Caspase mRNA expression in a rat model of focal cerebral ischemia

Molecular Brain Research 89 (2001) 133–146 www.elsevier.com / locate / bres

Research report

Caspase mRNA expression in a rat model of focal cerebral ischemia David C. Harrison a , *, Robert P. Davis a , Brian C. Bond b , Colin A. Campbell a , Michael F. James a , Andrew A. Parsons a , Karen L. Philpott a b

a Department of Neurology, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5 AW, UK Department of Statistical Sciences, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5 AW, UK

Accepted 6 February 2001

Abstract Proteins of the caspase family are involved in the signalling pathway that ultimately leads to programmed cell death (apoptosis), which has been reported to occur in some experimental models of stroke. In a previous paper we used quantitative reverse transcription and polymerase chain reaction (RT-PCR) to characterise changes in the mRNA expression of one member of this family, caspase-3, in a rat model of permanent focal ischemia. Here we have used this technique to study the expression of a further three caspases which are involved in different aspects of caspase signalling. Caspase-8, involved in Fas-mediated apoptosis, was upregulated in the cortex of ischemic rats. Caspase-11, which leads to the synthesis of the functional form of the cytokine interleukin-1b, also showed increased expression, but with a different temporal profile from caspase-8. In contrast, caspase-9, which forms part of the pathway signalling through the mitochondria, showed a decrease in expression. The expression of a further four caspases (1, 2, 6 and 7) has also been characterised in a simpler experiment. These caspases all showed distinctive patterns of expression following the induction of ischemia. These data lead us to conclude that caspase expression as a whole is under very strict transcriptional control in this model. Certain elements of caspase signalling, such as the Fas-induced pathway and the events upstream of IL-1b processing, are upregulated, while others are not. This may be due to some form of genetic program activated in response to ischemia in the brain and may highlight which biological pathways are modulated.  2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Caspase; Ischemia; Gene expression; Apoptosis

1. Introduction The loss of cells following an ischemic insult to the brain results from a wide range of noxious stimuli in and around the affected area of tissue. These stimuli include deprivation of oxygen and glucose necessary for normal cellular survival, increased concentrations of excitotoxic amino acids and release of cytotoxic mediators such as cytokines [36]. Despite the variety of death-promoting stimuli, cell death is considered to occur by only two basic mechanisms: necrosis and apoptosis, which can be distinguished by their distinctive morphological and biochemical features, most notably the laddering which results *Corresponding author. Tel.: 144-1279-622-728; fax: 144-1279-622371. E-mail address: david c [email protected] (D.C. Harrison). ] ]

from internucleosomal cleavage of DNA in apoptosis. This, and other features of apoptosis, are observed in brain tissue from animal models of focal ischemia, and while there is widespread necrosis occurring within the area of injury, apoptosis is also thought to play an important role in ischemic cell death in the brain [38,40,44]. In C. elegans the CED-3 protein is essential for apoptosis occurring during normal development. The ced-3 gene was found to have homology to mammalian interleukin-1b converting enzyme (ICE), an enzyme responsible for the generation of the pro-inflammatory cytokine interleukin-1b (IL-1b) from its precursor [66]. Subsequently, ICE has been found to belong to a family of at least 12 related proteases, known as caspases, which share an active site, QACXG, and the ability to cleave substrates at sites with aspartate in the P1 position. A main functional role of caspases is to form a signalling cascade

0169-328X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00058-4

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between death-inducing stimuli such as activation of the death receptor Fas, and cleavage of protein substrates targeted during apoptosis [7,12,51]. In a variety of animal models of cerebral ischemia, including the model employed in this study, increased caspase activity has been observed [6,17] and caspase inhibitors can reduce the infarct volume [14,23,43]. Lesion volumes are also reduced in animals with mutations or deletions of caspase genes [22,55]. Apoptosis requires de novo synthesis of mRNA and protein to take place [46], although there is an overall decrease in RNA transcription [10]. Taken together, these observations suggest that there may be a distinct genetic program involving selective transcription of certain critical genes required for apoptosis to proceed [15]. There is increasing evidence of an upregulation of caspases at a transcriptional level during apoptosis in vitro [5,13,54,65]. Increased levels of caspase expression have also been demonstrated in several animal models of cerebral ischemia [1,4,27,29,32,49]. However, while most members of the caspase family have been studied to some extent in in vitro systems, until now work in in vivo systems has principally focussed on the more extensively studied members of the family, namely caspases-1, -2 and -3. Recent advances in the use of fluorogenic probes in conjunction with PCR have enabled the measurement of an accumulating PCR product in real time [19,26,39]. This allows the rapid generation of quantitative data showing changes in transcript numbers in tissue samples. An increase in throughput not only allows a greater number of transcripts to be studied, but also allows a greater number of animals and experimental conditions to be included in a study, enabling a more sensitive and reliable statistical analysis of the data. We have previously used this technique to study the expression of caspase-3 following permanent occlusion of the middle cerebral artery in rats, a severe model of cerebral ischemia [24]. In the present study we have carried out further analysis of caspase-3 expression and assessed the pattern and time course of expression of a further seven caspases in this model. We found marked differences in the expression profiles of the various caspases following induction of ischemia, with caspases-1, -3, -6, -7, -8 and -11 showing increased expression, although with differing time courses. In contrast, caspase-2 expression did not change and caspase-9 mRNA was reduced in the ischemic cortex. Taken together these data suggest that the caspases have functionally distinct roles in the development of the lesion, and give an indication of which caspases may be regulated in this process. This in turn may shed light on possible genetic programs activated in the ischemic brain. Increased understanding of both of these areas is of potential value in the search for therapeutic agents for stroke. Individual caspases may be viewed as potential

drug targets themselves. Alternatively, by understanding possible transcriptional programs activated in the ischemic brain, it may be possible to target signalling mechanisms upstream of transcription and inhibit the execution of such a program as a whole.

