Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy

ORIGINAL ARTICLES

Gene Therapy with HSP72 Is Neuroprotective in Rat Models of

Stroke and Epilepsy Midori A. Yenari, MD,*tJ Sheri L. Fink, PhD,$I1 Guo Hua Sun, MD, PhD,*J Louis K. Chang, BS,llS

Maitraya K. Pate1,ll David M. Kunis, BA,*J: David Onley, BA,** Dora Y. Ho, PhD,I$ Robert M. Sapolsky, PhD,$" and Gary K. Steinberg, MD, PhD*$S

Brain areas damaged by stroke and seizures express high levels of the 72-kd heat shock protein (HSP72). Whether HSP72 represents merely a marker of stress or plays a role in improving neuron survival in these cases has been debated. Some induced tolerance experiments have provided correlative evidence for a neuroprotective effect, and others have documented neuroprotection in the absence of HSP72 synthesis. We report that gene transfer therapy with defective herpes simplex virus vectors overexpressing hsp72 improves neuron survival against focal cerebral ischemia and systemic kainic acid administration. HSP72 overexpression improved striatal neuron survival from 62.3 to 95.4% in rats subjected to 1 hour of middle cerebral artery occlusion, and improved survival of hippocampal dentate gyrus neurons after systemic kainic acid administration, from 21.9 to 64.4%. We conclude that HSP72 may participate in processes that enhance neuron survival during transient focal cerebral ischemia and excitotoxin-induced seizures.

Yenari MA, Fink SL, Sun GH, Chang LK, Patel MK, Kunis DM, Onley D, Ho DY, Sapolsky RM, Steinberg GK. Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy. Ann Neurol 1998;44:584-591

In models of experimental cerebral ischemia and kainic acid (KA)-induced seizures, neurons have been observed to mount a typical stress response. Although protein synthesis is generally halted during cell stress, the transcription of certain stress response genes is up- regulated.' Among these stress response genes are the heat shock proteins (HSPs). The 72-kd HSP (HSP72; also known as inducible HSP70) has been extensively studied and is thought to be induced through activa- tion of heat shock factors by denatured proteins, thiol- disulfide redox states, oxidative stress, or HSP72 may remove denatured proteins from a cell and assist in new protein synthesis.23325 HSP72 is highly induced in rat brain after a variety of insults including cerebral ischemia and seizures.'

The pattern and time course of HSP72 expression has been studied in experimental cerebral ischemia and KA models, frequently with the aim of addressing whether HSP72 is expressed in cells that will die or survive after brain insults. In ischemia, some research- ers have found that HSP72 is expressed only in cell populations that will ultimately recover from cerebral ischemia,'-' whereas others have found expression in

From the Departments of *Neurosurgery and ?Neurology, and $Stanford Stroke Center, Stanford University Medical Center, and $Program in Neurosciences and "Department of Biological Sciences, Stanford University, Stanford, CA.

SPresent address: Washington University School of Medicine, St Louis, M O 63 110.

cell populations destined to infarct."," In either case, several studies have shown that stress protein induction is a sensitive indicator of injury, regardless of

HSP72 induction after various cellular stresses led investigators to study its potential neuroprotective effects. "Ischemic tolerance," where a prior sublethal insult leads to protection against a subsequent severe insult, was thought to be mediated through stress proteins. 13-" However, because the preinduction causes a host of changes in protein expression and me- tabolism rather than only increasing HSP72 synthesis, the role of HSP72 in mediating the protective effects has been far from decisive. Indeed, protection in tolerance experiments has been noted even when HSP synthesis is b l ~ c k e d , ' ~ suggesting that "induced tolerance" does not always require new HSP production.

To directly test HSP72's role in improving cell survival, neurons can be made to selectively overexpress the protein. This may be accomplished by viral- mediated gene transfer or the production of transgenic animals. Thus far, published experiments transferring bp72 to brain cells have assessed its effects in tissue culture models. In our previous study," we showed

Received Nov 25, 1997, and in revised form Feb 25 and Apr 8, 1998. Accepted for publication Apr 13, 1998.

