Neuroprotective effects of HSP70 overexpression after cerebral ischaemia—An MRI study

Experimental Neurology 195 (2005) 257 – 266 www.elsevier.com/locate/yexnr

Regular Article

Neuroprotective effects of HSP70 overexpression after cerebral ischaemia—An MRI study Louise van der Weerda,b,*, Mark F. Lythgoea, Romina Aron Badina,b, Lauren M. Valentimb, Mohammed Tariq Akbarc, Jackie S. de Bellerochec, David S. Latchmanb, David G. Gadiana a

RCS Unit of Biophysics, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK b Medical Molecular Biology Unit, Institute of Child Health, University College London, UK c Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Faculty of Medicine, Charing Cross Hospital, Imperial College London, UK Received 3 February 2005; revised 4 April 2005; accepted 7 May 2005 Available online 4 June 2005

Abstract Heat shock proteins (HSPs) have been reported to increase cell survival in response to a wide range of cellular challenges. However, the role of HSP70 overexpression is still a matter of debate, with some reports showing protection and others not. In order to resolve these discrepancies and further investigate the action of these proteins in vivo, transgenic mice overexpressing HSP70 have been compared to wildtype mice in a middle cerebral artery occlusion model of permanent cerebral ischaemia. Previously, the effect of HSP70 was assessed histologically postmortem. In this report, magnetic resonance imaging (MRI) was used to assess the mice in vivo after the onset of stroke. The lesion volume, as measured at 24 h using T2-weighted MRI, was significantly smaller in HSP70 transgenic mice compared with wildtype mice. The smaller lesion size in HSP70 transgenic mice could not be attributed to differences in vascular anatomy or in cerebral blood flow during occlusion. Additionally, the apparent diffusion coefficient showed different spatial and temporal patterns between the groups, suggesting that the damage within the lesion may be less severe for HSP70 transgenic mice. Thus, we conclude that overexpression of HSP70 reduces the overall lesion size and may also limit the tissue damage within the lesion. D 2005 Elsevier Inc. All rights reserved. Keywords: Focal ischaemia; Heat shock protein; MRI; Apparent diffusion coefficient

Introduction Heat shock protein 70 (HSP70) is one of the major inducible stress proteins that are upregulated in response to a wide range of cellular challenges, including ischaemia. The molecule is thought to act as a molecular chaperone, assisting in the refolding of aberrant proteins or targeting them for disposal into aggresomes. A neuroprotective role for HSP70 has been shown both in vitro and in vivo for a number of conditions (e.g., hypoxia, ischaemia, nutrient deprivation; see Giffard and Yenari, 2004). Recently, a

* Corresponding author. Fax: +44 20 7905 2358. E-mail address: [email protected] (L. van der Weerd). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.05.002

number of transgenic mice models have been developed to assess the effect of HSP70 expression in more detail. However, the results from these studies are not clear-cut. Some studies report a significant reduction in lesion size for HSP70-overexpressing mice after permanent or transient middle cerebral artery occlusion (MCAO) (Rajdev et al., 2000; Tsuchiya et al., 2003). In line with these reports, HSP70 knockout mice show a larger infarct area after transient MCAO (Lee et al., 2001b). In contrast, a study by Lee et al. (2001a) does not show any reduction of the lesion area in HSP70-overexpressing mice, whereas Plumier et al. (1997) report an increase in neuronal survival, though no difference in lesion size. All these studies used histological assessment of tissue damage, usually between 6 and 24 h after the initial insult.

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However, physiological changes, such as cerebral hypoperfusion and cytotoxic oedema, occur much earlier. The resulting change in intra- and extracellular environments may provide an opportunity to monitor cells destined for infarction using magnetic resonance imaging (MRI) parameters, in particular using the apparent diffusion coefficient of water (ADC). ADC decreases occur within minutes following stroke, and are closely associated with cytotoxic oedema and impaired energy metabolism; at later stages, the ADC pseudonormalises or increases, which is attributed to vasogenic oedema and the loss of cellular barriers (Busza et al., 1992; Moseley et al., 1990; Van Bruggen et al., 1994). ADC is particularly useful when used in conjunction with other magnetic resonance techniques, such as perfusion measurements, which yield information on the cerebral blood flow, and T1 and T2 measurements (Calamante et al., 1999; Van der Weerd et al., 2004). At subacute stages, increases in the latter parameters have been shown to correlate well with tissue water content and the development of vasogenic oedema (Van Bruggen et al., 1994). In our study, transgenic mice overexpressing HSP70 (HSP70tg) (Angelidis et al., 1991; Plumier et al., 1995) have been compared to wild-type (WT) mice in an MCAO model to further examine the role of HSP70 in cerebral ischaemia. We focused on the overall lesion volume and on several MRI parameters to assess the severity of injury. The total lesion size was estimated after 24 h of ischaemia using a multi-slice T2-weighted scan. Multi-parameter MRI at several time points was used to compare the temporal and spatial evolution of ADC, perfusion, T2 and T1 for HSP70tg and WT mice.

Materials and methods Animals All animal care and procedures were in accordance with the UK Animals (Scientific Procedures) Act 1986. The transgenic mice were a kind gift from Dr. Angelidis, University of Ioannina, Greece. The mice (HSP70tg) were produced in the Hellenic Pasteur Institute’s Facilities from F1 mice derived from crossing between original inbred CBA and C57B1/6 mice. The transgenic construct contains the human inducible hsp70 gene under the regulation of the h-actin promoter (Angelidis et al., 1991; Plumier et al., 1995). Homozygous offspring of the founder mouse 2960 were selected by Southern blot or slot blot hybridisation for transgene expression. CBA  C57B1/6 F1 crossings were used as controls (WT).

