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Research Report

Prior deafferentation confers long term protection to CA1against transient forebrain ischemia and sustains GluR2expression

Jaspreet Kaura, Zonghang Zhaoa, Rose M. Geransarb,Michalis Papadakis c, Alastair M. Buchana,c,⁎aHotchkiss Brain Institute and Calgary Stroke Program, Department of Clinical Neurosciences, University of Calgary,157-3330 Hospital Drive NW, Calgary, AB, Canada T2N 2T8bDepartment of Biochemistry, University of Calgary, Calgary, AB, CanadacNuffield Department of Clinical Medicine, University of Oxford, Oxford, UK

A R T I C L E I N F O

⁎ Corresponding author. Hotchkiss Brain InsCalgary, 157-3330 Hospital Drive NW, Calgar

E-mail address: abuchan@ucalgary.ca (A.

0006-8993/$ – see front matter © 2005 Elsevidoi:10.1016/j.brainres.2005.12.123

A B S T R A C T

Article history:Accepted 15 December 2005Available online 9 February 2006

Hippocampal CA1 pyramidal neurons undergo delayed neurodegeneration after transientforebrain ischemia, and the phenomenon is dependent upon hyperactivation of L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of glutamate receptors,resulting in aberrant intracellular calcium influx. The GluR2 subunit of AMPA receptors iscritical in limiting the influx of calcium. The CA1 pyramidal neurons are very sensitive toischemic damage and attempts to achieve neuroprotection, mediated by drugs, have beenunsuccessful. Moreover, receptor antagonism strategies in the past have failed to providelong-term protection against ischemic injury. Long-term protection against severe forebrainischemia can be conferred by fimbria–fornix (FF) deafferentation, which interrupts theafferent input to CA1. Our study evaluated the long-term protective effect of FFdeafferentation, 12 days prior to induction of ischemia, on vulnerable CA1 neurons. Ourresults indicate that at 7 and 28 days post-ischemia, prior FF deafferentation protected 60%of neurons against ischemic cell death. Furthermore, we sought to evaluate whether FFdeafferentation also sustained GluR2 levels in these neurons. GluR2 protein and mRNAexpression were sustained by deafferentation at 70% of control following ischemia.Correlation studies revealed a positive correlation between GluR2 protein and mRNAlevel. These results demonstrate that protection conferred by FF deafferentation was long-term and related to sustained GluR2 expression.

© 2005 Elsevier B.V. All rights reserved.

Keywords:DeafferentationNeuroprotectionIschemiaAMPA receptorGluR2 receptorHippocampus

titute and Calgary Stroke Program, Department of Clinical Neurosciences, University ofy, AB, Canada T2N 2T8. Fax: +1 403 283 3572.M. Buchan).

er B.V. All rights reserved.

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Abbreviations:FF, Fimbria–fornixAMPA, L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate4-VO, Four-vessel occlusionPBS, Phosphate-buffered salineNeuN, Neuron specificnuclear proteinMAP2, Microtubule associatedprotein 2FITC, Fluorescein IsothiocyanateDAPI, 4′,6-Diamidino-2-phenylindoleDAB, DiaminobenzidineHRP, Horseradish peroxidasePCR, Polymerase chain reactionRFE, Relativefluorescence expressionkDa, Kilo daltonsNMDA, N-methyl-D-aspartateRT-PCR, Real-time polymerase chainreactionROS, Reactive oxygen speciesSOD, Superoxide dismutaseBDNF, Brain derived nervegrowth factorNRSE, Neuron-restrictivesilencer elementREST, Neuronal repressor element-1silencing transcription factorSCI, Spinal cord injuryALS, Amyotrophic Lateral Sclerosis

1. Introduction

Pyramidal neurons in the hippocampal CA1 region undergodelayed neurodegeneration after transient, but severe, fore-brain ischemia. Previous studies from our laboratory demon-strated that selective lesions of the FF fibers protected CA1neurons against ischemic damage (Buchan and Pulsinelli,1990). This neuroprotection was observed when ischemia wasinduced at 9 and 12 days following deafferentation.