Address correspondence to Dr Yenari, Stanford Stroke Center, 701 Welch Road, Building B, Suite 325, Palo Alto, CA 94304.

584 Copyright 0 1998 by the American Neurological Association

that a herpes simplex virus (HSV) vector delivering hsp72 and the marker gene, lac2, protected cultured neurons from the protein denaturing stress of severe heat shock but failed to protect against direct application of the excitotoxin glutamate or the energetic toxin 3-nitropropionic acid. Overexpression of HSP72 by a retrovirus vector was protective against combined oxygen-glucose deprivation and isolated glucose deprivation in murine astrocyte c u l t ~ r e s ’ ~ , ~ ~ and hyper- thermia and ischemic stress in cultured peripheral neurons.21’22 At the whole animal level, strains of transgenic mice that overexpress HSP72 have been developed and found to be resistant to myocardial is- hernia.'^-'^ Hippocampal injury is attenuated after ir- reversible focal cerebral ischemia in these mice.26

We now report, in genetically normal animals, in vivo evidence that HSP72 improves neuron survival against experimental stroke and seizures, using gene transfer with defective HSV vectors.

Materials and Methods Materia Is Male Sprague-Dawley rats were obtained from Charles River (Wilmington, MA) for the ischemia experiments, and from Harlan Sprague Dawley (Indianapolis, IN) for the KA experiments. Vero cells (African green monkey kidney cells; ATCC CCL81) were obtained from American Type Culture Collection (Rockville, MD). Lipofectamine was from Life Technologies (Gaithcrsburg, MD). Vectastain ABC kits (peroxidase mouse IgG and peroxidase rabbit IgG), biotinylated horse anti-mouse antibody, and 3,3’-dianinobenzadine tctrahyrochloride (DAB) kit were from Vector Labora- tories (Burlingame, CA). 5’-Bromo-4-chloro-3-indolyl-P-~- galactopyranoside (X-gal) was from Molecular Probes (Eu- gene, OR). Monoclonal antibody against HSP72 was from Amersham (Arlington Heights, IL). KA, cresyl violet acetate, and chemicals for making buffers and salt solutions were obtained from Sigma Chemical (St Louis, MO). 3-0 nylon monofilament suture was from Ethicon (Somerville, NJ). phsp-8 was provided by Drs S. M. Massa and F. R. Sharp (University of California, San Francisco, CA). HSV mutant d120 and cell line E15 were provided by Dr N. A. DeLuca (University of Pittsburgh, Pittsburgh, PA). The Instat 2.01 statistics program was from GraphPad Software (San Diego, CA), and Statview 512+ was from Brain Power (Calabasas, CA) .

Generation of HSV Vectors The construction of amplicons pa22pgala4hsp72 and pa22pgal has been described el~ewhere.’~~~’ In brief, the amplicon plasmid pa22pgala4hsp72 contained the rat hsp72 gene (ph~p-8)~’ and the Escberichia coli lacZ gene under the control of the HSV tr4 and a22 promoters, respectively. The HSV oriS and the “a” sequence were also included to pro- vide the necessary cis-signals for replication and packaging of the amplicon DNA. pa22Pga1, which lacks the bsp7.2 sequence, was used to generate control vectors. Proto- cols for generating viral vectors have been described in de-

