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(Stroke. 1995;26:1670-1675.)
© 1995 American Heart Association, Inc.

Articles

Expression of bcl-2 From a Defective Herpes Simplex Virus–1 Vector Limits Neuronal Death in Focal Cerebral Ischemia

Matthew D. Linnik, PhD; Peter Zahos, MD; Michael D. Geschwind, MS Howard J. Federoff, MD, PhD

From the Department of Neurosurgery, University of Cincinnati (Ohio) College of Medicine, Marion Merrell Dow Research Institute (M.D.L.), and the Departments of Medicine and Neuroscience (P.Z., M.D.G., H.J.F.) and Neurosurgery (P.Z.), Albert Einstein College of Medicine, Bronx, NY.

Correspondence to Howard J. Federoff, MD, PhD, Department of Neurology, Box 673, University of Rochester School of Medicine, 601 Elmwood Ave, Rochester, NY 14642.

	Abstract

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Background and Purpose A process resembling programmed cell death appears to contribute to postischemic neuronal loss in several models of stroke. Because the expression of the bcl-2 gene has been shown to rescue neurons from programmed cell death due to other causes, we determined whether it would be similarly neuroprotective in stroke.

Methods Replication defective herpes viral vectors that transduce bcl-2 (HSVbcl2) or Escherichia coli lacZ (HSVlac) were injected into two sites in the rat cerebral cortex 24 hours before induction of neocortical focal ischemia by tandem permanent occlusion of the right middle cerebral artery and ipsilateral common carotid artery. Local ischemic damage was determined 24 hours after occlusion by staining with 2% 2,3,5-triphenyltetrazolium chloride.

Results Expression of bcl-2 in cerebral cortex was confirmed by immunohistochemistry in animals injected with the HSVbcl2 expression vector. Viable tissue was significantly increased at the injection sites in HSVbcl2- but not HSVlac-injected animals. The protection observed in the HSVbcl2 animals was localized to the injection sites.

Conclusions These data indicate that bcl-2 expression protects neurons in vivo from ischemic injury and suggest the feasibility of gene therapy for stroke and perhaps other neurological diseases in which programmed cell death is involved.

Key Words: apoptosis • cerebral ischemia • genetics • herpes simplex • neuronal death

	Introduction

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Disruption of blood flow to the brain initiates a cascade of events that produces neuron death and leads to neurological dysfunction. A number of extracellular mediators are implicated in ischemic cell death, including excitatory amino acids, free radicals, and calcium.^{1 2} Recently, several studies have examined the intracellular events that accompany ischemic cell death to determine whether neurons are passively killed or actively participate in self-destruction. These studies indicate that some neurons contribute to their own demise through a series of biochemical events similar to programmed cell death.^{3 4 5 6 7 8 9}

Programmed cell death is a phylogenetically conserved and genetically specified process by which redundant neurons are eliminated during development.^{10 11 12} Recent observations have begun to define the proteins that are important in programmed cell death, including those that activate and prevent the process. One of the gene products, bcl-2, has been shown to prevent cell death triggered by free radicals, glucose deprivation, excess glutamate, and growth factor deprivation.^{13 14 15 16} These data suggested the possibility that expression of bcl-2 could protect neurons from ischemic death. This notion is supported by the recent demonstration of smaller infarct sizes in transgenic mice that constitutively overexpress bcl-2.¹⁷ To address this hypothesis, we used a viral vector to express bcl-2 within the rat cortex and demonstrate that its expression protects neurons in brain tissue subjected to an ischemic insult.

Replication defective HSV-1 vectors achieve efficient transduction and expression of heterologous genes within the nervous system^{18 19 20 21 22 23 24 25 26} and have been used as vehicles in experimental gene therapy studies.^{19 27} In the present study, we constructed a plasmid-based defective HSV-1 vector, an amplicon, that expresses the human bcl-2 gene. Herein, we demonstrate that delivery of this vector can protect neurons against subsequent ischemic injury.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vector Construction, Virus Packaging, and Titering
The vector was constructed by cloning of the human bcl-2 gene as an EcoR1 fragment (a gift of Dr Arnon Rosenthal) into HSVprPUC,^{19 28 29 30 31 32} thereby placing it under the transcriptional control of the strong HSV immediate early 4/5 promoter (HSVbcl2; Fig 1A). An identical vector expressing the Escherichia coli ß-galactosidase gene was used as a negative control (HSVlac). These vectors were packaged into virus, purified, concentrated, and stored at -70°C until use.^{33 34} In brief, the RR1 cells³⁵ were used for packaging HSV amplicons along with D30 EBA helper virus.³⁶ Packaging was performed by plating 3x10⁶ RR1 cells plated in medium containing 10% FCS and 4 hours later transfecting them by adding 40 µL of lipofectin (Gibco-BRL), waiting 5 minutes, and then adding vector DNA solution dropwise (30 µg at 1 µg/µL in Dulbecco's modified Eagle medium). Six hours later, plates were fed with medium containing 5% FCS. Approximately 20 hours after transfection, D30 EBA virus was added to achieve a multiplicity of infection (MOI) of 0.2. Five milliliters of complete medium with 5% FCS was added to each plate after 1 hour. HSV-1 vector stocks were harvested 2 days later and repassaged on fresh RR1 cells (4x10⁶ cells/60-mm plate). Two days later the stocks were harvested. The amplicon component of virus stocks was titered by an expression or slot blot assay. HSVlac stocks were titered by measuring the number of X-gal+(blue)–forming units per milliliter. The HSVbcl2 amplicon virus was titered by immunostaining infected NIH 3T3 cells with an antibody against human bcl-2 (see below for conditions) and HSVlcb (reverse orientation of bcl-2) was titered by slot blot assay to determine genome content as described previously.³³ The D30 EBA helper virus in each stock was titered by plaque assay on RR1 cells.³³ Helper virus titers used in this study ranged from 5x10⁶ to 6x10⁷. Virus stocks were 100-fold concentrated by centrifugation as described previously.³³