2. Materials and methods Animal procedures were performed in accordance with the Home Office regulations as outlined in the Animals (Scientific Procedures) Act 1986. All procedures were critically reviewed by colleagues experienced in animal research, in accordance with company regulations. All animals were housed under standardised environmental conditions (12 h light / dark cycle, 21618C and 5565% humidity) and allowed free access to food and water for at least 5 days acclimatisation prior to use. Permanent middle cerebral artery occlusion (MCAO) was carried out according to the method of Zea-Longa et al. [68]. Male Sprague Dawley rats weighing 300–350 g were anesthetised with halothane. The left middle cerebral artery was occluded by an intraluminal filament at its origin from the circle of Willis. The rats were euthanased 3, 6, 12 or 24 h after surgery by halothane overdose. Parallel groups of rats either received sham surgery, in which an identical procedure was followed but without inserting the filament, or no treatment at all prior to ¨ rats, four sham and four MCAO euthanasia (n 5 2 naıve for 3, 6 and 12 h time points, three sham and five MCAO for the 24 h time point). A neurological assessment of motor and behavioural changes was carried out on all sham-operated and MCAO animals 1 h after surgery (to determine success of occlusion), and immediately prior to sacrifice. Rats were scored on the following six point scale: 0, no deficit; 1, failure to extend right forepaw fully; 2, decreased resistance to lateral push, or decreased grip of right forelimb while tail pulled; 3, spontaneous circling or walking to contralateral side; 4, walks only when stimulated; 5, unresponsive to stimulation with a depressed level of consciousness. The inclusion of an animal in the study was dependent on a neurological score of $3 for MCAO rats or 0 for shamoperated rats 1 h after surgery. The left and right cerebral cortices were dissected from each rat, snap frozen in liquid nitrogen and stored at 2808C. The frozen tissue samples were homogenised in TRIzol reagent (Life Technologies, Gaithersburg, MD, USA) using 1 ml of TRIzol per 50 mg of tissue. Total RNA was extracted from the tissue according to the manufacturer’s suggested protocol with the addition of an extra chloroform extraction step and phase separation, and an extra wash of the isolated RNA in 70% ethanol. The RNA was resuspended in PCR grade water and the concentration calculated by A 260 measurement. RNA

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quality was assessed by electrophoresis on a 1% agarose gel. An aliquot of each RNA sample was taken and equal quantities of RNA were pooled from all rats according to treatment group, time point and tissue. Data was generated for all of the caspases in the study using these pooled RNA samples. In addition, RNA samples from individual rats were used for caspases-3, -8, -9 and -11. For this latter group of caspases, only data from individual rats is reported here. First strand cDNA was synthesised from 1 mg of each RNA sample; 0.01 M DTT, 0.5 mM each dNTP, 0.5 mg oligo(dT) primer, 40 U RNAseOUT ribonuclease inhibitor (Life Technologies), 200 U SuperscriptII reverse transcriptase (Life Technologies). Triplicate reverse transcription reactions were performed along with an additional reaction in which the reverse transcriptase enzyme was omitted to allow for assessment of genomic DNA contamination in each sample. The resulting cDNA products were divided into 20 aliquots using a Hydra 96 robot (Robbins Scientific, Sunnyvale, CA, USA) for parallel Taqman PCR reactions using different primer and probe sets for quantification of multiple cDNA sequences. Where pooled RNA was used, all cDNAs were accommodated on one 96 well plate, however individual rat samples were assayed on four plates, with one time point on each plate, together with ¨ animals to control for replicate samples from the naıve plate effects. Taqman PCR was carried out using an ABI prism 7700 sequence detector (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) on the cDNA aliquots; 2.5 mM MgCl 2 , 0.2 mM dATP, dCTP, dGTP and dUTP, 0.1 mM each primer, 0.05 mM Taqman probe, 0.01 U AmpErase uracil-N-glycosylase, 0.0125 U Amplitaq Gold DNA polymerase (all reagents from Applied Biosystems) 508C for 2 min, 958C for 10 min followed by 40 cycles of 958C for 15 s, 608C for 1 min. Additional reactions were performed on each 96 well plate using known dilutions of rat genomic DNA (Clontech Laboratories, Palo Alto, CA, USA) or in the case of caspases-2 and -9, where the amplicon lies across an intron / exon boundary, plasmid DNA. This provided a PCR template to allow construction of a standard curve relating threshold cycle to template copy number. Primer and probe sets for quantitative RT-PCR were designed using Primer Express v.1.0 software (Applied Biosystems). For caspases-1, -2, -3 and -11 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the rat mRNA sequence was obtained from the GenEMBL database. For caspases-6, -7, -8 and -9 no published rat sequence was available. In these cases, small fragments of rat sequence were generated by PCR using rat brain cDNA together with primers designed from published human or mouse sequences. The PCR products were sequenced, and the partial rat caspase sequences thus generated were sufficient for the design of rat-specific primers and probes for quantitative RT-PCR. Primer and