tail? In brief, vectors were generated by transfection of pa22pgala4hsp72 or pa22pgal into E5 cells using Lipo- fectamine according to the manufacturer’s protocol. Twenty- four hours after transfection, the cells were superinfected with helper virus d120 (HSV-1 strain KOS)30 at a multiplic- ity of infection of 0.03, 0.1, or 0.3. The cells were harvested when 100% cytopathic effect developed. Some stocks were further purified by centrifugation at 1,800 g for 10 minutes, and the supernatants were spun at 70,000 g for 18 hours through a 25% sucrose cushion in phosphate-buffered saline (PBS) by using an SW41 rotor. The resulting pellets were resuspended in PBS. The amplicon plasmids were denoted with the prefix p , and the defective vectors thus generated were denoted with the prefix u. The titers of helper virus were determined on E5 cells by using a standard plaque assay. The titers of amplicon vectors were determined on Vero cells by quantifying the number of P-galacatosidase @gal; the gene product of hcZ) expressing cells. For va22pgala4hsp72 (HSP72 vector), titers were as follows: 1.1 to 3.3 X 10’ amplicons/ml and 6.6 X 10‘ to 6.8 X lo7 helper virus titer/ml. For va22Pgal (control vector), titers were 5.1 to 8.1 X 10‘ amplicon/ml and 2.6 to 5.0 X 10’ helper virus titer/ml.

Focal Cerebral Ischemia Experiments VECTOR DELIVERY. Animals were anesthetized with 1 to 2% halothane by face mask and placed in stereotaxic frames. Injection sites were identified over each hemisphere. Coordi- nates from bregma were: AP = 0, ML = 3.5 mm with two injection sites at DV = 4.5 and 3.5 mm. Each injection consisted of 3 pl of vector. Either HSP72 or control vectors were directly injected bilaterally into the striata of rats 12 hours before middle cerebral artery (MCA) occlusion. The striatum was chosen for injection as this region consistently shows signs of injury in this model.

TRANSIENT FOCAL ISCHEMIA MODEL. Rats weighing 290 to 310 g were anesthetized by face mask with 2% halothane plus oxygen and air supplied in a ratio of 0.2 L/min:0.8 L/min. Once surgical levels of anesthesia were attained (assessed by absence of hind leg withdrawal to pinch), halothane was decreased to 1 to 1.5% and anesthetic levels were reassessed every 15 minutes throughout the remainder of the procedure. Ischemia was induced by using an occluding in- traluminal A cervical midline incision was made and the left carotid artery and branches were isolated. The common carotid artery, external carotid, and pterygopalatine were identified and ligated. An aneurysm clip was placed on the proximal internal carotid artery and an arteriotomy was made on the distal common carotid artery. An uncoated 30- mm-long segment of 3-0 nylon monofilament suture with the tip rounded by a flame was inserted into the arteriotomy. The aneurysm clip was removed and the suture was ad- vanced under direct visualization into the internal carotid artery approximately 19 to 20 mm from the bifurcation in order to occlude the ostium of the MCA.

The occluding suture was kept in place for 60 minutes. At the end of the ischemic period, the suture was removed and the surgical incisions were closed. The animal was allowed to recover, then transported to the intensive care unit at the

Yenari et al: In Vivo Gene Transfer of HSP72 585

animal facility for postoperative monitoring. Forty-eight hours later, the animal was killed by barbiturate overdose, then perfused transcardially with heparinized saline followed by 3% paraformaldehyde. Brain sections were prepared for histological analysis.

HISTOPATHOLOGY/CELL COUNTS. After postfixing in 3% paraformaldehyde/20% sucrose solution for 1 or 2 days, 25-pm frozen sections in the coronal plane were taken at 100-pm increments 0.5 mm anterior and 0.5 mm posterior to the infusion sites. Sections were costained with X-gal (to identify vector-infected neurons expressing Pgal) and cresyl violet (to define cellular morphology). The number of transfected neurons was countcd at 4 0 X magnification by an in- vestigator blinded to treatment. Although these vectors preferentially infect neurons, the vector can also be taken up by ependymal and endothelial cells; therefore, cells were counted only if they were contained within the striatum, were X-gal- positive, and possessed characteristic neuronal morphology. The number of surviving neurons was expressed as the ratio of positive blue neurons in the ischemic striatum compared with the contralateral nonischemic striaturn.