Koizumi model (Koizumi et al., 1986). Briefly, mice were anaesthetised with 2.5% isoflurane and maintained on 1.75% isoflurane with pure oxygen. The rectal temperature was maintained at 36.8 T 0.3-C throughout the procedure. A ventral neck incision was made to expose the right carotid artery. A 6-0 monofilament suture (Monocryl; Ethicon, UK) with a 180- to 200-Am rounded tip was advanced from the common carotid artery into the internal carotid artery past the MCA junction (approximately 9 mm). The incision was sutured, after which the animals were immediately transferred to the MRI scanner to confirm the occlusion by ADC and cerebral blood flow (CBF) measurements. After all scans were completed, the animals were recovered and transferred to the animal house, with free access to food and water. At 24 h, the mice were re-anaesthetised and scanned again. Two HSP70tg animals and one WT animal did not show any CBF or ADC reduction in the ipsilateral hemisphere immediately after MCAO and were excluded from the experiment, which left 6 animals in both groups. Postmortem examination of the excluded animals confirmed that the intraluminal suture was not in the correct position. MRI Coronal images were obtained approximately 0.5 mm from bregma at 1 h, 2 h and 24 h after the onset of stroke. MRI measurements were performed on a 2.35-T horizontal bore magnet (Oxford Instruments, Oxford, UK) interfaced to a SMIS console (Guildford, UK). The radio frequency (RF) pulses were transmitted with a volume coil of 6.5 cm length. The signal was received with a separate 1-cmdiameter surface coil. The following imaging parameters were used for all measurements: FOV 32  16 mm, 2 mm slice thickness, and 128  64 pixels. After MCAO, the animals were transferred to the magnet and imaged using the following protocol: 1h 2h

24 h

ADC (trace-weighted SE-EPI, with b values of 38 and 1187 s/mm2; TR 1500 ms; TE 58 ms; 64 averages) ADC as before CBF (continuous arterial spin labelling, inversion slice positioned in the neck, 1.5 mm from the back of the cerebellum; inversion time 3 s; delay time 500 ms; TR 1000 ms; TE 36 ms; 44 averages; see Alsop and Detre, 1996) T1 (IR-EPI, 8 inversion times between 200 and 3500 ms; TR 2000 ms; TE 18 ms; 22 averages) T2 (SE-EPI, 2 echoes with TE 24 and 66 ms; TR 1500 ms; 16 averages) ADC, CBF, T1, T2 as before Anatomical image (multi-slice T2-weighted SE; FOV 20 mm; slice thickness 1 mm; 9 slices; TR 1500 ms; TE 120 ms; 8 averages)

MCAO model Image processing Adult male (24 – 28 g; 10– 13 weeks old) HSP70tg mice (n = 8) and WT mice (n = 7) were subjected to permanent MCAO using an intraluminar suture method based on the

Quantitative ADC, T1 and T2 maps were calculated using dedicated algorithms, written in IDL (Research Systems

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Inc., Boulder, CO, USA). Perfusion maps were obtained by subtracting the labelled image from the control image. The T1 data were used to calculate the cerebral blood flow (CBF) based on the equation in Alsop and Detre (1996). For the calculation of CBF, the following values were used: T1a = 1.5 s, k = 0.9, efficiency of the spin labelling pulse = 0.71, tissue transit time = 1 s. The lesion area was determined manually by an observer blinded to the experiment, using the multi-slice anatomical T2-weighted scan, in the SMIS image analysis program. The relative infarcted area per slice was defined as the ratio of the lesion area to the size of the contralateral hemisphere. Images were analysed on a region-of-interest (ROI) basis. Ten ROIs were placed in the medial cortex, the dorsolateral cortex, the ventrolateral cortex, the lateral caudate putamen and the medial caudate putamen of each hemisphere (Fig. 1). The ADC, T1, T2 and CBF of the affected hemisphere were expressed relative to the contralateral side. Relative ADC maps were generated by determining the average ADC of the contralateral hemisphere, and subsequently normalising the ADC maps using this value. The normalised ADC maps were segmented into 6 pixel classes: >90%, 80 –90%, 70 – 80%, 60– 70%, 50 –60% and <50% of contralateral ADC values. Cerebrovascular anatomy To determine whether the groups differed with respect to vascular anatomy, 4 naı¨ve HSP70tg and 4 naı¨ve WT mice were randomly selected. The animals were anaesthetised with 2.5% isoflurane in 100% O2. The animals were transcardially perfused with approximately 1 ml Indian ink until the tongue turned black. The thoracic aorta was clipped to prevent perfusion of the rest of the body. The brains were removed and fixed in 4% paraformaldehyde. The cerebrovascular anatomy, in particular the MCA territory, was inspected with a surgical microscope. The appearance of the proximal part of the MCA was rated based on the branching patterns of the extending surface branches, with score 0 when no proximal surface branches were present, score 1 for one or two small surface branches and score 2 for one or more large surface branches, similar to the characterisation used by Niiro et al. (1996). The presence of posterior communicating arteries (PCommA) was assessed using a

Fig. 1. T2-weighted scan showing the selected regions of interest.