The rate of CA1 cell death and the degree of neuronal loss iscorrelated to the duration of ischemia (Pulsinelli et al., 1982;Colbourne et al., 1999). This is probably due to hyperactivationof glutamate receptors (Pollard et al., 1993) which results inaberrant amounts of calcium influx and excitotoxic cell death(Lu et al., 1995).

Glutamate-triggered increases in intracellular calciuminitiate degeneration by several pathways, including activa-tion of proteases and generation of free radicals (Choi, 1987). Inparticular, hyperactivation of post-synaptic AMPA receptorshas been implicated in calcium-mediated neurotoxicity(Weiss et al., 1994; Itoh et al., 1988; Choi, 1994). This can betransiently reversed by receptor antagonism (Colbourne et al.,1999; Buchan et al., 1991; Sheardown et al., 1993).

AMPA receptors, a subtype of excitatory ionotropicglutamate receptor family, are heteromeric or homomericassemblies comprised of subunits GluR1-4 (Hollmann and

Heinemann, 1994). Based on the type of subunits present inthe receptor assembly, a wide range of ion selectivity, iondependency, and channel kinetics is exhibited (Hollmann etal., 1991; Verdoorn et al., 1991). The GluR2 subunit limits thepermeability of the receptor to calcium (Gorter et al., 1997).AMPA receptors expressed in CA1 pyramidal neurons exhibitproperties of calcium-impermeable, (i.e., GluR2 containing)receptors.

Transient global ischemia has been shown to cause adelayed, but specific, decrease in GluR2 protein expression inCA1 pyramidal neurons (Sommer and Kiessling, 2002). Thisresults in an increase in the permeability of AMPA channels tocalcium, leading to elevated intracellular calcium concentra-tions and a higher vulnerability to ischemia. In situ hybrid-ization studies have shown that experimental cerebralischemia induces a reduction in the GluR2 mRNA (Pollard etal., 1993; Pellegrini-Giampietro et al., 1992; Colbourne et al.,2003; Alsbo et al., 2000). Other studies, however, havedemonstrated unchanged levels of GluR1, 2 and 3 mRNA(Friedman et al., 2000).

mRNA levels do not necessarily correlate with the amountof protein present in the cell. In particular, if the proteinsynthesis machinery is impaired after ischemia, there may bea discrepancy in the levels of mRNA and the correspondingprotein levels in the hippocampal CA1. Therefore, both theprotein and the mRNA levels need to be assessed in order to

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ascertain whether the level of GluR2 protein or mRNA canserve as a predictor of neuronal survival and/or death.

This study evaluated whether the duration of FF deaf-ferentation-induced neuronal protection against ischemia islong-term.We also tested if this protection sustained both theGluR2 protein and mRNA expression levels. Hitherto, therelationship of GluR2 expression to deafferentation-conferredprotection has been assessed at the protein or at the mRNAlevel (Pollard et al., 1993; Sommer and Kiessling, 2002;Pellegrini-Giampietro et al., 1992; Colbourne et al., 2003;Alsbo et al., 2000; Friedman et al., 2000). Therefore, we carriedout a thorough study to investigate the correlation of GluR2protein to GluR2 mRNA levels in the ischemic CA1 ofdeafferented and non-deafferented hemisphere.

2. Results

2.1. The protection conferred to CA1 pyramidal neuronsagainst transient forebrain ischemia by prior FFdeafferentation is long-term

Previous studies by our laboratory have demonstrated that 10min of 4-VO leads to selective cell death in hippocampal CA1pyramidal neurons and that the injury was complete at 7 dayspost-ischemia (Pulsinelli and Buchan, 1988). This finding wasreplicated in the present study (Fig. 1C(i–iv)). Deafferentationsurgery conferred a robust protective effect in the ipsilateralCA1 (Fig. 1B(i, ii)), whereas complete neuronal loss wasobserved in the contralateral CA1 (Fig. 1B(iii, iv)).

Although previous studies have shown the protectiveeffect of FF deafferentation following global ischemia (Buchanand Pulsinelli, 1990), we examined whether this effect wasmaintained long-term. Neurons exhibiting normal morphol-

Fig. 1 – FF deafferentation confers long term protection againststained coronal sections (10 μm) showing normal morphology of(A ii, iv) high magnification. (B i, ii) CA1 of deafferentated hemispon both right (C i, ii) and left (C iii, iv) sides at 7 days post-ischem[Scale bar: 10× = 10 μm; 40× = 1 μm].

ogy in CA1 of non-ischemic control animalswere counted. At 7days post ischemia, histological assessment of CA1 ofischemic deafferented hemisphere showed that the percent-age of live pyramidal neurons was significantly higher than inthe contralateral side (63.7% ± 16.9% vs. 26.7% ± 13.8%,P = 0.014) (Fig. 1E).

At 28 days post-ischemia, the percentage of survivingneurons on CA1 ipsilateral to deafferentation was significant-ly higher as compared to that observed on the CA1 of non-deafferented contralateral hemisphere (58.3% ± 18.4% vs.11.6% ± 10.1%, P = 0.003) (Fig. 1E). These results demonstratethat prior deafferentation was successful in conferring long-term protection to CA1 pyramidal neurons against ischemiccell death.

The percentage of neurons in the ischemic CA1 of deaf-ferented or non-deafferented hemisphere of the 7 day survivalgroup did not differ significantly from their counterparts at the28 day time point (ipsilateral: 63.7% ± 16.9% vs. 58.3% ± 18.4%,P = 0.652; ns; contralateral: 26.7% ± 13.8% vs. 11.6% ± 10.1%,P = 0.08; ns). These results demonstrate that the protectionconferred by deafferentation is long term and that the injurydoes not progress further after 7 days.

We did not observe statistically significant differencebetween the percentage of surviving neurons in the CA1 ofanimals subjected to sham-ischemia, with or without deaf-ferentation surgery (Fig. 2B). Therefore, deafferentation didnot have an effect on the viability of neurons in animals thatwere not subjected to ischemia. No statistically significantdifference in percentage of healthy neurons was observedbetween CA1 of normal control and deafferentation + shamischemia group (Fig. 2B). Overall, these results show that FFdeafferentation prior to forebrain ischemia significantlyprotects the otherwise vulnerable CA1 neurons and this effectis long-term.

ischemia to CA1 pyramidal neurons. Hematoxylin and eosinCA1 pyramidal neurons of control animals at (A i, iii) low andhere and (B iii, iv) CA1 of non-deafferented side. CA1 neuronsia. (D i–iv) CA3 is unaffected by ischemia in all paradigms

Fig. 2 – (A) Neuroprotective effect is compared at differentsurvival times following 10min of global ischemia performed12 days after deafferentation at 7 (*P = 0.014) and 28 days(*P = 0.003). (B) Percentage of surviving neurons is comparedbetween ischemia only, deafferentation + ischemia, shamdeafferentation + ischemia, deafferentation + sham ischemiaand in normal animals. Significant differenceswere observedin the deafferentation + ischemia group only (*P = 0.0001).

Fig. 3 – Ischemia results in loss of GluR2 and MAP2immunoreactivities. Coronal sections (30 μm) were stainedwith rabbit anti-GluR2 and mouse anti-MAP2 antibodies.Normal level of (A1a) GluR2, (A2a) MAP2 immunoreactivitiesin non-ischemic control CA1. Partial loss of (A1b) GluR2, (A2b)MAP2 immunoreactivity in CA1 at 48 h post-ischemia.Significant decrease in immunolabeling of (A1c) GluR2 and(A2c) MAP2 in CA1 pyramidal neurons after 7 days post-ischemia. In CA3, immunoreactivities of (B i) GluR2, and (B ii)MAP2 remain unaffected after 7 days post-ischemia.

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2.2. Ischemia causes delayed neurodegeneration of CA1neurons and a corresponding decrease in GluR2 and MAP2immunoreactivities and deafferentation prior to ischemiasustained these proteins

Ten minutes of transient forebrain ischemia causedselective degeneration of CA1 pyramidal cells. To demon-strate a decrease in expression levels of GluR2 protein inCA1, we used immunohistochemical techniques. Coronalsections incubated with GluR2 antibodies showed a robustimmunoreactivity in the cytoplasm, as well as in thedendrites of CA1 pyramidal neurons of normal animals(Fig. 3A(1a)). MAP2 was used to confirm somatic anddendrosomatic labeling in neuronal cells, and to check theviability of CA1 neurons. Strong dendrosomatic MAP2 (Fig.3A(2a)) immunolabeling was detected in CA1 of normalcontrols. Ischemia caused loss of GluR2 immunoreactivityin the cytoplasm as well as in the dendrites of the CA1pyramidal neurons projecting to the dentate gyrus. Similar

loss of cytoplasmic immunolabeling for MAP2 also oc-curred after ischemia, illustrating the neurodegeneration ofCA1.

A partial loss of immunoreactivity for GluR2 proteinwas observed in the ischemic CA1 at 48 h (Fig. 3A(1b)),and a complete loss occurred 7 days post ischemia (Fig.3A(1c)). Simultaneous, progressive loss of immunoreactiv-ities at the above mentioned survival time points werealso observed for MAP2 (Fig. 3A(2b, c)). The hippocampalCA3 region, however, was unaffected by ischemia andshowed robust immunolabeling for GluR2 and MAP2proteins even at 7 days post ischemia (Fig. 3B(i, ii)).These results confirm that, following ischemia, selective,delayed neuronal degeneration of CA1, and not CA3,occurs and that the expression of GluR2 relates toobserved cellular degeneration.

Double labeling for GluR2 and MAP2 using immuno-fluorescent immunocytochemistry was utilized to observewhether the neurons sustained by deafferentation wereviable and were expressing both GluR2 and MAP2 proteins.After 7 days of transient forebrain ischemia, fluorescentimmunocytochemistry of coronal sections showed an in-tense cytoplasmic labeling for GluR2 (Figs. 4A, C) and strongdendritic MAP2 immunoreactivity in the CA1 ipsilateral todeafferentation surgery (Figs. 4B, C). Dramatic loss of

Fig. 4 – FF deafferentation sustains GluR2 and MAP2 immunoreactivities in CA1 pyramidal neurons after ischemia.Coronal sections (10 μm) were costained with rabbit anti-GluR2 (A, D) and mouse anti-MAP2 (B, E) and visualized byimmunofluorescentmicroscopy. (C, F) Merged images. (G) Optical intensity of GluR2 fluorescence in the CA1 of deafferented andnon-deafferented side [*P = 0.002; Scale bar: 40× = 1 μm].

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immunolabeling occurred for both GluR2 (Figs. 4D, F) andMAP2 (Figs. 4E, F) in the ischemic CA1 of non-deafferentedhemisphere. Significantly higher optical intensities wereobserved on the CA1 ipsilateral to FF deafferentation thanthose on the contralateral CA1 for GluR2 (81.2% ± 12.3% vs.48.8% ± 11.0%, P = 0.002) (Fig. 4G), thus demonstrating theprotective effect of prior FF deafferentation against loss inGluR2 protein.

2.3. Western blotting confirmed sustained levels of GluR2protein in ischemic CA1 of deafferented hemisphere

Since immunofluorescent cytochemistry cannot reliablyrepresent the actual protein content, Western blot analysiswas carried out to enable densitometric quantification ofGluR2 protein. Immunoreactive band intensities revealedstrong bands for GluR2 protein (Mr, 110 kDa) in the CA1 ofcontrol animals (Fig. 5A(i)). No difference in the immuno-reactive intensities of GluR2 was observed between CA1 ofleft and right hemispheres. However, significant loss ofGluR2 protein in CA1 was observed at 7 days post-ischemia(Fig. 5A(iii)).

Immunoblot analysis of GluR2 protein showed sustainedlevels in the CA1 of deafferented hemisphere as comparedto that of non-deafferented side at 7 days post-ischemia(Fig. 5A(ii)). Significantly higher GluR2 expression levelswere observed in the ischemic CA1 of deafferented ascompared to those in the contralateral hemisphere

(69.5% ± 22.8% vs. 35.5% ± 5.8%; P = 0.028). Bands of similarintensities for loading control, actin (Mr, 42 kDa), wereobserved in the CA1 of animals subjected to ischemia aloneas well as in the ischemic CA1 of deafferented and non-deafferented hemispheres (Fig. 5A(i–iii)). Since the assess-ment of GluR2 protein by immunoblotting was controlledfor cellular degeneration by measuring the amount ofprotein and by labeling for actin, the reduction of GluR2immunoreactivity in the contralateral side is independent ofcell death and is thus a cellular response per se. Theseresults are suggestive of GluR2 being a mediating factor fordeafferentation induced cellular protection. Therefore, ourresults show that deafferentation sustained GluR2 proteinlevel to about 70% of control values, whereas in the non-deafferented hemisphere, protein level decreased to approx-imately 36% of control (Fig. 5B).