Because the extent of vector-mediated infection is limited to only a few hundred striatal neurons within 500 p m of the injection site, it is not expected that overall infarct size would be affected. O n the other hand, improvement in vector- infected striatal neuron survival could be explained by smaller infarcts in one group compared with the other. To confirm that improved striatal neuron survival was not due to an imbalance between groups with regard to the severity of ischemia, the cresyl violet-stained sections were inspected for injury both by gross visual inspection and examination of brain sections at high power (40X objective). Brains were assigned a numeric score on a scale of 0 to 3 with O representing no damage and 3 representing severe damage with involvement of the striatum and surrounding cortex. Animals with no visible damage (score of 0) were excluded from the analysis.

Kainic Acid Experiments VECTOR DELIVERY. Rats (275 to 340 g) were anesthetized with a mixture of ketamine 100 mg/ml/acepromazine 10 mg/ml/xylazine 100 mglrnl (10:2:1, by volume) given intraperitoneally. Vector was stereotaxically microinfused into both hippocampi of each rat (coordinates from lambda: AP, +4.1; ML, 22.0; DV, -3.0; with bregma = lambda). Each rat was microinfused with 2 p1 of inoculum containing HSP72 vector on one side and control vector on the contralateral side.

KAINIC ACID SEIZURE MODEL. At 15 to 17 hours postinfection, rats received 8 to 10 mg/kg KA intraperitoneally. At 24 hours after KA injection, rats were killed and 30 p m thick brain sections werc prepared as before, taking every third section for analysis. These sections were also costained with X-gal and cresyl violet. Brains of rats that died before this time were not analyzed.

ANALYSIS OF VECTOR EFFECT. Brains in which there was no detectable injury, using light microscopy, were excluded

from analysis, as were brains in which vector targeting (identified by X-gal staining) was not contained within the hippocampal formation. Brains with the most severe injury were included. The effect of the HSP72 versus control vector on neuronal survivorship was assessed in the dentate gyrus. This area was chosen for study because seizure discharges from dentate granule neurons may be the primary cause of KA- induced injury32 and because our vector preferentially infects the dentate The number of surviving, vector- infected cells were counted and expressed as a percentage of the average number of surviving neurons in sham control rats, which received the corresponding vector but not KA. The average survival was then calculated and then compared between groups.

HSP72 Protein Expression in Hippocampal and Striatal Neurons Vector (HSP72 or control) was injected into the striatum or hippocampus at the coordinates described for the above experiments. For studies of striatal neuron HSP72 expression, animals were also subjected to 1 hour of MCA occlusion at 12 hours postinjection. At various times postinjection, rats were killed and brain slices were either immunostained to identih HSP72 protein expression, or processed with X-gal to identify Pgal expressing neurons. Frozen sections (30 p m thick) were prepared as described above and stained with X-gal and cresyl violet. The intervening slices were collected in PBS-filled wells of a 24-well tissue culture plate. Free- floating slices were treated in 0.5% Triton X-100 with 30% hydrogen peroxide (HZ02) in PBS for 20 minutes, then blocked for 1 hour in 1.5% horse serum or 0.5% milk in PBS and incubated with anti-HSP72 mouse monoclonal antibody (1:500) for 1 hour at room temperature. Slices were rinsed and incubated with secondary antibody (biotinylated horse anti-mouse, 1 :200), then treated with Vectastain ABC reagent and DAB.

Statistical Analysis Standard statistical methods were used to analyze data. For both the ischemia and the KA models, differences between groups were determined by using Student’s t test. Nonpara- metric tests (eg, Mann-Whitney) were used to compare differences between HSP72 and control groups with respect to numeric infarct scores for the ischemia study. Statistical sig- nificance was determined at the p < 0.05 level. All data are presented as mean 2 SEM values.

Results Protection Against Focal Ischemia A total of 22 animals were studied with 3 excluded because of no infarct. Two had received HSP72 vector and 1 had received control vector. Among the 19 animals included in this analysis, infarct grades were no different between treated and untreated groups, imply- ing that the severity of ischemia was similar between groups (mean infarct scores for HSP72: 1.8 5 0.3, n = 11; vs control vector: 2 2 0.5, n = 8; p = 0.38).