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semiquantitative score based on that used by Murakami et al. (1998). A score of 2 indicated a normal PCommA, 1 an arthretic PCommA and 0 an absent PCommA. In situ hybridisation For in situ hybridisation studies, naive untreated animals were killed and brains were rapidly removed then frozen on dry ice. Sections were cut (10 Am on a cryostat; Bright Instruments), thaw-mounted onto Superfrost slides (VWR) and stored at 80-C until use. For detection of human HSP70 mRNA, a 30-mer antisense oligonucleotide (SigmaGenosys Ltd.) complementary to nucleotides 850– 879 of the human HSP70 gene was used (Hunt and Morimoto, 1985). Oligonucleotide probes were 3V end-labelled using terminal deoxynucleotidyl transferase (Promega) and [35S]dATP (Amersham Pharmacia Biotech). Cryostat sections were processed and in situ hybridisation was carried out as previously described (Akbar et al., 2003). In brief, slides were fixed in 4% paraformaldehyde in PBS (4-C) for 10 min, rinsed in PBS for 2  5 min and then treated with 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl for 10 min. Following dehydration in increasing concentrations of ethanol, the sections were delipidated in chloroform for 5 min, rinsed in ethanol and allowed to air dry. Sections were then hybridised overnight at 42-C with 2  106 c.p.m. of labelled probe in 100 Al of hybridisation buffer (5 Denhardt’s [0.02% Bovine Serum Albumin, 0.02% Ficoll and 0.02% Polyvinylpyrrolidone], 4 standard saline citrate [SSC], 50% formamide, 10% dextran sulphate, 200 Ag/ml polyadenylic acid, 200 Ag/ml sheared single-stranded salmon sperm DNA, 50 mM phosphate buffer, 100 Ag/ml tRNA and 100 mM dithiothreitol). The following day, sections were stringently washed (1 SSC at room temperature for 30 min, 1 SSC at 55-C for 30 min, 0.5 SSC at 55-C for 30 min, 0.1 SSC at 55-C for 30 min and 0.1 SSC at room temperature), dehydrated in an ascending series of ethanols and air-dried. When dry, sections were exposed to X-ray film (BioMax MR, Eastman Kodak Co.) for 17 days and developed. Western blots Overexpression of HSP70 was checked by Western blot. The brains from 3 HSP70tg and 3 WT naı¨ve animals were removed and the basal ganglia, the dorsolateral cortex and the ventrolateral cortex were dissected and homogenised in 0.1% Tween extraction buffer. The same was done for 3 out of the 6 mice in each group that underwent MCAO. Following SDS-polyacrylamide gel electrophoresis, monoclonal anti-HSP70(human) primary antibody (1:1000, clone number G92F3A-5, Stressgen) and anti-mouse HRP secondary antibody (1:2000, DAKO) were used to measure relative levels of HSP activation in the two selected areas. The signal was visualised by an enhanced chemoluminescence kit (ECL, Amersham Biotechnologies) and exposing

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the membranes to X-ray film. The bands were quantified in a GS-800 calibrated densitometer using QuantityOne Version 4.2.1 software (BioRad, USA). Statistics The lesion volume per slice was compared using a degrees of freedom adjusted repeated measures ANOVA. The CBF and relaxation time data were evaluated for every time point. The ADC data were analysed using the difference between time points as a summary measure; a two-way repeated measures mixed model ANOVA was used (SPSS 12.0.1, SPSS Inc., Chicago, USA). For each analysis, the appropriate within-subject variables and between-subject factors were adopted. Differences were considered significant when the P value was less than 0.05. The HSP70 expression levels, as determined by Western blots, were analysed using standard t tests. Wherever a multi-comparison correction was appropriate, a Bonferroni correction of the significance level was performed, as stated in the text. All data are presented as mean T SD.

Results Characterisation of HSP70tg mice Previous reports showed no obvious effects of the genetic modification on the development or survival of the mice (Plumier et al., 1995, 1997). Gross observation of HSP70tg mouse brains did not show any obvious differences in size or structure when compared to the control group. India ink studies did not reveal any significant differences in PCommA between the groups (Table 1). Scores 0 and 1 can be classified as a hypoplastic Pcomm artery, whereas score 2 indicates a normal, and functional artery; all mice tested showed an incomplete circle of Willis, with one or both posterior communicating arteries missing. The proximal branching patterns of the MCA were assessed as well. There is a large variation in MCA branching Table 1 Rating of the cerebrovascular appearance after India ink perfusion Animal

WT

1 2 3 4

Lesion volume

HSP70tg

Pcomm

MCA

Pcomm

between individual animals, but there are no significant differences between the transgenic and the control group. Furthermore, the T2-weighted MRI data after focal ischaemia were used to quantify the brain volume for each animal, and no differences were found between the groups (368 T 25 mm3 and 398 T 48 mm3 for WT and HSP70tg mice, respectively). The expression of the human HSP70 transgene was characterised by in situ hybridisation. The oligonucleotide probe used in the present study has been previously characterised in rat CNS tissue (Akbar et al., 2001) and has 100% nucleotide identity to the human HSP70 gene. The hybridisation signal obtained was RNase-A sensitive (data not shown) and was absent from sections of naı¨ve, unstressed wild-type mice lacking the human HSP70 transgene (Figs. 2A, B). Expression of human HSP70 mRNA was extensive throughout cerebral cortex, cerebellum and particularly intense in CA1 and CA3 of hippocampus, dentate gyrus, striatum, septum, paraventricular thalamic nucleus anterior, medial habenula nucleus and dorsomedial hypothalamic nucleus (Figs. 2C, D). Moderate levels of expression were seen in the inferior colliculus and periaqueductal grey. Less expression was seen in thalamus and white matter (Figs. 2C, D). Therefore, these transgenic mice clearly contain high levels of inducible HSP70 mRNA in the naı¨ve state. A separate group of naı¨ve mice was used to determine HSP70 protein levels using Western blots. Dorsolateral cortex, ventrolateral cortex and basal ganglia were dissected, and HSP70 protein levels were determined for all regions (Fig. 3). No significant differences were found between the dissected areas, so all areas were grouped together for further analysis. HSP70tg mice clearly showed higher expression levels, with mean expression levels being approximately 10 times higher for HSP70tg animals ( P = 4  10 8). Three animals out of each MRI group were used for Western blot analysis after MCA occlusion. MCAO caused a 4-fold increase of HSP70 in the WT animals ( P = 0.0001). After ischaemia, the mean HSP70 expression levels for the transgenic increased by similar amounts in absolute terms, but this increase does not reach significance ( P = 0.1).