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probe sequences together with Genbank accession numbers where applicable are shown in Table 1. The way in which particular transcripts change between treatment groups and their behavior over time was assessed by analysis of variance (ANOVA) on log transformed data accommodating for expression of mRNA for the housekeeping gene GAPDH by incorporating this as a covariate. ANOVAs of data from individual rats were split according to the hierarchical random error structure (i.e. between animals, between samples within animals and between RT replicates within sample) and treatment factors tested against the appropriate error term. Due to pooling of samples, ANOVAs of the remaining data had a simpler structure with only one error term (i.e. between RT replicates within sample). Resulting treatment means from analyses are adjusted for the housekeeping gene. Pairwise ¨ comparisons of each treatment group relative to the naıve group of animals were made at each time point and the 95% (for individual data) or 99% (for pooled data) confidence interval for the fold difference was calculated using the appropriate pooled estimates of variability from the ANOVA. Error between RT replicates is naturally smaller than that between animals and between samples, and hence the necessity to use this in the calculation of confidence intervals for pooled data was compensated by the calculation of 99% confidence intervals. In addition, for caspases where data from individual rats was used, comparisons were made between ipsilateral and contralateral cortex of MCAO rats, and also ipsilateral cortex of MCAO rats and the corresponding (left) cortex of shamoperated rats. Three variance components analyses were used to investigate the magnitude of the different sources of random variability in the two experiment for each gene. The first two analyses were performed on the individual data where available. One on the sham and MCAO data ¨ rats alone. together and the other on the data from naıve ¨ The analysis of the naıve data enabled the investigation of plate to plate variability, which was aliased with the time factor in the sham and MCAO data. The third variance component analysis was performed on the pooled data. These are presented in the original units and additionally as percentages of the total amount of random variability in that particular gene (Table 2).

3. Results We have carried out a quantitative analysis of caspases8, -9 and -11 in the permanent MCA occlusion model using measurements taken from individual rats. For comparison, further analysis has also been performed on the data for caspase-3 reported previously [24]. Covariate analysis has been used to normalise data using values obtained for mRNA expression of the housekeeping gene GAPDH. This allows one to correct for fluctuations in

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136 Table 1 Primer and probe sequences Caspase-1

U14647 Rat

Caspase-2

U34684 Rat

Caspase-3

U49930 Rat

Caspase-6

AF025670 Rat

Caspase-7

U67321 a Mouse

Caspase-8

AF067834 a Mouse

Caspase-9

U60521 a Human

Caspase-11

Y13089 a Mouse

GAPDH

U75401 Rat

Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe Forward primer Reverse primer Taqman probe

59-GACAGGTCCTGAGGGCAAAG 59-AAAAGTTCATCCAGCAATCCATTT 59-TTGTCCCTACACTCACTGAGTTGATAAATTGCTT 59-CTCTTCAAGCTTTTGGGCTACAA 59-TTCTGAAGTTTCTCTTGCATTTCCT 59-TCCATGTGCTGTATGACCAGACTGCA 59-AATTCAAGGGACGGGTCATG 59-GCTTGTGCGCGTACAGTTTC 59-TTCATCCAGTCACTTTGCGCCATG 59-AGCATGACGTGCCATTGGT 59-ACGTTGTCGTCCAGCTTGTCT 59-TCTGATGATCCACCACGTCCAGAGG 59-GTCCTTGCCATGCTCATTCAG 59-CCCAGGGAAAGGGCTCCT 59-TTGTGCAGGCCCTCTGCTCCAT 59-TAAGACCTTTAAGGAGCTTCATTTTGA 59-AGGATACTAGAACCTCATGGATTTGAC 59-ATCGTGTCCTTCAGTGATTGCACAGCA 59-GAGGGAAGCCCAAGCTGTTC 59-GCCACCTCAAAGCCATGGT 59-TTTCTGCTCACCACCACAGGCCTG 59-CAGACAGTCACATTCCTGGTGTTAA 59-GCTTCACTGTGCATTGTTCCA 59-CTCATGGCACATTGTCAGGGCCTT 59-GAACATCATCCCTGCATCCA 59-CCAGTGAGCTTCCCGTTCA 59-CTTGCCCACAGCCTTGGCAGC

a

In the case of caspases-7, -8, -9 and -11, these orthologs were used to design primers for the generation of a fragment of rat mRNA sequence by RT-PCR from rat brain mRNA.

RNA quality and efficiency of the reverse transcription reaction. We have previously demonstrated that GAPDH mRNA is unaltered in this model [24].