Sttiatal neuron survival was improved among animals receiving HSP72 vector compared with animals

586 Annals of Neurology Vol 44 No 4 October 1998

Fig 1. Several surviving vector targeted, X-gal-stained neurons (arrows) are observed within thhr nonischemic (A) and ischemic (B) striata of an animal injected with HSP72 (72-kd heat shock protein) vector (va22Pgah4hsp72). In the striata o f an animal given control vector (va22pgal), X-gal-stained neurons are seen within targeted striatal neurons on the nonischemic side (C), whereas fewer X-gal-stained cells are observed on the ischemic side (0). (Scale bar = 100 pm.)

receiving control vector. Within nonischemic striata, the number of X-gal-positive neurons was similar between groups, with an average of 306 -t 54 positive neurons among the group given the HSP72 vector and 343 2 65 among those given the control vector. Within the ischemic striata, there were 285 -t 65 X- gal-positive neurons among the HSP72 group, and 206 ? 36 among controls. The percent survivorship of striatal neurons transfected with the HSP72 vector was 95.4 2 10.9% of the stained neurons in the control striatum, whereas among control vector-treated animals, only 62.3 2 5.5% of the stained striatal neurons remained ( p < 0.05) (Figs 1 and 2).

Protection Against Kainic Acid-Induced Hippocampal Damage Rats given KA developed characteristic staring spells, masticatory movements, and wet-dog shakes followed by convulsive seizures 1 hour postinjection. When brains were examined 24 hours after KA administration, there was widespread damage to the hippocampus, including nearly total ablation of CA3 with decreased density of CA1 and dentate granule cells. After

excluding hippocampi with no detectable injury or poor vector targeting, 8 HSP72 vector-injected and 6 control vector-injected hippocampi were available for analysis. Among sham control animals not receiving KA, 12 HSP72 vector-injected and 9 control vector- injected hippocampi were available for comparison.

Treatment with the HSP72 vector attenuated cell death in hippocampal dentate neurons of KA-receiving rats (Fig 3 ) . Survival of control vector-infected hippocampal neurons was only 21.9 * 9.1% in rats that received KA (compared with the sham controls). In contrast, survival of HSP72 vector-infected hippocampal neurons was 64.4 ? 15% ( p = 0.04, Student's t test).

HSP72 and Pgal Coexpression and Temporal Profile We have shown previously that infection with HSP vector led to coexpression of HSP72 and @gal in cultured hippocampal neurons.18 We wished to confirm these observations in vivo, and establish whether the control vector induced HSP72. HSP72 vector injection into the hippocampus led to coexpression of HSP72 and Pgal in the hippocampus 12 hours postinfection


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Fig 2. HSP72 (72-kd heat shock protein; Hsp72) overexpression improves striatal neuron survival in focal cerebral ischemia. Injection of HSP72 vector (va22Pgah4hsp72) improves striatal neuron survival compared with injection of control vector (va22&al) in rats subjected to I hour o f middle cerebral artery occlusion followed by 47 hours of reperjuion. Data represent the percentages of X-gal-stained neurons remaining in the ischemic (L) striatum relative to the contralateral nonischemic (R) striatum (*p = 0.03).

(Fig 4) . After control vector injection, pgal expression was observed, but not HSP72 (Fig 5) .

Expression was also assessed in nonischemic striatum at 13, 18, 36, and 60 hours posttransfection with HSP72 vector. Pgal expression was observed with X-gal staining at all time points. In contrast, HSP72 protein expression persisted as late as 18 hours after injection, but was no longer observed at 36 and 60 hours posttransfection (data not shown).

HSP72 protein expression within ischemic striata was also studied. Again, infected cells stained positive for X-gal at all time points. HSP72 immunostaining was also observed at all time points; however, endogenous HSP72 induction in ischemic striata was also observed. By using these immunocytochemical tech- niques, it was not possible to distinguish vector-driven versus endogenous expression.