View larger version (97K): [in this window] [in a new window]   Figure 1. Schematic and photographs show construction and expression of HSVbcl2. A, The HSVbcl2 construct. HSVbcl2 also contains an HSV origin of replication (HSV oriS) and an HSV cleavage and packaging site (HSV pac site), as well as an SV40 poly A site positioned after the bcl-2 gene. B, Western blot of HSVbcl2-transduced cells. HSVbcl2 was packaged with a rapid deletion packaging method.34 Lane 1, cells transduced with HSVlac virus; lanes 2 and 3, cells transduced with HSVbcl2 virus from two different viral stocks. C and D, Immunostaining of vector-transduced cells. NIH 3T3 fibroblasts were transduced at an MOI of 0.5 with HSVbcl2 (C) or HSVlac (D). Cells were immunostained with an antibody against human bcl-2.

Expression Studies
Amplicon stocks (HSVbcl2 and HSVlac) contained between 0.5x10⁷ and 1x10⁷ infectious particles per milliliter and were used to transduce NIH 3T3 fibroblasts (5x10⁶ cells/60-mm plate) at an MOI of approximately 0.5. After 24 hours, cells were harvested, washed in ice-cold PBS, and lysed in SDS–polyacrylamide gel electrophoresis loading buffer. Approximately 25 µg of each protein lysate was loaded per lane, electrophoresed under denaturing conditions, transferred to nitrocellulose, probed with a monoclonal antibody against human bcl-2 (Dako Corp; 1:500), and developed with Amersham ECL reagents. For immunocytochemistry, fibroblasts (5x10⁶ cells/60-mm plate) were transduced at an MOI of 0.5, and after 24 hours they were washed in PBS three times, fixed in 4% paraformaldehyde, immunostained with a monoclonal antibody against human bcl-2 (Dako Corp; 1:1000), and visualized with a secondary antibody (goat and mouse; 1:300, HRP-linked) with diaminobenzidine used as a chromagen. For immunocytochemistry studies in vivo, spontaneously hypertensive rats (Harlan Sprague-Dawley) were anesthetized with chloral hydrate (500 mg/kg IP, with supplemental doses as needed) and each given a 2-µL injection containing approximately 10⁵ infectious particles of HSVbcl2, HSVlac, or HSVlcb. Twenty-four hours after injection, animals were anesthetized (chloral hydrate, as above), killed, and perfused transcardially with 100 mL 10% formalin in PBS. Brains were postfixed in formalin for 18 hours and stored in 70% ethanol. They were dehydrated through a graded series of ethanol solutions, embedded in paraffin, cut into 5-µm sections, rehydrated, microwaved at 800 W until the bathing 10 mmol/L citrate buffer boiled, allowed to slowly reach room temperature, incubated with a monoclonal antibody against human bcl-2 (Dako Corp; 1:500), developed with avidin-biotin complex and peroxidase, and counterstained with hematoxylin.

Neuroprotection Protocol
Virus injection site coordinates were selected by use of data from control animals to locate the injections approximately 0.5 to 1 mm from the medial edge of the infarction. Anesthesia was performed with chloral hydrate as described above. A preliminary study in control animals indicated that the medial edge of the infarction was at 3.06±0.02 mm (mean±SEM) lateral to midline (range, 2.6 to 3.4 mm; n=5) at -3 mm to bregma. Therefore, we chose two injection sites at the following coordinates relative to bregma: 1.7 mm anterior, 3.8 mm lateral, and 2.8 mm deep and 3.3 mm posterior, 3.8 mm lateral, and 2.3 mm deep.³⁷ Each site received a 2-µL injection (approximately 10⁵ infectious particles/site) continuously delivered at 0.25 µL per minute. Syringes were maintained in the needle tract for 5 minutes after injections were completed. Access holes were sealed with bone wax, the incision was sutured closed, and the animals were allowed to recover.