MCA

L

R

L

R

L

R

L

R

0 1 1 1

0 1 2 0

2 1 1 1

2 2 0 1

0 2 1 0

1 1 0 0

2 0 2 1

1 1 2 2

The presence of posterior communicating arteries (PCommA) was assessed using a semiquantitative score. A score of 2 indicated a normal PCommA, 1 an arthretic PCommA and 0 an absent PCommA. The appearance of MCA was rated based on the branching patterns of the extending surface branches, with score 0 when no proximal surface branches were present, score 1 for one or two small surface branches and score 2 for one or more large surface branches.

To determine whether HSP70 overexpression confers protection against ischaemic injury, HSP70tg (n = 6) and WT (n = 6) mice were subjected to permanent MCA occlusion. The lesion size was evaluated using a multi-slice T2-weighted anatomical MRI scan after 24 h (Fig. 4A). This type of occlusion induced an ischaemic region in the ipsilateral hemisphere, including the basal ganglia and a large part of the cerebral cortex. A blinded observer quantified the lesion size in all slices. At 24 h after permanent MCA occlusion, the mean lesion area per slice was reduced for the HSP70 mice. Statistical analysis using a

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Fig. 2. Representative in situ film autoradiographs showing expression of human HSP70 mRNA in horizontal (A, C) and coronal (B, D) sections from wild-type mice (A, B) and transgenic mice overexpressing human HSP70 (C, D). There is widespread expression of human HSP70 mRNA throughout the brain in mice expressing the transgene. Scale bar = 2 mm.

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Fig. 4. (A) Representative multi-slice T2-weighted images obtained from one animal 24 h after onset of stroke. The lesion area appears as a bright signal. (B) Effect of HSP70 overexpression on the lesion area after permanent middle cerebral artery occlusion. The lesion area per slice was calculated relative to the contralateral hemispherical area. Mixed model ANOVA showed a significant difference in lesion area between the HSP70tg mice and the WT mice. No interaction was found between the animal groups and the different slices.

mixed model ANOVA indicated that this was a significant difference between the HSP70tg mice and the WT mice ( P < 0.05, Fig. 4B). No interaction was found between the animal groups and the different slices. Overall, the lesion volume was 89 T 13 mm3 for the WT mice compared to 59 T 18 mm3 for the HSP70tg mice; these values were significantly different ( P = 0.008).

Apparent diffusion coefficient

Fig. 3. (A) Representative Western blots showing HSP70 protein levels in WT and HSP70tg mice. Naı¨ve mice were taken for controls. The post-MCAO samples were taken from 3 animals within each MRI group. (B) Quantitative levels of HSP70 expression for individual animals, based on the Western blots. The symbols depict different brain regions (> = dorsolateral cortex; g = ventrolateral cortex; ‚ = caudate putamen).

Apparent diffusion coefficients (ADC) were obtained for one slice, centred around the MCA region at 1 h, 2 h and 24 h after the onset of stroke. As early as 1 h after occlusion, a distinct ischaemic lesion was detected on the ADC images, corresponding topologically to the lesion area as observed in the T2-weighted scan after 24 h. In all animals, occlusion of the MCA produced a marked reduction of the ADC in a large part of the ipsilateral hemisphere, particularly in the dorsolateral cortex and the basal ganglia. All ADC maps were segmented into different classes of ADC reduction to define areas of increasing injury (Fig. 5A). At the acute stage of ischaemia (1 h and 2 h), the WT and HSP70tg mice showed very similar patterns of ADC reduction to 60– 70% of the contralateral value. However, a qualitative inspection shows distinct differences between the groups in the temporal evolution of the lesions; the WT animals show a partial renormalisation of ADC at 24 h, whereas the ADC in large parts of the lesion HSP70tg animals decreases further. To quantify the temporal pattern, 5 regions of interest were selected in each hemisphere (Fig. 1), and the average ADC was calculated for each of these regions. No significant trends were found for the early time points (1 and 2 h) for either group, so the data for these time points were averaged to condense the data into two time points

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Fig. 5. (A) ADC segmentation into regions with different degrees of injury. On the right-hand side, a spin echo (SE) anatomical image is shown for comparison. (B) Absolute ADC values at 1 – 2 and 24 h for the different regions of interest. (C) Difference in relative ADC values at 1 – 2 and 24 h for the different regions of interest. The ADC values are expressed relative to the mean ADC of the contralateral hemisphere.