Discussion HSP72 appears to be up-regulated in a temporal and graded fashion after cerebral ischemia721o and KA ad- mini~ t ra t ion .~~ With global cerebral ischemia, HSP72 expression is highest within cell populations most vulnerable to ischemic injury. After brief periods of focal cerebral ischemia, expression occurs within potential penumbral zone.?"35 but is absent after infarction,9.36,37 HSP72 is induced after KA administration within brain regions known to be injured in this mod-

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Hsp72 Control

Fig 3. HSP72 (72-kd heat shock protein; Hsp72) overexpression improves hippocampal dentate neuron survival against systemic kainic acid (IO)-induced injury. Injection of HSP72 vector (va22Pgah4hsp72) improves hippocampal dentate neuron survival compared with injection o f control vector (vcu22Pgal) in animals given 8 to 10 mg/kg k2 intraperitoneally. Data represent the numbers of X-gal-stained neurons remaining in the injured hippocampus among animals receiving KA compared with others that did not receive KA (*p = 0.04).

e1.38,39 HSP72 expression has also been found to be significantly decreased in vulnerable brain regions after global i ~ c h e m i a ~ , * ~ and prolonged limbic seizures.39 Others have also found expression regardless of the fate of the cell," leading many to believe that HSPs were little more than markers of stressed cells.

Using gene transfer, we now show that viral vector- mediated overexpression of HSP72 improves neuron survival against transient focal cerebral ischemia and KA-induced injury. These findings are consistent with those of others who found that in vitro HSP72 over-

and transgenic animals overexpressing HSP7224-26 were protected against various ischemic insults. Our observations are also consistent with studies where preischemic induction of the stress response led to protection against both f ~ c a l ' ~ ' * ~ and global 16,4243 cerebral ischemia. Chen and associates15 showed that the temporal pattern of tolerance to focal cerebral ischemia correlated with the temporal expression of HSP72. Because they studied correlation rather than causation, it is possible that other factors could have contributed to their observations.

HSP72 vector infection attenuated injury to dentate gyrus neurons in a model of KA-induced seizures. This is in contrast to our previous results in culture that showed that glutamate-induced toxicity in cultured hippocampal neurons was not affected by HSP72 over-


Fig 4. Coexpression of pgal (p-gahctosidase) and HSP72 (72-kd heat shock protein) occurs 12 hours after vector injection. Hippocampus from an animal injected with HSP72 vector ( v a 2 2 ~ g a h 4 h ~ 7 & demonstrates coexpression o f pgal (A) and HSP72 protein (B) 1 2 hours later. Positive X-gal (a chromogenic substrate of pgal) staining of hippocampal dentate neurons is observed (A) with an adjdcent section (B) showing positive HSP72 protein immunostaining o f neurons in the same distribution.

expression, using an identical vector.18 This may be due to the in vitro model being even more severe than the present one, with direct application of an excitotoxin. Cells in culture are directly exposed to high con- centrations of glutamate, resulting in overwhelming and acute excitotoxin exposure, whereas KA administered intraperitoneally does not completely cross the blood-brain barrier.44 An alternate possibility is that injury in the KA model may have been due to physi- ological disturbances from the seizures, rather than direct effects of the excitotoxin.

We chose to assess the number of hippocampal dentate granule neurons in the KA experiments, because our vector tends to infect dentate gyrus neurons after hippocampal The preferential transfection of dentate neurons may be due to specific cell re- ceptors that aid in the binding of the virus.46 Systemic KA administration in our model led to diffuse injury to the hippocampal formation, including the dentate

Fig 5. Control vector does not induce HSP72 (72-kd heat shock protein) expression. Brain sections j$om an animal were injected with control vector (vcu2Zpgal). Vector-targeted hip- pocarnpal neurons stain positive f i r X-gal (A) but do not stain f i r HSP72 protein (B) 12 hours after transfection.

gyrus, with decreased transfected neuron survival to 22% among controls. As the dentate gyrus has projec- tions to CA3/4 neurons, and presynaptic dentate neurons contribute to KA-induced damage, we hypothe- sized that increased survival of dentate gyrus neurons conferred by HSP72 overexpression might also lead to protection of the CA3/4 neurons. In this study, we did not find any changes in overall survival of CA3/4 neurons (data not shown), although this may be due to the small number of neurons infected by the vector.