3.1. Caspase-3 Caspase-3 mRNA expression, as reported previously [24], underwent a steady increase over the time course following MCA occlusion (Fig. 1a). This increased expression was significant (P , 0.05) at 6, 12 and 24 h post¨ rats MCAO when compared to expression levels in naıve (Fig. 1b), representing increases of 41, 46 and 220%, respectively. By contrast, there was no significant alteration in expression levels in the contralateral cortex in MCAO rats or in either cortex in sham rats (Fig. 1b). In this study, further analysis was carried out to compare mRNA in the ipsilateral cortex of MCAO rats to the other control groups, namely the ipsilateral (left) cortex of shamoperated rats, and the contralateral cortex of MCAO rats. Caspase-3 mRNA was found to be significantly elevated at 6, 12 and 24 h, but not 3 h, in ipsilateral MCAO cortex when compared with contralateral MCAO (Fig. 1c) and ipsilateral sham (Fig. 1d) cortices, showing that increases were infarct dependent.

3.2. Caspase-8 Caspase-8 mRNA expression underwent a steady in-

crease over the time course following MCAO which was specific to the ipsilateral cortex of the ischemic animals ¨ rats the mRNA was elevated (Fig. 2a). Compared to naıve by 65, 65 and 223% in ipsilateral MCAO cortex at 6, 12 and 24 h, respectively. No such changes were observed in the contralateral cortex from MCAO rats or in shamoperated rats (Fig. 2b). That these changes are infarct dependent is indicated by comparison with the other control groups; a significant increase in caspase-8 mRNA was observed at 12 and 24 h, but not at 6 h, in contralateral cortex from MCAO rats (Fig. 2c), and at 6, 12 and 24 h compared to ipsilateral cortex in sham-operated rats (Fig. 2d). At the 3 h time point the mRNA for caspase-8 was reduced significantly in both sides of the cortex in MCAO rats, but not in sham-operated rats when compared to the ¨ group (Fig. 2b). There was no difference in caspasenaıve 8 mRNA between ipsi- and contralateral MCAO cortex at this time point.

3.3. Caspase-11 Caspase-11 mRNA was also increased in the ipsilateral cortex of ischemic rats, but the changes have a somewhat different profile from those observed for caspases-3 and -8. The largest increase in expression occurred between 3 h, when the RNA level is not significantly different from that ¨ rats, and 6 h where an increase of 101% observed in naıve

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137

Table 2 Variance components analysis a Caspase-1 pooled Variance components Between plates Between animals within plates Between samples within animal Between RT replicates Total

0.0077 0.0077

Caspase-2 pooled

0.0061 0.0061

Variance components (%) Between plates Between animals within plates Between samples within animal Between RT replicates Total

Caspase-3 Individual

¨ Indiv. naıves

0.0037 0.0084 0.0037 0.0158

0.0042 0.0053 0.002 0.0022 0.0137

Variance components (%) Between plates Between animals within plates Between samples within animal Between RT replicates Total

31 39 15 16 100

Pooled

Individual

¨ Indiv. naıves

0.0016 0.003 0.0028 0.0074

0.0212 0.0145 0 0.0031 0.0388

0.0163 0.0163

Caspase-11

Individual

¨ Indiv. naıves

0.0077 0.0039 0.0097 0.0213

0.073 0.0108 0 0.0097 0.0935

36 18 46 100

0.0049 0.0049

23 53 23 100 Caspase-9

Variance components Between plates Between animals within plates Between samples within animal Between RT replicates Total

Pooled

Caspase-6 pooled

78 12 0 10 100

0.0021 0.0021

22 41 38 100

Caspase-7 pooled

0.0054 0.0054

Caspase-8 Individual

¨ Indiv. naıves

Pooled

0 0.0176 0.0088 0.0264

0.0523 0.0067 0.0077 0.0059 0.0726

0.0088 0.0088

0 67 33 100

72 9 11 8 100

GAPDH

55 37 0 8 100

1

2

Pooled

Individual

¨ Indiv naıves

Pooled

Pooled

0.0075 0.0075

0.0027 0.0015 0.0036 0.0078

0.0044 0.0018 0 0.0019 0.0081

0.004 0.004

0.0033 0.0033

35 19 46 100

54 22 0 23 100

a

Notes: time and its interactions are aliased with plate and its interactions in individual analyses. All samples in pooled analyses are on one plate. ¨ Individual and pooled analyses do not include naıves. Between replicates variance component is very consistent within a gene whether or not samples have ¨ been pooled. A large proportion of the variability in the naıves comes from between plates (30–78%).

was measured. This increase continued at 12 h (123%) and at 24 h (168%) (Fig. 3a and b). This increase was infarct dependent, because it was significant when compared to contralateral MCAO cortex and sham-operated cortex (Fig. 3c and d). ¨ rats, none of the other groups Compared with naıve showed any changes except for a small increase of 31% which was detected in the contralateral cortex of MCAO rats at 24 h (Fig. 3b).

3.4. Caspase-9 Of the caspases analysed using data from individual rats, caspase-9 was the only one for which MCAO treatment did not result in an increase in mRNA expression (Fig. 4a). In fact, a trend towards decreased expression was observed, although compared to the other caspases the changes were small. Ipsilateral caspase-9 mRNA was reduced significantly (P , 0.05) against all groups at 12 h, where the greatest decrease (45%) was observed (Fig. 4b–d). None of the other tissues showed any significant ¨ rats (Fig. 4b). change in relation to naıve

3.5. Caspases-1, -2, -6 and -7 In Fig. 5b, d, f and h the error bars indicate 99% confidence intervals for the differences between ipsi- and ¨ samples contralateral MCAO and sham cortices and naıve using data from RT-PCRs carried out in triplicate. Since the mRNAs encoding all of these caspases were measured using pooled samples only the variation between RT replicates can be estimated and hence assessment of treatment effects using this source of variation alone will be overly optimistic, hence the use of a more cautious confidence limit. The mRNA expression of caspase-7, like caspases-3, -8, and -11, increases from 6 h after MCAO in the ipsilateral cortex (Fig. 5a and b). Caspase-7 mRNA also appears to increase in the contralateral MCAO cortex at 24 h. By contrast, the largest increase in caspase-6 mRNA was observed at 6 h post-MCAO (Fig. 5e and f). Subsequently, the level of expression returned towards the ¨ basal value observed in the cortex of naıve rats. This transient increase in expression was confined to the ipsilateral MCAO cortex. The results for caspase-1 mRNA (Fig. 5c and d) indicate

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Fig. 1. Expression of mRNA encoding caspase-3 in the cerebral cortex following permanent occlusion of the middle cerebral artery. (a) mRNA expression in ipsi- and contralateral cortex of MCAO and sham-operated rats plotted against time. Mean expression in each group is shown as a fold increase ¨ rats which has been ascribed an arbitrary value of 1 as indicated by the horizontal line. (b) Mean expression levels at compared to mean expression in naıve ¨ rats. A change in expression from the each time point with error bars representing 95% confidence intervals for the comparison of each group with naıve ¨ level is indicated by error bars not crossing the horizontal line. (c) Comparison of mRNA expression in ipsi- and contralateral cortex of MCAO rats naıve at each time point. Contralateral expression is given an arbitrary value equal to 1, and ipsilateral expression is shown as a fold increase over contralateral. (d) Comparison of mRNA expression in ipsilateral (left) MCAO cortex with the left cortex in sham-operated rats. Expression in sham-operated rats is given an arbitrary value equal to 1, and MCAO expression is shown as a fold increase over sham.

that its increase was delayed compared with caspases-3, -7, -8 and -11. Of the caspases investigated, only caspase-2 showed very little change in expression at any of the time points tested (Fig. 5g and h). Variance components analyses indicate a consistent contribution by the RT replication to the variability, regardless of whether samples are from individuals or pooled (Table 2). Values range between 0.0028 (caspase11, individual samples) and 0.0163 (caspase-6, pooled). There are some differences in the contribution of RT replication between genes, but even these are within the same degree of magnitude. The largest contribution to the variability, however, arose from plate effects, as demon¨ rats. strated in the analysis on the data from naıve

4. Discussion In this study we have examined the expression of mRNA encoding eight members of the caspase family in a

rat model of permanent focal ischemia. Some of the data for caspase-3 have been published previously [24]. The stroke model used in this study involved the permanent occlusion of the middle cerebral artery at its origin from the circle of Willis by an intraluminal filament [68]. This procedure is widely used and has been shown to give a highly reproducible effect as assessed by a variety of criteria, including reduction of cerebral blood flow [67], infarct volume and position [53,67] and motor impairment [53]. The infarct produced by this procedure, at its greatest extent involves the ipsilateral cortex and striatum, corresponding to the territory perfused by the middle cerebral artery. In this study we confined our analysis of gene expression to the cerebral cortex. The caspase family has been subdivided according to different parameters such as sequence homology, substrate specificity and structure [7,12,51]. On the basis of their structure, the caspases fall into two groups according to the size of the N-terminal prodomain. Type I caspases have large prodomains which typically contain motifs that allow the binding of procaspases to other molecules to form signalling complexes. These include the death effector

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139

Fig. 2. Expression of mRNA encoding caspase-8 in the cerebral cortex following permanent occlusion of the middle cerebral artery. (a) mRNA expression in ipsi- and contralateral cortex of MCAO and sham-operated rats plotted against time. Mean expression in each group is shown as a fold increase ¨ rats which has been ascribed an arbitrary value of 1 as indicated by the horizontal line. (b) Mean expression levels at compared to mean expression in naıve ¨ rats. A change in expression from the each time point with error bars representing 95% confidence intervals for the comparison of each group with naıve ¨ level is indicated by error bars not crossing the horizontal line. (c) Comparison of mRNA expression in ipsi- and contralateral cortex of MCAO rats naıve at each time point. Contralateral expression is given an arbitrary value equal to 1, and ipsilateral expression is shown as a fold increase over contralateral. (d) Comparison of mRNA expression in ipsilateral (left) MCAO cortex with the left cortex in sham-operated rats. Expression in sham-operated rats is given an arbitrary value equal to 1, and MCAO expression is shown as a fold increase over sham.

domain (DED) of caspase-8 (and caspase-10) which is involved in the formation of a death-inducing signalling complex (DISC), together with the adapter molecule FADD and the death receptor Fas [28]. Similarly, the caspase recruitment domain (CARD) of caspase-9 allows the formation of the apoptosome, together with the molecules Apaf-1 and cytochrome c [69]. Activation of type I caspases occurs upon formation of such complexes, which autocatalyse cleavage of the caspase to initiate further caspase signalling. In contrast, type II caspases possess a much shorter prodomain. Activation of these caspases occurs due to cleavage, principally by other caspases. While there may be commonality in the signalling pathway at the level of type II caspases, the type I caspases utilise distinct pathways involved in the initiation of apoptosis. mRNA expression of all eight caspases was examined using pooled RNA samples, in the interests of establishing a rapid method for screening mRNA expression in disease models. In addition, we studied the mRNA expression of four caspases in detail, using RNA samples derived from individual rats. Three of these (caspases-8, -9 and -11) were chosen as representatives of the principal caspase

signalling pathways, and one, caspase-3, because it is thought to be a key effector caspase acting downstream. Data from pooled RNA samples for these caspases is not reported. Table 2 shows the sources of random variability arising between animals, between samples within animals and between RT-PCR replicates from the same sample. These data are compared with variability between replicates from pooled RNA samples. It is critical to assess changes over time in sham and ¨ group as strong plate MCAO groups relative to the naıve effects were observed for most genes (Table 2). This enables the time effect to be studied independently of any plate effects. When designing studies which require the division of samples between multiple plates, it is essential that this is taken into consideration. Naturally, additional variation is introduced between animals and between samples within animals. These are not insubstantial (Table 2) and therefore the appropriate error term incorporating these must be utilised. For example, the different experimental procedures applied to individual animals should be assessed relative to error composed of all three types of variation.

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Fig. 3. Expression of mRNA encoding caspase-11 in the cerebral cortex following permanent occlusion of the middle cerebral artery. (a) mRNA expression in ipsi- and contralateral cortex of MCAO and sham-operated rats plotted against time. Mean expression in each group is shown as a fold ¨ rats which has been ascribed an arbitrary value of 1 as indicated by the horizontal line. (b) Mean expression increase compared to mean expression in naıve ¨ rats. A change in expression levels at each time point with error bars representing 95% confidence intervals for the comparison of each group with naıve ¨ level is indicated by error bars not crossing the horizontal line. (c) Comparison of mRNA expression in ipsi- and contralateral cortex of from the naıve MCAO rats at each time point. Contralateral expression is given an arbitrary value equal to 1, and ipsilateral expression is shown as a fold increase over contralateral. (d) Comparison of mRNA expression in ipsilateral (left) MCAO cortex with the left cortex in sham-operated rats. Expression in sham-operated rats is given an arbitrary value equal to 1, and MCAO expression is shown as a fold increase over sham.

4.1. Caspase-3 Previously, we have shown that caspase-3 mRNA is upregulated following pMCAO in rats when compared ¨ rats. Here we have taken with the expression seen in naıve the analysis further to show that there is also a significant upregulation in caspase-3 mRNA in ischemic cortex versus the contralateral side, and the cortex of sham-operated animals. Changes in the expression of some genes, for example the death receptor TR3, at the level of messenger RNA have been observed following the sham operational procedure in this model [25]. In view of the invasive nature of the sham surgical procedure, which is likely to result in considerable perturbations in cerebral blood flow, some changes in gene expression are to be expected, and for this reason it was decided to use a number of different control tissues for comparison. Caspase-3 is the mammalian caspase with the closest homology to the C. elegans CED-3 protein. As a type II

caspase it is activated by a number of other upstream caspases and is responsible for the cleavage of a large number of substrates whose proteolysis occurs in many apoptotic systems. As such it may be regarded as one of the key executioners of apoptosis [7]. Its importance in the CNS is demonstrated by the profound hyperplasia during brain development observed in caspase-3 deficient mice [34] and the continued high level of expression of caspase3 in the adult brain [50]. Increased caspase-3 activity has been demonstrated in vulnerable cells, for example CA1 hippocampal neurons, following transient global ischemia [6,20,48], and increased neuronal caspase-3 expression is observed in transient [49] and permanent [32] focal ischemia. There is a body of evidence, therefore, to suggest that caspase-3 is upregulated and activated in neurons following cerebral ischemia. In a permanent model of focal ischemia [32], increased procaspase-3 immunoreactivity was observed in microglia and astrocytes. This may be due either to induction of caspase-3 or proliferation of caspase-3 ex-

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141

Fig. 4. Expression of mRNA encoding caspase-3 in the cerebral cortex following permanent occlusion of the middle cerebral artery. (a) mRNA expression in ipsi- and contralateral cortex of MCAO and sham-operated rats plotted against time. Mean expression in each group is shown as a fold increase ¨ rats which has been ascribed an arbitrary value of 1 as indicated by the horizontal line. (b) Mean expression levels at compared to mean expression in naıve ¨ rats. A change in expression from the each time point with error bars representing 95% confidence intervals for the comparison of each group with naıve ¨ level is indicated by error bars not crossing the horizontal line. (c) Comparison of mRNA expression in ipsi- and contralateral cortex of MCAO rats naıve at each time point. Contralateral expression is given an arbitrary value equal to 1, and ipsilateral expression is shown as a fold increase over contralateral. (d) Comparison of mRNA expression in ipsilateral (left) MCAO cortex with the left cortex in sham-operated rats. Expression in sham-operated rats is given an arbitrary value equal to 1, and MCAO expression is shown as a fold increase over sham.

pressing cells. A similar mechanism could also partially be responsible for the increase in caspase-3 mRNA observed in this study.