(Fig. 5B). This plot clearly shows that the actual ADC is reduced throughout the lesion area in both groups for both time points, whereas the contralateral side does not show any significant changes. To analyse the temporal evolution of the ADC, the difference in ADC (DADC) was calculated between 1– 2 and 24 h, relative to the contralateral side for each individual animal (Fig. 5C). A negative value in this plot signifies that the ADC decreased further between 1 – 2 and 24 h compared to the contralateral side, whereas a positive value indicates renormalisation of the ADC between these two time points. The plot shows a striking difference between the groups ( P < 0.001 using a repeated measures two-way mixed model) without a significant interaction between the factors. Cerebral blood flow Successful occlusion of the MCA resulted in a severe reduction of CBF in a large part of the ipsilateral hemisphere, in agreement with the reduced ADC in this area (Figs. 6A, B). In the regions of interest within the ischaemic lesion (2– 5), the CBF fell to 2 T 3% of the

contralateral values after 2 h of occlusion, and decreased even further to undetectable levels after 24 h. There were no significant differences in CBF reduction within the lesion area (regions 2– 5) between the groups at 2 or at 24 h after stroke. Note that the medial cortex, which is primarily supplied by the anterior cerebral artery, also showed a reduction in CBF, which was partially restored after 24 h. However, this reduction did not result in ADC changes in this area (Fig. 5B). Relaxation times After 2 h, ipsilateral T2 values were not significantly different from contralateral values in any of the selected regions (Figs. 6C, D). After 24 h, both groups showed a fairly uniform increase in T2 throughout the dorsolateral cortex and the caudate putamen (regions 2, 4 and 5) of 58 T 15% and 67 T 7% for the HSP70tg and WT mice, respectively. The large standard deviation for the HSP70tg mice reflects the heterogeneity in the lesion area and severity between animals, with not all mice exhibiting lesions that did encompass all selected ROIs. No significant

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Fig. 6. (A, C, E) Typical examples of quantitative CBF, T2 and T1 maps, respectively, at 2 and 24 h after stroke. (B, D, F) Relative CBF, T2 and T1 values for the selected regions of interest. The ipsilateral values are expressed relative to the contralateral values for the corresponding regions of interest.

differences in T2 were found between the WT and HSP70tg mice after Bonferroni multiple comparison correction of the significance level. In all lesion areas, T1 started to rise after 2 h of ischaemia (Figs. 6E, F). This increase continued in the next 24 h, after which the T1 of the ipsilateral ROIs within the lesion (regions 2 –5) was on average 36 T 10% higher for the HSP70tg group and 38 T 7% for the WT group when compared to the contralateral side. There were no significant differences between the groups, or between the selected regions of interest within the groups.

Discussion Constitutive overexpression of the human HSP70 gene in the mouse brain was associated with a marked reduction of lesion volume in a model of permanent focal ischaemia; MRI showed a lesion volume of 89 T 13 mm3 for the WT mice compared to 59 T 18 mm3 for the HSP70tg mice. This is the first study using MRI to evaluate the effects of HSP70 overexpression. Several studies have evaluated the correlation between histological lesion detection, e.g., with TTC staining, and MRI-visible lesions, as seen on T2weighted images or DWI in other mouse and rat models. (Kazemi et al., 2004; Li et al., 2000; Van Bruggen et al., 1994). A close correlation was found between the different techniques, justifying the use of MRI to determine lesion progression in vivo. The protective effect of HSP70 seen in this study is in agreement with several papers demonstrating the cytoprotective effects of HSP70 overexpression in the brain, either by transgenic overexpression or induced by a viral vector

(Hoehn et al., 2001; Kelly et al., 2001; Rajdev et al., 2000; Tsuchiya et al., 2003; Yenari et al., 1998). However, not all reports on HSP70 transgenic mice show obvious protective effects after cerebral ischaemia (Lee et al., 2001a; Plumier et al., 1997). The reasons for the discrepancies between these studies could be due to the severity of the insult and the level of HSP70 overexpression within the different strains. Rajdev et al. (2000) and Tsuchiya et al. (2003) used a strain where HSP70 levels in the brain were 5- to 10-fold higher than normal, resulting in a reduction in lesion size of at least 60% after 24 h of permanent or transient MCAO. A different strain, with expression levels only 2 times higher than normal, was developed by Lee et al. (2001a), and they could not find any protective effects for their transgenic mouse strain after permanent MCAO. Our study used the same strain as Plumier et al. (1997); our in situ hybridisation data showed that, in this strain, human HSP70 mRNA was abundant in the cerebral cortex, hippocampus, striatum and cerebellum of the transgenic mice. HSP70 protein levels are about 10 times higher than in WT mice, i.e., comparable to the Raidev and Tsuchiya strains that showed protection. In an earlier study of the same strain (Plumier et al., 1997), the lesion size was not altered after 24 h of permanent occlusion, though they did observe an improved hippocampal survival. However, their lesion areas were small for a permanent MCA occlusion (21.6 T 18.6% of the coronal slice at bregma 0.3 mm for control mice). Assuming that the area of both hemispheres is equal, that would translate to an infarct size of approximately 43% for the central slice, compared to 76% for our control mice, suggesting that their model actually produced a much milder insult than in our case. A possible cause for this difference may be the smaller suture they used to occlude the MCA. Overall, the emerging

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picture seems to be that the level of HSP70 overexpression strongly influences the amount of protection. The actual HSP70 protein levels in the two groups of mice were determined in different brain areas (dorsolateral cortex, ventrolateral cortex and basal ganglia) using Western blots. The HSP70 antibody detects both human and mouse HSP70; therefore, we cannot distinguish between endogenous and human expression. After ischaemia, the mean HSP70 expression levels for the transgenic and WT animals increased by similar amounts in absolute terms. However, this increase does not reach significance in the transgenic mice ( P = 0.1), possibly because in these animals the endogenous HSP70 is only a fraction of the total HSP70 protein level. There is still disagreement about the extent to which cell death during stroke involves apoptosis or necrosis. The general view seems to be that ischaemic cell death in the brain is a mixture of both processes, and the role of apoptosis may be more pronounced than presumed traditionally (MacManus and Buchan, 2000). HSP70 is known to protect from both necrotic and apoptotic cell death, though the exact mechanisms by which HSP70 protects against cerebral ischaemia are still not resolved (Giffard and Yenari, 2004). Among the protective mechanisms is the function of HSP70 as a molecular chaperone, restoring and stabilising denatured proteins (Beaucamp et al., 1998). Furthermore, HSP70 is a potent antiapoptotic protein that can influence the apoptotic cascade at multiple sites (Beere and Green, 2001; Gabai et al., 1998; Mosser et al., 1997). Recent work shows that HSP70 also influences the mitochondrial apoptotic pathway by regulating cytochrome c release and subsequent caspase-3 activation after cerebral ischaemia (Lee et al., 2004; Tsuchiya et al., 2003). In addition to this upstream inhibition, other studies in tumor cell lines report that, in these cells, HSP70 exerts its protective effect downstream of caspase activation, indicating that the Fpoint of no return_ in apoptosis signalling may, in certain cell types, be later than sometimes thought (Ja¨a¨ttela¨ et al., 1998). Despite these recent insights into the way HSP70 functions, it is still not clear which of its multiple tasks are directly involved in brain protection. The CBF measurements showed a similar decrease in perfusion after MCAO for HSP70tg and WT mice, implying that differences in blood flow cannot account for the difference in lesion size between HSP70tg and WT animals. Some concern has been raised about differences in susceptibility to ischaemic insults for different mouse strains, due to differences in the MCA territory, most frequently in the posterior communicating arteries (Maeda et al., 1998). Our transgenic mouse strain has been derived from a CBA and C57B1/6 crossing, but the mice in this study are several generations removed from the original F1 crossing. Since the transgenic strain is homozygous, the closest control strain we could use was a F1 CBA  C57B1/ 6 cross, but evidently there may be genetic differences other than HSP70 present between the groups. Therefore, we

compared the cerebrovascular anatomy between the groups using Indian ink perfusion. The fact that we did not find any significant differences between Wt and transgenic animals suggests that the protective effect found in the transgenic mice is not due to differences in vascular anatomy. This is corroborated by the CBF measurements, since we would expect the perfusion to reflect differences in vascular territories between the groups. The ADC shows a heterogeneous spatial and temporal response to permanent MCA occlusion. The most profound absolute changes in ADC occurred within the dorsolateral cortex and the lateral caudate putamen for both groups of animals. In this study, the ADC decreased to about 60% of contralateral values, with obvious differences in the time course of the ADC changes between the groups. We originally chose the early ADC time points of 1 and 2 h post-ischaemia based on the hypothesis that HSP70 overexpression would alter the early stress response, and therefore the early time course of ADC reduction. However, no differences in ADC were found between the two time points or the different groups. Therefore, we summarised the diffusion data into relative changes in ADC between 1– 2 and 24 h. The HSP70 mice showed a decrease in ADC within the lesion between 1 –2 and 24 h, whereas for the WT mice, the ADC increases again or remains constant between those two time points. To interpret these time course differences correctly, it is important to take the additional information from CBF, T1 and T2 into account. ADC reflects the dynamics of specific stages of the ischaemic cascade and histomorphological development (Fiehler et al., 2002; Knight et al., 1994). Early ADC changes are thought to be related to an osmotically driven shift of extracellular water to intracellular compartments due to a disrupted ion homeostasis, thus reflecting cytotoxic oedema and impaired energy metabolism (Busza et al., 1992; Moseley et al., 1990). At subacute stages, the ADC pseudonormalises, usually between a day and a week after onset of stroke (Jiang et al., 1993; Knight et al., 1994). At later time points, the diffusion might show a subsequent increase above normal values, or go onto a secondary decrease. Pseudonormalisation and increase of ADC above control values are attributed to the loss of cellular barriers combined with an excessive accumulation of water due to vasogenic oedema (Pierpaoli et al., 1993; Verheul et al., 1992). Pseudonormalisation can be distinguished from ADC normalisation due to tissue recovery using additional relaxation data; the former is accompanied by an increase in T1 and T2 (Gill et al., 1995; Knight et al., 1994; Rudin et al., 2001). Vasogenic oedema typically develops during subacute and chronic stages; correspondingly, increases in relaxation times are predominantly associated with late and irreversible damage. Knight et al. (1994) studied the time dependence of ADC changes, demonstrating that tissues with the most severe histological damage showed the fastest ADC decrease and subsequent normalisation of ADC values, combined with a more dramatic increase in T1 and