The HSP72 vector used for this experiment contains sequences for hsp72 and IacZ under two separate promoters. Our study showed that HSP72 and Pgal were coexpressed in vector-infected neurons in vivo for at least 18 hours postinjection. This is consistent with previous in vitro results showing coexpression with this vector for at least 40 hours18 and in vivo results with similar vectors coexpressing Pgal and glucose transporter or BCL-2 for at least 24 h o ~ r s . ~ ~ , ~ ’ It is inter- esting that by 36 hours, we no longer detected HSP72 expression immunohistochemically, whereas infected cells continued to display Pgal staining for at least 60


hours. This could be explained in the following several ways: (I) Pgal may have a longer tissue half-life compared with other proteins such as HSP72. Indeed, @gal has been known to persist in eukaryotic cells long after protein translation has ceased.4824‘ (2) HSP72 protein detection by immunohistochemistry may have failed. This is unlikely, as HSP72 protein immunostaining (most likely endogenous) was detected within ischemic regions even when nonischemic, vector-targeted regions failed to stain in the same brain sections. (3) The du- ration of expression by the two promoters may be different. However, both the a4 and a22 promoters are immediate early promoters of HSV, and would be expected to show similar, although not identical, kinetics of expre~sion.~’ It is also conceivable that improvement in neuron survival is due to other genes expressed by d120; however, these genes are not represented in the plasmid constructs. The control vector contains the same genes as the HSP72 vector minus the hsp72 sequences; therefore, the improved survival with the HSP72 vector is in addition to any beneficial effects of the control vector.

Others have shown that endogenous HSP72 protein expression begins 4 to 6 hours after i n s ~ l t . ’ , ~ ~ ~ ~ ’ ~ ~ 1,52

In the present study, ischemic and excitotoxic insults were applied 12 hours after vector injection, and experiments showed coexpression of HSP72 and Pgal in vector-infected neurons at that time, and for a least 6 more hours. Therefore, vector-mediated HSP72 overexpression was present before endogenous HSP72 induction. This may be a critical period during which stress proteins are needed to permit cell survival.

These studies show that preinsult overexpression of HSP72 improves cell survival in stroke and epilepsy models. For gene transfer therapy to have significant clinical relevance, future studies should explore whether the HSP72 overexpression can protect neurons when administered after insult. This has already been studied by using vectors expressing the protooncogene bcl-2. Gene transfer of bcl-2 was neuroprotective against glutamate toxicity in cultured neurons when given 8 hours later,53 and against MCA occlusion when given 1.5 hours later.54,55

Although we show that gene transfer therapy is fea- sible, it is currently limited by the regions that these viral vectors can infect and the route of administration. It is not possible for vector-mediated HSP72 overexpression to attenuate overall infarct size or alter overall hippocampal neuron densities and is likely due to the relatively small number of neurons infected by our vectors. Future experiments should explore vectors that can be purified to higher titers or improved vector delivery systems. In the meantime, viral vector-mediated gene transfer offers a unique tool for answering impor- tant biological questions.

This study was supported by NIH NINDS grants RO1 NS 27292 (G.K.S.) and KO8 NS01860 (M.A.Y.), an MSTP grant from the National Institute for General Medical Sciences (S.L.F.), a summer research fellowship from the Howard Hughes Foundation (L.K.C.), the Adler Foundation (R.M.S.), and Bernard and Ronni Lacroute (G.K.S.).

We thank Drs S. M. Massa and F. R. Sharp for providing the phsp-8 clone, and Dr N. A. DeLuca for d120. We are grateful to A. Iyer, A. Gupta, T. Park, J. McLaughlin, and H. Karadjuzovic for technical assistance.

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Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy