, including superoxide ²⁸. As our excitotoxicity model involves the use of NMDA to induce neurodegeneration, it is possible that prevention of ROS accumulation may account for at least some of the protective effects exhibited by EIAV-Bcl-2 in this model.

Neuroprotection in a cerebral ischemic model by GDNF has been demonstrated in rats with transient bilateral common carotid artery ligation and right middle cerebral artery ligation ^4,10. Recombinant GDNF has also been shown to protect against NMDA-induced excitotoxicity in hippocampal slice cultures ²⁹. GDNF is a secreted factor and therefore has the propensity to exert its protective effects on a larger number of cells compared to a cellular protein, for example Bcl-2. The precise mechanism of GDNF protection against excitotoxicity is less clear. Wang et al. ³⁰ demonstrated that heterozygous mice lacking in the GDNF receptor -1 (GFR1) were more vulnerable to focal cerebral ischemia. GFR1 is expressed in the CA1–CA3 subfields of the hippocampus and in the dentate gyrus and is found in 72% of parvalbumin-immunoreactive CA1 interneurons ³¹. In cortical murine cultures, GDNF exerts neuroprotective activity against NMDA-induced neuronal death, via its GFR1 receptor, by modulating NMDA receptor function, thereby reducing its activity in stimulating Ca²⁺ influx and restoring Ca²⁺ homeostasis ³². The modulation of NMDA receptor activity occurred through the activation of the extracellular signal-regulated kinase pathway ³². Given the presence of GFR1 expression in the hippocampus, it is possible that the mechanism of neuroprotection by EIAV-GDNF against NMDA toxicity in our study could occur by this pathway. However, other mechanisms by which GDNF may provide neuroprotection, for example promoting the expression of antiapoptotic factors such as X-linked inhibitor of apoptosis, Bcl-2, and Bcl-xL ^33,34 or by reducing intracellular free radical production, cannot be excluded.

Using Bcl-2 and GDNF as examples of neuroprotective targets this study has provided proof-of-principle data that EIAV vectors are ideal tools for gene therapy in disease models involving excitotoxicity, for example cerebral ischemia. These vectors may also be utilized as expression tools for target validation in gene discovery programs that identify neuroprotective targets in ischemia models ³⁵. EIAV vectors have the advantage of possessing a large cloning capacity and therefore can accommodate the expression of most genes. Furthermore, compared to adenoviral ^36,37 and herpes simplex viral ³⁸ systems, EIAV vectors do not mount a large immune response in the central nervous system and therefore can mediate long-lasting transgene expression in transduced cells ¹⁵. The lack of expression of potentially immunogenic viral accessory proteins by EIAV vectors enhances the utility of these vectors for gene therapy in cerebral ischemia, as inflammation often exacerbates injury in ischemia. EIAV vectors can therefore be exploited to express novel neuroprotective targets in the brain to minimize cellular damage and promote neuronal survival after a cerebral infarct.

For therapy in cerebral ischemia, administration of these vectors in the early phases of stroke may have to be performed to prevent excitotoxic cell death and apoptosis in insulted neurons. Alternatively, in cases of predisposition to multiple strokes therapeutic vectors can be applied after the initial stroke to avoid further damage from subsequent stroke incidents. The use of lentiviral vectors to express neuroprotective genes thus represents a potential strategy for preventing secondary damage after the primary ischemic insult, thus improving functional outcome after an ischemic event.

Materials and Methods

Lentiviral vector construction and production

The cDNAs encoding LacZ, human Bcl-2, and GDNF were subcloned into the same EIAV transfer vector (pONY8 series), which has been described previously ^15,39. Briefly, the respective genes were placed under the control of a minimal hCMV promoter in the pONY8 vector containing a 5' cPPT element. The resultant vectors were pseudotyped with envelope glycoproteins derived from the Evelyn–Rokitnicki–Abelseth strain of rabies virus (rabies-G). Viral vector stocks were produced by transient transfection of human embryonic kidney 293T cells plated on 10-cm dishes with FuGENE 6 (Roche, UK). Three DNA components were transfected into the cells: 2 g of vector plasmid encoding the gene of interest, 2 g of gag/pol plasmid (pONY3.1), and 1–2 g of plasmid encoding the envelope glycoprotein. After transfection for 16 h, sodium butyrate was added to a final concentration of 10 mM. Supernatants were harvested 24–42 h after transfection and filtered through a 0.45-m filter. Concentrated viral preparations were produced by an initial low-speed ultracentrifugation at 6000g at 4°C for at least 18 h, followed by ultracentrifugation at 50,000g at 4°C for 90 min. The virus was resuspended in formulation buffer containing 19.75 mM Tris–HCl, pH 7.0, 40 mg/ml lactose, 37.5 mM sodium chloride, 1 mg/ml human serum albumin, and 5 l/ml protamine sulfate for 2–3 h at 4°C, aliquoted, and stored at -80°C. Biological titers of the control (LacZ) viral preparations, expressed as number of transducing units per milliliter (TU/ml), were determined by transducing canine osteosarcoma (D17) cells in limiting dilutions in the presence of Polybrene (8 g/ml). After 2–3 days incubation, the cells were incubated in 5-bromo-3-indolyl--D-galactosidase (X-gal) solution and blue colonies were counted. The titers of EIAV-LacZ, EIAV-Bcl-2, and EIAV-GDNF preparations were calculated by determining the number of viral RNA genomes per milliliter of viral stock solution using real-time PCR analysis (ABI 7700; PE Applied Biosystems) and comparing it to a bank viral preparation of known biological titer, as described in Rohll et al. ⁴⁰ and Martin-Rendon et al. ⁴¹. Titers of the vectors were as follows: EIAV-LacZ, 4 10⁹ TU/ml; EIAV-Bcl-2, 1 10⁹ TU/ml; EIAV-GDNF, 9 10⁸ TU/ml.

Dissociated hippocampal cultures

Hippocampi were dissected out of embryonic E18 rat pups in Hanks' balanced salts medium (Gibco BRL, UK) under a dissecting microscope. The hippocampi were washed five times in Hanks' medium after which trypsin was added and incubated for 10 min at 37°C. After the hippocampi were washed in Hanks' basal medium, they were washed in Neurobasal medium (Gibco BRL) and triturated to break up the cells. The cells were counted and plated in a 24-well plate at a density of 7.5 10⁴ cells/well and subsequently cultured in Neurobasal medium (Gibco BRL) supplemented with B-27 (Gibco BRL). The following day, transduction of hippocampal neurons was carried out at a m.o.i. of 10 in a volume of 300 l medium at 37°C in quadruplicate. Five days after transduction glutamate (Sigma, UK) was added to the cultures to a final concentration of 100 M or 1 mM for 1 h, after which the glutamate-containing medium was replaced with fresh conditioned medium. Cell viability was determined by detection of mitochondrial dehydrogenase activity using thiazolyl blue tetrazolium blue (MTT) according to the manufacturer's instructions (Sigma).

Delivery of EIAV into the hippocampus

All surgical procedures were approved by the local veterinarian and ethical committee and were carried out according to UK Home Office Regulations. Stereotaxic administrations were performed on adult male Wistar rats under Hypnorm and Hypnovel anesthesia using a 10-l Hamilton syringe with a 33-gauge blunt-tip needle. Approximately 1.5 10⁶ TU in a volume of 3 l of each vector (n = 10 EIAV-LacZ, n = 7 EIAV-Bcl-2, n = 4 EIAV-GDNF) was slowly infused, at a speed of 0.2 l/min using an infusion pump (World Precision Instruments, Inc., Sarasota, FL, USA), into the hippocampus using the stereotaxic coordinates anteroposterior -3.3 mm, mediolateral 2 mm, dorsoventral 1.5–2.5 mm (from the dura). After viral vector injections, the needle was left in place for approximately 5 min before being withdrawn. The skin was closed using a 5-O Vicryl suture and following surgery, animals were kept warm until recovery was complete. After 3 weeks, the rats were again anesthetized and 0.5 l of PBS or 0.5 l of 5 mM NMDA was injected into the left or right hippocampus, respectively, using stereotaxic coordinates anteroposterior -3.3 mm, mediolateral 2 mm, dorsoventral 2.5 mm. Following recovery the rats were sacrificed 72 h later by an ip injection of Euthatal (Rhone Merieux, USA) and transcardially perfused with 4% (w/v) paraformaldehyde containing 2 mM MgCl₂ and 5 mM ethylene glycol bis (-aminoethyl ether)-N,N,N',N',-tetraacetic acid and brains were harvested. Rat brains were cryoprotected in 30% sucrose, after which they were frozen in Tissue-Tek OCT (Sakura Finetek). Forty-micrometer coronal sections were obtained on a CM3050 cryostat (Leica, UK) and collected into wells containing PBS. Immunohistochemistry was performed as described below.

Immunohistochemistry

Immunohistochemistry was performed on transduced hippocampal neurons or brain sections using the following antibodies made up in 10% goat or donkey serum: rabbit anti--gal (1:300 made up in 10% normal goat serum; R&D Systems, UK), anti-Bcl-2 (1:200; DAKO), anti-GDNF (1:250; R&D Systems), anti-NeuN (1:300; Chemicon, UK), anti-GFAP (1:200; Chemicon), and anti-MAP2 (1:200; Santa Cruz Technologies, USA). Briefly, cells or sections were washed twice in PBS and blocked in 10% normal goat or donkey serum for 1 h at room temperature. Incubation with primary antibody at 4°C was for at least 24 h. After three PBS washes, cells or sections were incubated with secondary antibody for at least 2 h at room temperature. For fluorescence immunohistochemistry, species-specific secondary antibodies Cy3 or Texas red (1:200; Jackson Laboratories) and Alexa 488 (1:200; Molecular Probes) were utilized. For DAB visualization, sections were incubated in biotinylated secondary antibody (Vector Laboratories, UK) for 1 h, followed by ABC reagent (Vector Laboratories) for 1 h, after which they were incubated in DAB for 5–10 min (Vector Laboratories). After PBS washes, cells or sections were mounted in Vectashield containing DAPI (Vector Laboratories) for fluorescence immunohistochemistry or in DPX for DAB visualization. Images were captured using the AxioVision software (AxioVision Systems, UK). Fluorojade-B (Histo-Chem, Inc., USA) staining was performed according to Schmued and Hopkins ²¹. For X-gal staining, sections were incubated in X-gal solution for 3–4 h at 37°C. Cell counts for transduced hippocampal neurons in vitro were performed on at least five different fields under 20 magnification. For quantification of lesion size in vivo, the length of the intact/lesioned CA1 region, as observed by presence/lack of NeuN staining, was measured by a blind observer in every fifth brain section spanning the dorsal hippocampus from bregma -3.0 to -4.6 mm anterioposteriorly using AxioVision software. In total at least 10 brain sections were measured under 10 magnification. The lesion size was expressed as a percentage of the total length of CA1 on each side of the hippocampus. All statistical analyses were carried out using two-tailed unpaired Student's t test and a P value of <0.05 was considered significant.

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Acknowledgements

The authors thank Nicole Deglon for the kind gift of the GDNF cDNA; Mimoun Azzouz, Robert Barber, and Kyriacos Mitrophanous for scientific discussion; and all at Oxford BioMedica who have contributed to the production of the viral vectors.