Neocortical focal cerebral ischemia was produced by permanent tandem occlusion of the right middle cerebral artery and right common carotid artery as previously described.⁴ In brief, rats were reanesthetized with chloral hydrate 24 hours after injection of the virus and anesthesia was maintained with supplemental doses as needed. The right common carotid artery was isolated by a midline incision, ligated in two places, and severed. The middle cerebral artery was exposed through a 3x3 mm craniotomy, raised 0.5 to 1 mm above the cortical surface, and severed by electrocautery at a site within 2 mm of the inferior cerebral vein. Animals were maintained at 36°C to 37.5°C throughout recovery by means of a rectal thermistor connected to a heating lamp directed at the head of the animal until the righting reflex returned. The effect of virus injection on ischemic damage was evaluated 24 hours after middle cerebral artery occlusion by whole brain staining with 2% TTC in PBS for differentiation of live from dead tissue.^{37 38} Animals were killed by decapitation and the brains were rapidly removed and immersed in TTC for 30 minutes at 37°C. The amount of viable tissue was determined at both injection sites and at a site equidistant between the injection sites by measurement of the distance from the longitudinal fissure to the medial edge of the infarction with a digital micrometer. Three sequential determinations were made at each of the site s and the mean of these observations was used for statistical analysis. The brains were then sliced into 2-mm sections and stained in TTC for an additional 15 to 20 minutes to get adequate staining of subcortical tissue. The slices were fixed in 10% phosphate-buffered formalin and the volume of the infarction was determined by computer-assisted image analysis. Comparisons between treatment groups were by two-tailed unpaired Student's t test with P<.05 considered significant. Data are expressed as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A replication defective HSV-1 amplicon vector was constructed in which the human bcl-2 gene was placed under the transcriptional control of HSVbcl2 (Fig 1A). An identical vector expressing the E coli ß-galactosidase gene was used as a negative control (HSVlac). These vectors were packaged into virus, purified, concentrated, and stored at -70°C until use.^{33 34} Transduction of fibroblasts with the HSVbcl2 virus (Fig 1B, lanes 2 and 3, and Fig 1C) but not HSVlac (Fig 1B, lane 1, and Fig 1D) resulted in the expression of appreciable quantities of bcl-2 in most cells observed in every field.

Stereotaxic delivery of the HSVbcl2 vector resulted in expression of the transduced gene in cells surrounding the injection site. Immunohistochemical staining revealed many immunostained cells (Fig 2A). Some stained cells had large cell bodies consistent with neuronal somata (Fig 2B). Specific immunostaining was absent in sections from animals that had been injected with HSVlac (expresses ß-galactosidase) or an HSV vector containing the bcl-2 gene inserted in the reverse orientation (Fig 2C and 2D).

View larger version (0K): [in this window] [in a new window]   Figure 2. Photomicrographs show expression of bcl-2 within the cortex. Rats were injected with HSVbcl2, HSVlac, or HSVlcb, a vector in which the bcl-2 gene is in the reverse orientation. Twenty-four hours after the injection, brains were removed and processed for bcl-2 immunocytochemistry. Low-power (x80) (A) and high-power (x800) (B) micrographs show cortical tissue near the site of HSVbcl2 virus injection. Many immunoreactive cells are seen, some with the characteristic somata of cortical neurons. Low-power (x80) (C) and high-power (x800) (D) micrographs show cortical tissue near the site of HSVlcb virus injection. Note that only a few cells with scant immunostaining are present.

To assess neuroprotective effects, the HSV-1 vector constructs were stereotaxically microinjected into rat cerebral cortex in two locations 24 hours prior to induction of an ischemic insult by tandem permanent occlusion of the right middle cerebral and ipsilateral common carotid arteries in the spontaneously hypertensive rat.^{39 40} Injection site coordinates were selected by use of data from control animals to locate the injections approximately 0.5 to 1 mm from the medial edge of the infarction. Each site received approximately 10⁵ infectious particles.

Neuroprotection was evaluated 24 hours after the occlusion by measurement of the extent of viable tissue from the longitudinal fissure to the medial edge of the infarction (Fig 3). Significantly more viable tissue was present at both the anterior and posterior injection sites in HSVbcl2- (Fig 3, sites 1 and 2) but not in HSVlac-injected animals (Fig 3, sites 1 and 2). No protection was observed in HSVbcl2 animals at a site (Fig 3, midpoint) equidistant between the two virus injection sites. The amount of viable tissue at the midpoint in HSVbcl2-injected animals was comparable to that at sites 1 and 2 in HSVlac-injected (Fig 3) and in uninjected control animals (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic and bar graph show that transduction with bcl-2 protects against subsequent ischemic injury. Rats were injected with HSV-1 vector and after 24 hours they were subjected to permanent tandem occlusion of the right middle cerebral artery and right common carotid artery. After an additional 24 hours, brains were removed and analyzed by staining with TTC for differentiation of live from dead tissue.38 Left, The distance from the midline of the brain to the medial edge of the infarct was determined at each injection site (sites 1 and 2) and at a site equidistant between the two injection sites (midpoint). Right, The extent of viable tissue from the longitudinal fissure to the medial edge of the infarction along a line perpendicular to the midsagittal plane was measured with a digital micrometer in animals that received HSVlac (open bars) and HSVbcl2 (shaded bars). Significant protection (*P<.05 by two-tailed unpaired Student's t test) was observed at sites 1 and 2 in HSVbcl2 animals compared with their midpoint values or with any site in HSVlac animals. Bars represent mean±SEM for six rats per group.

Immediately after measurements were made on the cortical surface, the brain was sliced into 2-mm coronal sections and stained for an additional 15 to 20 minutes in TTC to delineate subcortical ischemic tissue. Measurements of brain volume (1681±44 mm³ and 1583±55 mm³ for HSVbcl2 and HSVlac, respectively) and infarct volume (167±12 mm³ and 169±11 mm³ for HSVbcl2 and HSVlac, respectively) revealed no significant dif-ferences as determined by quantitative image analysis. The lack of an effect on total infarct volume was not unexpected considering the relatively small number of HSVbcl2 virions injected and the cell-autonomous action of bcl-2.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanism by which bcl-2 inhibits programmed cell death is not fully understood. bcl-2 has a single membrane attachment region at its carboxy terminus that promotes localization of the gene product to the nuclear envelope, endoplasmic reticula, and mitochondria.^{42 43 44 45} Membrane localization appears to increase the protein's ability to suppress programmed cell death.^{43 46} Recent studies suggest that bcl-2 functions by attenuating the generation of reactive peroxide species and limiting their toxicity.^{13 47}

Stroke elicits a number of injurious stimuli that may act singly or in aggregate to trigger programmed cell death in susceptible neurons. That bcl-2 can inhibit cell death initiated by some of these stroke-associated stimuli, including excitatory amino acid excess,^{14 15} free radicals,^{13 47 48} and increased intracellular calcium,⁴⁸ without specifically affecting their formation or interfering with their proximate molecular targets suggests that it exerts its action more distally, within a common final pathway.¹⁴ In this conceptual framework, it is unclear whether susceptibility to programmed cell death is a property intrinsic to all neurons with differences due to graded thresholds for activation. Alternatively, programmed death could be a characteristic of some neurons that constitute a biologically vulnerable subpopulation.

In our study, the expression of bcl-2 prior to induction of stroke was found to be neuroprotective. The effect is likely to reflect expression of bcl-2 in neurons, although it is possible that expression in glia could have contributed to the neuroprotection. Subsequent studies will evaluate the critical timing parameters between bcl-2 expression and stroke induction and their outcome in terms of neuroprotective efficacy. Recent data from phenomenological studies suggest that there is a window of opportunity between the initiation of ischemic injury and irreversible commitment to programmed cell death.^{6 7 49 50} The clinical implications of this observation for the development of gene therapy for stroke should stimulate further experimentation.

The conservation of programmed cell death across diverse species and among different cell types underscores its importance in cellular and organismal homeostasis. Given the ubiquity of the process, it is likely to be a component of pathogenetic mechanisms, perhaps those involved in chronic neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Moreover, a shared final common pathway suggests that similar therapeutic strategies targeted to interrupt programmed cell death may be effective in delaying the progression of these diseases.

	Selected Abbreviations and Acronyms

 

FCS = fetal calf serum HSV-1 = herpes simplex virus type-1 HSVbcl2 = replication-defective herpes viral vector that transduces bcl-2 HSVlac = replication-defective herpes viral vector that expresses the Escherichia coli ß-galactosidase gene MOI = multiplicity of infection TTC = 2,3,5-triphenyltetrazolium chloride X-Gal = 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside

	Acknowledgments

 
The authors thank Ms Diane Edelstein, Lauren R. Montgomery, and Cynthia D. Wallace for expert technical assistance. We also thank Dr Michael Brownlee and Dr Richard Kitsis for critical reading of this manuscript. This work was supported by grants from the Public Health Service to H.J.F. (HD31300, HD27116). M.D.G. was supported by Public Health Service grant GM7288-16.

Received February 9, 1995; revision received April 14, 1995; accepted April 25, 1995.

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