4.2. Caspase-8 Caspase-8 can be activated through the Fas and TNF receptors binding to their ligands at the cell surface [28]. Two studies, in models similar to the one we have employed, have shown that caspase-8 is found constitutively in neurons, and has increased expression following ischemia [32,60]. Increased expression of caspase-8 may increase the availability of protein to be recruited to form DISCs, thus contributing to an increase in apoptotic signalling. Significantly, mRNAs for both Fas and the type I TNF receptor are also upregulated in this model [25]. In this study, caspase-8 shows an early increase in expression with a similar time course to that which we have previously shown for caspase-3 [24]. However, there is some evidence to suggest that increased caspase-3 and caspase-8 expression occur in different sub-populations of neurons [60]. We also observe a transient decrease in caspase-8

mRNA expression at 3 h which, as it occurs in both sides of the cortex, suggests that it is due to an acute after-effect of the MCAO procedure, such as a stress response, although the mechanism involved is unclear. It is worth noting that this global effect would not have been detectable had either contralateral cortex or sham-operated animals been used alone as controls.

4.3. Caspase-11, caspase-1 Caspase-11 was originally identified and characterised in mice and has closest homology to human caspase-4 [59,62]. Like caspase-4 in humans, it is essential for the activation of caspase-1 [63]. Caspase-1, originally termed interleukin-1b converting enzyme (ICE), is involved in the processing of interleukin-1b from its precursor. Thymocytes derived from caspase-1 deficient mice, while responding normally to ultraviolet radiation and dexamethasone, are resistant to apoptosis induced by Fas antibody [33]. Thus, under certain circumstances caspase-1 is required for apoptosis, however caspase-1 and caspase11 knockout mice develop normally [33,37,63]. Elevated expression of the pro-inflammatory cytokine

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Fig. 5. Expression of mRNA encoding caspase-7 (a,b), caspase-6 (c,d), caspase-1 (e,f) and caspase-2 (g,h) in the cerebral cortex following permanent occlusion of the middle cerebral artery. (a,c,e,g) mRNA expression in ipsi- and contralateral cortex of MCAO and sham-operated rats plotted against time. ¨ rats which has been ascribed an arbitrary value of 1 as indicated by Expression in each group is shown as a fold increase compared to expression in naıve the horizontal line. (b,d,f,h) Mean value arising from three independent RT-PCRs at each time point with error bars representing 99% confidence intervals ¨ rats. for the comparison of each group with naıve

IL-1b is seen in ischemia [41] and is localised in microglia and macrophages [9], and ischemic brain injury is reduced by exogenous application of the endogenous IL-1 receptor antagonist [52]. Brain injury is also reduced in ischemic

mice lacking caspase-1 [55] or expressing a dominant negative mutation of caspase-1 [18]. This may be due to the removal of either the injurious effects of IL-1b or to a reduction in apoptosis, or both. In the normal brain the

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localisation of caspase-1 and IL-1b has been shown to reside in cells of the vascular system [64] and in neurons. Following transient global ischemia in gerbils, a model where neuronal death is observed, microglial expression of caspase-1 is induced, but neuronal expression does not change [4]. Microglial caspase-1 immunoreactivity has also been described in the corpus callosum, but not in the infarct or penumbra in a permanent model of focal ischemia [32]. Again, this points to the major effect of the caspase-11 / caspase-1 pathway being pro-inflammatory in the ischemic brain. The increase in contralateral expression of caspase-11 at 24 h is in keeping with this hypothesis. Elevated levels of IL-1b protein start to appear in the contralateral hemisphere at a late stage in this model. Increased caspase-1 and caspase-11 would be expected for the processing of this molecule. Again, detection of this contralateral change is only made possible by inclusion of all of the appropriate controls in the study.

4.4. Caspase-9 Caspase-9 can be activated by apoptotic stimuli which do not activate caspase-8; cells derived from mice lacking caspase-9 undergo cell death triggered by Fas, but are resistant to apoptosis induced by dexamethasone or gamma radiation [21]. Caspase-9 knockout mice display CNS malformations and caspase-9 is constitutively expressed in neurons both during development and in the adult, indicating an important role for caspase-9 in neuronal apoptosis [21,35]. We have detected a decrease in caspase-9 expression following cerebral ischemia. If caspase-9 is activated under ischemic conditions, the levels of this protein normally found in cells undergoing apoptosis must be sufficient for apoptosome formation and for signalling to occur normally. The decrease observed here suggests either a loss of caspase-9 expressing cells in the region affected, or a relative decrease in its expression. The appearance of decreased caspase-9 as early as 3 h post-MCAO, when there is little evidence of any major changes in the cellular composition of the infarct, would point to the latter scenario. If this caspase has a role in apoptosis in cerebral ischemia it appears that this employs the caspase-9 already present, and that little or no de novo synthesis of caspase-9 is required.

4.5. Caspases-6 and -7 Caspases-6 and -7 are both type two caspases with close homology to caspase-3. Both have been reported to be expressed at very low levels, if not absent, in normal brain [59], therefore any involvement of these caspases is likely to require de novo synthesis of mRNA and protein. Our data show increased transcription of both caspases, but with different time courses.

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Caspase-6 has been shown to activate caspase-3 [42], and in Jurkat cells, caspase-9 dependent activation of caspases-8 and -10 is blocked by inhibition of caspase-6 [57]. Thus the role of caspase-6 may not simply be as a late stage ‘executioner’ caspase, but may be more complex. An increased requirement for caspase-6 at an early time point may indicate its involvement in such a mechanism. Interestingly, a transient increase in caspase-6 immunoreactivity in astrocytes has been reported in permanent focal ischemia [32]. This effect did not occur until 12 to 24 h after surgery, however the model employed was less severe than the one in our study and it is therefore not possible to compare the two time courses directly. Caspase-7 has not been reported to participate in any feedback mechanism in the same manner as caspase-6. Its late increase in expression would suggest that its role in ischemia induced apoptosis is limited to cleavage of proteins downstream of the caspase pathway, or that it is found in a different population of cells whose requirement for caspase transcription occurs at a different stage in the overall progression of the developing lesion.

4.6. Caspase-2 Our data in this study show no overall changes in the expression of mRNA for caspase-2. This protein exists in two isoforms, long and short, encoded by alternately spliced variants of the mRNA. The long and short forms of caspase-2 act as positive and negative regulators of apoptosis, respectively [61]. The primers and probe we used are specific for the long, pro-apoptotic form of caspase-2. In models of both focal [1] and global ischemia [29], a transient increase in mRNA for both isoforms of caspase-2 mRNA has been reported. The different results seen here may be due to the precise model employed or, in the case of the focal ischemia study, to the tissues sampled; our study was restricted to the cerebral cortex, whereas Asahi et al. sampled the whole cerebral hemisphere. Since different regions of the brain are prone to apoptosis to differing degrees it is likely that changes in caspase expression will also differ between one region and another. Kinoshita et al. [29], using a gerbil model of transient global ischemia, report a greater increase in the CA1–4 fields of the hippocampus which readily undergo apoptosis, than in cortex which is more resistant to cell death [30]. In caspase-2 deficient mice there is no reduction in neurological damage in models of either permanent focal or transient ischemia when compared with wild type littermates [3]. When evaluating changes in gene expression in an in vivo situation such as the one described in this study it is important to consider any changes taking place in cellular composition of the tissue. Following focal cerebral ischemia a number of substantial changes take place in the affected area. Neurons and oligodendrocytes die within the ischemic infarct [2,45], particularly after 12 h of ischemia.

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Astrocytes and microglia are decreased in number in the core region of the lesion and proliferation of both of these cell types occurs in the marginal areas [8]. Macrophages and polymorphonuclear leukocytes invade the margins of the lesion at a late stage, after around 24 h of ischemia [8,31]. These changes become most pronounced only after 12 to 24 h of ischemia. At earlier time points, therefore, changes in observed mRNA expression are likely to be representative of regulation of gene expression within normal tissue, whereas later differences in mRNA levels are more likely to be due to overall changes in the cellular composition of the tissue sample. Studies which examine expression at the mRNA level suffer from the inherent disadvantage that changes seen in mRNA do not necessarily correspond to changes seen in protein expression. Furthermore, it has been suggested that the coupling of mRNA transcription to protein synthesis is in some way disrupted under ischemic conditions. While there can be no doubt that normal physiological function is disturbed following ischemia, protein synthesis may still be important because its inhibition blocks the development of the ischemic infarct [40]. Our results suggest that the transcriptional activation patterns of the eight caspases studied fall into three (or possibly four) groups. In the first group, a modest increase in expression is observed at 6 h post-MCAO and continues to rise. This pattern includes caspases-3, -7 and -8 and -11. Whereas caspases-3 and -7 are thought to represent type II caspases, caspase 8 (and possibly caspase-11) are type I caspases. In fact, the activation mechanism of caspase-11 is unknown, although its large prodomain suggests a type-I mechanism. Caspase-1 may also fit into this group, although its increase in expression is delayed compared with caspases-3, -7, -8 and -11. Caspase-6 appears to be separately grouped because its mRNA increased only transiently at 6 h. Caspases-2 and -9 form a third grouping in that their mRNA was unchanged or even reduced during the time course of the experiment. These results could suggest that the transcription of these enzymes is under the control of distinct mechanisms. In view of the finding that mRNA transcription and translation are required for apoptosis to take place [46], these findings raise interesting possibilities regarding possible genetic programs activated in this model. Caspases-8 and -10 are the key initiator caspases in TNF and Fas-induced apoptosis [28]. Caspases-3 and -7 can both be activated by caspase-8 [58] and caspase-10 [16]. Furthermore, both of these enzymes are activated themselves during TNF and Fas-induced apoptosis [11,56]. mRNA for Fas and TNF is substantially increased in focal ischemia [25] and Fas has been shown to play an important role in neuronal apoptosis in vivo and in focal ischemia [47]. The concurrent increases in the mRNA for caspases8, -3 and -7 may represent an apoptotic program for a pathway that clearly has an important role in this model. Caspases-1 and -11 may also be part of this program,

although their importance as mediators of inflammation via IL-1b would suggest a different or additional role. Caspases-2 and -9, which lie on separate pathways, would appear not to be under control of such a program, although the lack of increased transcription does not rule out an important role for these enzymes in this model of ischemia. In conclusion, we have presented a detailed analysis of the temporal changes in caspase mRNA expression following permanent occlusion of the middle cerebral artery. This has revealed distinct patterns of transcription pointing to the roles of different pathways and individual caspases during the development of the lesion. Such information provides a platform for the development of novel drug targets and gives insight into time windows when compounds designed against such targets could be effective.

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