2.4. Real-time PCR (RT-PCR) studies reveal reduced GluR2transcript levels in ischemic CA1 of non-deafferented side

The results showed that GluR2 protein was sustained inischemic CA1 of deafferented side and decreased in CA1 ofcontralateral side. We sought to investigate whether GluR2gene expression was also sustained by deafferentation.Expression level of GluR2 transcript was determined usingreal-time PCR, and RFE value was calculated. We observedthat in the ischemic CA1 of non-deafferented hemisphere,GluR2 gene expression decreased to 26% of control levels.

Fig. 5 – Deafferentation sustains GluR2 protein and mRNA expression in the CA1 of deafferented hemisphere. SDS-PAGEelectrophoresis of CA1 lysate (50 μg protein/lane) was performed and transferred overnight onto a nitrocellulose membrane.The amount of GluR2 protein was determined by quantitative Western blotting. (A i) Strong visualization of GluR2 and actinprotein immunolabeling in the left and right CA1 of control rats. (A iii) GluR2 and actin protein immunoreactivity in left and rightCA1 of animals 7 days after exposure to 10 min of 4-VO. (A ii) Level of GluR2 and actin in CA1 of deafferented (Deaff) andnon-deafferented (Non-Deaff) sides. (B) Histogram showing representative densitometric readings of the bands obtained forGluR2 (*P = 0.028). (C) Sustained relative fluorescence expression of GluR2, determined by real-time PCR, in the CA1 ofdeafferented side and non-deafferented ischemic hemispheres (*P = 0.03). Regression plot showing strong correlation betweenpercentage relative fluorescence expression and protein densitometric values of GluR2 in CA1 of (D) ipsilateral deafferented(r = 0.95) and (E) contralateral non-deafferented hemisphere (r = 0.90).

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Deafferentation, however, protected the ipsilateral ischemicCA1. Percentage of RFE for GluR2, sustained at almost 60%of control, was significantly higher than that observed inCA1 of contralateral side (58.5% ± 22.1% vs. 26.5% ± 17.1%;

P = 0.03) (Fig. 5C). This further illustrates that deaf-ferentation surgery helped to sustain GluR2 mRNA levelsand is in agreement with the results of Western blottingexperiments.

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2.5. GluR2 protein and transcript levels are positivelycorrelated

In this study, we also sought to investigate whether GluR2protein level correlated with GluR2 mRNA level in theischemic CA1 of deafferented and non-deafferented hemi-sphere. Pearson product moment analysis showed a positivecorrelation between GluR2 protein and RFE in the CA1 ofipsilateral deafferented (r = 0.95, P = 0.02) (Fig. 5D), as well as inthe CA1 of contralateral non-deafferented side (r = 0.90P = 0.01) (Fig. 5E), suggesting both protein and mRNA levelsare downregulated in ischemia and that deafferentation priorto induction of transient forebrain ischemia sustains bothGluR2 protein and transcript level.

3. Discussion

Previous studies using AMPA receptor antagonists to limitischemic injury were able to confer only a transientprotection to hippocampal CA1. The objective of this studywas to investigate whether the duration of protectionagainst ischemia to CA1 pyramidal neurons by prior FFpathway transection was long-term. Our results indicate thatFF deafferentation, 12 days prior to induction of transientforebrain ischemia, afforded long-term CA1 protection andare in keeping with studies done previously by our group(Buchan and Pulsinelli, 1990). Similar studies demonstratingprotection from ischemic damage by surgical transection ofCA3 inputs to CA1 protection have been reported (Kaplan etal., 1989). Kimura and Saji (1997) also showed protection ofCA1 from ischemic damage by blocking axoplasmic transportof neurons with colchicines. As well, Anzai et al. (2003)reported that reduction of glutamate mediated inputs to CA1confers protection against ischemia.

The protection by FF transection occurred only whenischemia was induced 12 days after deafferentation surgery,and is suggestive of a preconditioning-like effect, requiring 12days to mature. Cortical axotomy-induced neuronal apoptosishas been shown to be mediated by oxidative stress (Martin etal., 2003) and transgenic over-expression of antioxidativeenzymes such as superoxide dismutase-1 (SOD-1) providedsignificant protection (Martin et al., 2003). Oxidative stress cancontribute to adaptation of tissues as well as resistance tosubsequent ischemia (Baker, 2004). Preconditioning is alsoknown to improve the brain's ability to mobilize antioxidantsthat attenuate the massive reactive oxygen species (ROS)mediated oxidative stress (Glantz et al., 2005; Puisieux et al.,2004).

Our results, consistent with other reports, show delayeddegeneration of CA1 neurons following ischemia and areduction in GluR2 mRNA expression (Anzai et al., 2003;Calderone et al., 2004; Liu et al., 2004; Oguro et al., 2004).Furthermore, our results indicate that FF deafferentation, inaddition to the protective effect on pyramidal neurons,sustained the GluR2 protein and mRNA levels. The resultsare in accordance with reports of Colbourne et al. (2003) whoreported sustained levels of GluR2 mRNA after postischemichypothermia. The sustained GluR2 levels observed afterhypothermia by Colbourne et al. (2003) and after deafferenta-

tion during our studymay be a direct, rather thanmechanistic,effect of cell survival. It is well known that ischemia alters theexpression of stress proteins and neurotrophic factors invulnerable CA1 region (Nowak et al., 1993; Lindvall et al., 1994).Lowenstein et al. (1991, 1994) showed that recurrent seizuresmay result in acute activation of heat shock proteins invulnerable regions and can protect against glutamate toxicity.As well, Pellegrini-Giampietro et al. (1997) are of the opinionthat stress-related proteins may be involved in the down-regulation, as well as in the stability, of GluR2-containingreceptors.

During development, hippocampal neurons have beenshown to undergo dendritic sprouting and mice that wereGluR2-deficient lacked the ability to sprout (Swann et al.,1999). Friedman and Veliskova (1999), therefore, suggested atransient neurotrophic role of GluR2 during development andhypothesized that increased sprouting of inhibitory neuronscan counteract further excitotoxic damage (Friedman andVeliskova, 1999) to provide neuronal protection.

Various regulatory elements and transcription factorshave been identified that regulate GluR2 gene expression(Myers et al., 1998). Researchers have reported functionalpromoter studies that indicate that GluR2 gene is transcrip-tionally regulated by repressor element-1 silencing tran-scription factor (REST) (Myers et al., 1998; Huang et al., 1999).Transient forebrain ischemia increases the expression ofGluR2 promoter neuron-restrictive silencer element (NRSE)/neuronal (REST) that represses GluR2 gene expression in CA1(Tanaka et al., 2002; Calderone et al., 2003). The presentstudy shows that deafferentation provides cellular protec-tion against ischemia and sustains GluR2 protein andtranscript levels to about 60% of control. Colbourne et al.(2003) reported sustained levels of GluR2 mRNA afterpostischemic hypothermia and suggested that the point ofintervention of hypothermia induced protection of GluR2expression could be at the level of inhibition of NRSE/RESTtranscription factors. It can be safely argued that the point ofintervention of cellular protection and GluR2 maintenanceinduced by deafferentation could also be at the level oftranscription factor inhibition.

Our Western blot and RT-PCR results indicate that follow-ing ischemia, GluR2 proteins, as well as mRNA, are down-regulated and that deafferentation induces their sustenance.Furthermore, our study also revealed that changes in GluR2protein correlated to mRNA levels. Eastwood et al. (1995)reported a similar decrease in GluR2 mRNA in schizophrenicpatients that was accompanied by a loss of GluR2 protein.Spinal cord injury (SCI) has also been shown to decrease GluR2protein and mRNA levels in thoracic ventral horn motorneurons (Grossman et al., 2001).

The AMPARs are conferred distinctive kinetic propertiesnot only through subunit composition, but also through flip/flop exon usage that determines the total Ca2+ entry (Pulsinelliand Buchan, 1988; Mosbacher et al., 1994). In rats, the editedform of GluR2, GluR2(R)/flop predominates the unedited GluR2(Q)/flip form and conversion of flop to flip form results inlarger deactivation and desensitization that permits greaterinflow of Ca2+ through the channel (Sommer et al., 1990;Mosbacher et al., 1994; Geiger et al., 1995). Under normalphysiological conditions, a complete editing of the GluR2 Q/R

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site occurs that renders the receptor Ca2+ impermeable(Paschen et al., 1996). Under-editing of the Q/R site has beenshown to occur in a disease specific and region selectivemanner (Mosbacher et al., 1994). Comparable reductions inGluR1, GluR2 and GluR3 mRNA were reported in postischemicCA1 (Alsbo et al., 2000). However, increased GluR2 mRNA aswell as flop isoform expression has been reported in CA1neurons 3 days after tolerance induction (Alsbo et al., 2000),and supports increased expression of GluR2.

Yamaguchi et al. (1999) also reported that change in editingrate of GluR2 subunit isoform occurred mainly in the CA1regions when the animals were subjected to 2 min ofpreconditioning ischemia followed by a 5 min injuriousischemia on day 3. However, this change was not observedin animals that were not subjected to preconditioningischemia. Henceforth, many studies suggest that the modifi-cation of RNA editing of GluR2 rather than its expression levelsmay be crucial in ischemic tolerance (Alsbo et al., 2000;Yamaguchi et al., 1999). Ischemic rats injected with a GluR2(Q) cDNA prior to ischemic exposure showed an enhanced CA1damage that spread to relatively resistant CA2/CA3 areas aswell (Anzai et al., 2003). This strongly suggests that theexpression of Q facilitates the ischemic injury. GluR2 mutantmice showed increased kainite potency, a large kainiteactivated ion current as well as high Ca2+ conductance (Iiharaet al., 2001). Neurons overexpressing the Q isoform also showstrong Na+ inward rectification in addition to high Ca2+

permeability (Sudo et al., 1999).Defects in editing of GluR2 mRNA have been implicated in

neurodegenerative disease such as in the spinal motorneurons of patients with sporadic Amyotrophic LateralSclerosis (ALS) (Kawahara et al., 2004). Enhanced neuronaldeath causes premature death in mice with defective RNAediting of GluR2 (Brusa et al., 1995). Restoring the RNA editingfunction rescues these mice from premature demise (Higuchiet al., 2001). These studies furthermore indicate that GluR2editing is crucial for neuronal survival.

Mansour et al. (2001) have demonstrated that expression ofheteromeric assemblies of GluR1 and GluR2 are preferred andflip-flop differences can also play an important role in drivingthe preferred expression of subunit assemblies. Brorson et al.(2004) suggested that flip-flop isoform differences, in additionto subunit differences, can promote the assembly to hetero-meric subunits that result in the formation of more Ca2+-permeable AMPA receptors than can be predicted by thepresence of GluR2 subunit alone. Synergistic action of Ca2+

permeable Q isoform as well as GluR2 lacking AMPA receptorscan cause enhanced ischemic cell death (Anzai et al., 2003;Pellegrini-Giampietro et al., 1997).

The probability of forming toxic assemblies of Ca2+

permeable AMPA receptors as well as changes in RNA editingwithin the CA1 may be prevented by deafferentation of the FFpathway prior to ischemic exposure. The changes in RNAediting of GluR2 may explain to a certain degree thediscrepancies in the literature regarding selective or non-selective decrease in GluR2 expression (Friedman et al., 2000,2001) following ischemia and its probable link to neuronaldeath. It, therefore, seems relevant to explore themechanismsunderlying the dysfunction of RNA editing, editing sites aswell as enzymes involved in RNA editing for a potential

therapeutic strategy to counteract neuronal death in neuro-degenerative diseases (Kawahara et al., 2004).