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T2. In the light of their paper, our findings suggest that the damage within the lesion may be less severe for the HSP70 mice. This would indicate that overexpression of HSP70 not only reduces the overall lesion size, but may also limit the tissue damage within the lesion, which is in agreement with the findings of Plumier et al. (1997) on the same mouse strain who found improved neuronal survival within certain parts of the lesion in HSPtg animals. Histological examination of another highly expressing mouse strain that was subjected to transient MCA occlusion also showed improved neuronal survival within the striatum (Tsuchiya et al., 2003). In summary, we conclude that overexpression of HSP70 confers protection against cerebral ischaemia, resulting in a smaller lesion volume, and possibly also less cell damage within the lesion at 24 h. However, the question as to whether HSP70 actually prevents ischaemia injury, or merely delays it, remains to be resolved. The molecular mechanisms by which HSP70 provides protection against cerebral infarction are not fully understood at the moment, and further investigation is required to see whether HSP70 overexpression could play a clinical role in protecting the brain after injury. This study shows that MRI may play an important role to study the dynamics of the injury due to its non-invasive character. Acknowledgments We thank the Biotechnology and Biological Sciences Research Council and the Wellcome Trust for financial support of this project. We are especially grateful to Dr. Angelidis (University of Ioannina, Greece) who provided the transgenic mice. References Akbar, M.T., Wells, D.J., Latchman, D.S., de Belleroche, J., 2001. Heat shock protein 27 shows a distinctive widespread spatial and temporal pattern of induction in CNS glial and neuronal cells compared to heat shock protein 70 and caspase 3 following kainate administration. Mol. Brain Res. 93, 148 – 163. Akbar, M.T., Lundberg, A.M., Liu, K., Vidyadaran, S., Wells, K.E., Dolatshad, H., Wynn, S., Wells, D.J., Latchman, D.S., de Belleroche, J., 2003. The neuroprotective effects of heat shock protein 27 overexpression in transgenic animals against kainate-induced seizures and hippocampal cell death. J. Biol. Chem. 278, 19956 – 19965. Alsop, D.C., Detre, J.A., 1996. Reduce transit-time sensitivity in noninvasive magnetic resonance imaging of human cerebral blood flow. J. Cereb. Blood Flow Metab. 16, 1236 – 1249. Angelidis, C.E., Lazaridis, I., Pagoulatos, G.N., 1991. Constitutive expression of heat-shock protein 70 in mammalian cells confers thermotolerance. Eur. J. Biochem. 199, 35 – 39. Beaucamp, N., Harding, T.C., Geddes, B.J., Williams, J., Uney, J.B., 1998. Overexpression of HSP70 facilitates reactivation of intracellular proteins in neurones and protects them from denaturing stress. FEBS Lett. 441, 215 – 219. Beere, H.M., Green, D.R., 2001. Stress management: heat shock protein 70 and the regulation of apoptosis. Trends Cell. Biol. 11, 6 – 10.

265

Busza, A.L., Allen, K.L., King, M.D., Van Bruggen, N., Williams, S.R., Gadian, D.G., 1992. Diffusion-weighted imaging studies of cerebral ischemia in gerbils. Potential relevance to energy failure. Stroke 23, 1602 – 1612. Calamante, F., Thomas, D.L., Pell, G.S., Wiersma, J., Turner, R., 1999. Measuring cerebral blood flow using magnetic resonance imaging techniques. J. Cereb. Blood Flow Metab. 19, 701 – 735. Fiehler, J., Foth, M., Kucinski, T., Knab, R., von Bezold, M., Weiller, C., Zeumer, H., Rother, J., 2002. Severe ADC decreases do not predict irreversible tissue damage in humans. Stroke 33, 79 – 86. Gabai, V.L., Meriin, A.B., Yaglom, J.A., Volloch, V.Z., Sherman, M.Y., 1998. Role of HSP70 in regulation of stress-kinase JNK: implications in apoptosis and aging. FEBS Lett. 438, 1 – 4. Giffard, R.G., Yenari, M.A., 2004. Many mechanisms for HSP70 protection from cerebral ischemia. J. Neurosurg. Anesthesiol. 16, 53 – 61. Gill, R., Sibson, N.R., Hatfield, R.H., Burdett, N.G., Carpenter, T.A., Hall, L.D., Pickard, J.D., 1995. A comparison of the early development of ischaemic damage following permanent middle cerebral artery occlusion in rats as assessed using magnetic resonance imaging and histology. J. Cereb. Blood Flow Metab. 15, 1 – 11. Hoehn, B., Ringer, T.M., Xu, L., Giffard, R.G., Sapolsky, R.M., Steinberg, G.K., Yenari, M.A., 2001. Overexpression of HSP72 after induction of experimental stroke protects neurons from ischemic damage. J. Cereb. Blood Flow Metab. 21, 1303 – 1309. Hunt, C., Morimoto, R.I., 1985. Conserved features of eukaryotic HSP70 genes revealed by comparison with the nucleotide sequence of human HSP70. Proc. Nat. Acad. Sci. U. S. A. 82, 6455 – 6459. Ja¨a¨ttela¨, M., Wissing, D., Kokholm, K., Kallunki, T., Egeblad, M., 1998. HSP70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J. 17, 6124 – 6134. Jiang, Q., Zhang, Z.G., Chopp, M., Helpern, J.A., Ordidge, R.J., Garcia, J.H., Marchese, B.A., Qing, Z.X., Knight, R.A., 1993. Temporal evolution and spatial distribution of the diffusion constant of water in rat brain after transient middle cerebral artery occlusion. J. Neurol. Sci. 120, 123 – 130. Kazemi, M., Silva, M.D., Li, F., Fisher, M., Sotak, C.H., 2004. Investigation of techniques to quantify in vivo lesion volume based on comparison of water apparent diffusion coefficient (ADC) maps with histology in focal cerebral ischemia of rats. Magn. Reson. Imaging 22, 653 – 659. Kelly, S., Bieneman, A., Horsburgh, K., Hughes, D., Sofroniew, M.V., McCulloch, J., Uney, J.B., 2001. Targeting expression of HSP70 to discrete neuronal populations using the Lmo-1 promotor: assessment of the neuroprotective effects of HSP70 in vivo and in vitro. J. Cereb. Blood Flow Metab. 21, 972 – 981. Knight, R.A., Dereski, M.O., Helpern, J.A., Ordidge, R.J., Chopp, M., 1994. Magnetic resonance imaging assessment of evolving focal cerbral ischemia. Comparison with histopathology in rats. Stroke 25, 1252 – 1262. Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G., 1986. Experimental studies of ischaemic brain edema: 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn. J. Stroke 8, 1 – 8. Lee, J.E., Yenari, M.A., Sun, G.H., Xu, L., Emond, M.R., Cheng, D., Steinberg, G.K., Giffard, R.G., 2001a. Differential neuroprotection from human heat shock protein 70 overexpression in in vitro and in vivo models of ischemia and ischemia-like conditions. Exp. Neurol. 170, 129 – 139. Lee, S.H., Kim, M., Yoon, B.W., Kim, Y.J., Ma, S.J., Roh, J.K., Lee, J.S., Seo, J.S., 2001b. Targeted hsp70.1 disruption increases infarction volume after focal cerebral ischemia in mice. Stroke 32, 2905 – 2912. Lee, S.H., Kwon, H.M., Kim, Y.J., Lee, K.M., Kim, M., Yoon, B.W., 2004. Effects of hsp70.1 gene knockout on the mitochondrial apoptotic pathway after focal cerebral ischemia. Stroke 35, 2195 – 2199. Li, F., Silva, M.D., Sotak, C.H., Fisher, M., 2000. Temporal evolution of ischemic injury evaluated with diffusion-, perfusion-, and T2-weighted MRI. Neurology 54, 689 – 696.

266

L. van der Weerd et al. / Experimental Neurology 195 (2005) 257 – 266

MacManus, J.P., Buchan, A.M., 2000. Apoptosis after experimental stroke: fact or fashion? J. Neurotrauma 17, 899 – 914. Maeda, K., Hata, R., Hossmann, K.A., 1998. Differences in the cerebrovascular anatomy of C57black/6 and SV129 mice. NeuroReport 9, 1317 – 1319. Moseley, M.E., Cohen, Y., Mintorovitch, J., Chileuitt, L., Shimizu, H., Kucharczyk, J., Wendland, M.F., Weinstein, P.R., 1990. Early detection of regional cerebral ischemia in cats: comparison of diffusion- and T2weighted MRI and spectroscopy. Magn. Res. Med. 14, 330 – 346. Mosser, D.D., Caron, A.W., Bourget, L., Denis-Larose, C., Massie, B., 1997. Role of the human heat shock protein HSP70 in protection against stress-induced apoptosis. Mol. Cell. Biol. 17, 5317 – 5327. Murakami, K., Kondo, T., Kawase, M., Chan, P.H., 1998. The development of a new mouse model of global ischemia: focus on the relationships between ischemic duration, anesthesia, cerebral vasculature, and neuronal injury following global ischemia in mice. Brain Res. 780, 304 – 310. Niiro, M., Simon, R.P., Kadota, K., Asakura, T., 1996. Proximal branching patterns of middle cerebral artery (MCA) in rats and their influence on the infarct size produced by MCA occlusion. J. Neurosci. Methods 64, 19 – 23. Pierpaoli, C., Righini, A., Linfante, I., Tao-Cheng, J.H., Alger, J.R., Di Chiro, G., 1993. Histopathologic correlates of abnormal water diffusion in cerebral ischemia: diffusion-weighted MR imaging and light and electron microscopic study. Radiology 189, 439 – 448. Plumier, J.C., Ross, B.M., Currie, R.W., Angelidis, C.E., Kazlaris, H., Kollias, G., Pagoulatos, G.N., 1995. Transgenic mice expressing the human HSP70 have improved post-ischemic myocardial recovery. J. Clin. Invest. 95, 1854 – 1860. Plumier, J.C., Krueger, A.M., Currie, R.W., Kontoyiannis, D., Kollias, G., Pagoulatos, G.N., 1997. Transgenic mice expressing the human

inducible HSP70 have hippocampal neurones resistant to ischemic injury. Cell Stress Chaperones 2, 162 – 167. Rajdev, S., Hara, K., Kokubo, Y., Mestril, R., Dillmann, W., Weinstein, P.R., Sharp, F.R., 2000. Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction. Ann. Neurol. 47, 782 – 791. Rudin, M., Baumann, D., Ekatodramis, D., Stirnimann, R., McAllister, K.H., Sauter, A., 2001. MRI analysis of the changes in apparent water diffusion coefficient, T2 relaxation time, and cerebral blood flow and volume in the temporal evolution of cerebral infarction following permanent middle cerebral artery occlusion in rats. Exp. Neurol. 169, 56 – 63. Tsuchiya, D., Hong, S., Matsumori, Y., Kayama, T., Swanson, R.A., Dillman, W.H., Liu, J., Panter, S.S., Weinstein, P.R., 2003. Overexpression of rat heat shock protein 70 reduces neuronal injury after transient focal ischemia, transient global ischemia, or kainic aid-induced seizures. Neurosurgery 53, 1179 – 1188. Van Bruggen, N., Roberts, T.P.L., Cremer, J.E., 1994. The application of magnetic resonance imaging to the study of experimental cerebral ischaemia. Cerebrovasc. Brain Metab. Rev. 6, 180 – 210. Van der Weerd, L., Thomas, D.L., Thornton, J.S., Lythgoe, M.F., 2004. MRI of animal models of brain disease. Methods Enzymol. 386, 149 – 176. Verheul, H.B., Berkelbach van der Sprenkel, J.W., Tulleken, C.A., Tamminga, K.S., Nicolay, K., 1992. Temporal evolution of focal cerebral ischemia in the rat assessed by T2-weighted and diffusionweighted magnetic resonance imaging. Brain Topogr. 5, 171 – 176. Yenari, M.A., Fink, S.L., Sun, G.H., Chang, L.K., Patel, M.K., Kunis, D.M., Onley, D., Ho, D.Y., Sapolsky, R.M., Steinberg, G.K., 1998. Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy. Ann. Neurol. 44, 584 – 591.