Pharmacological blockage of AMPA receptors was tran-siently able to reduce Ca2+ influx into the cells following anischemic episode, thereby failing to afford long-term protec-tion to the vulnerable CA1 neurons (Colbourne et al., 1999;Buchan et al., 1991; Sheardown et al., 1993). Moreover, adverseside-effects and toxicity of the antagonists limits the neuro-protective ability of receptor antagonists (Bullock and Fuji-sawa, 1992; Fisher, 1995). Deafferentation, however, wassuccessful in conferring long-term neuroprotection by sus-taining the GluR2 expression and possibly limiting the amountof Ca2+ influx. Hypothermia is the only other successfulmeansof inducing long-term protection against ischemia (Corbett etal., 1997; Swain et al., 1991; Swann et al., 1999; TheHypothermia after Cardiac Arrest Study Group, 2002). Thesuccess of these paradigms is presumably because of theirdirect or indirect effects at the level of gene expression.

In conclusion, this study demonstrated that, unlike thetransient protection provided by AMPA receptor antagonists,the protective effect of FF deafferentation against forebrainischemia was long-term. Furthermore, our results indicatethat deafferentation sustains GluR2 protein and mRNAexpression and that GluR2 protein levels correlated withthose of mRNA level. This long-term protective effect may bemediated through multiple pathways, involvement of tran-scription factors and most importantly through the editing ofGluR2 Q/R site.

The present study focuses on the correlation of GluR2levels with cell survival, induced by deafferentation, againsttransient forebrain ischemia. However, in light of all thestudies challenging the “GluR2 hypothesis”, suggesting thatAMPA receptors lacking GluR2 subunit and the resultingcalcium permeability does not relate to cell death (Iihara etal., 2001; Jia et al., 1996; Hu et al., 2001), experiments usingantisense oligonucleotides against GluR2 have been plannedto establish a causal relationship of deafferentation-inducedcellular protection and sustained GluR2 levels. A moredetailed understanding of various pathways affected by deaf-ferentation and othermodalities, e.g., hypothermia (Corbett etal., 1997), at the level of inducing cellular protection againsttransient forebrain ischemia, will be crucial to the develop-ment of future neuroprotective drugs.

4. Experimental procedures

Adult male Wistar rats (200 g) were obtained from Charles RiverLaboratories. All procedures were conducted in strict accordancewith regulations of the Animal Care Committee at the Universityof Calgary. Animals were anesthetized with N2/O2/halothanemixture, (70%: 28%: 2%). The rectal temperature was maintainedat 37.5 ± 0.5 °C throughout the surgical procedure using a feedbackregulated heating lamp. A total number of 45 animals were used inthe present study.

4.1. Fimbria–fornix deafferentation

Twelve days prior to the induction of global ischemia, rats wereanesthetized and placed in stereotaxic ear bars. The skull wasexposed using sterile techniques. A cranial window was made

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over the right parietal cortex with a dental drill and surgicalmicroscope. The dura was resected and cortex and subcorticalwhite matter were aspirated with a 21-gauge Luer stub connectedto a vacuum pump. The FF pathway, connecting the septum to thedorsal hippocampus, was visualized with a microsurgical micro-scope and transected by aspiration with a 23-gauge needleconnected to a vacuum pump. This procedure also transectedthe septal pole of the hippocampus, the ventral commissure,some alvear fibers and a portion of the cingulum bundle. Thetotality of this aspiration lesion was verified by visualization witha surgical microscope. In the sham-deafferentation group, theprocedure was terminated after resection of the dura andaspiration of the cortex and subcortical white matter.

4.2. Four-vessel occlusion preparative surgeryand ischemia

Severe forebrain ischemia was produced by amodified four-vesselocclusion (4-VO) surgery (Pulsinelli and Buchan, 1988). Twelvedays after deafferentation surgery, on the thirteenth day, ratswere anesthetized, followed by electrocauterization of the verte-bral arteries. Common carotid arteries were exposed and 2-O silksuture was looped around them. An 18-gauge needle was used toguide a 1-O silk suture through the neck posterior to the trachea,esophagus, jugular veins, carotid arteries, and vagal nerves, butanterior to the cervical and paravertebral musculature. After 24 h,rats were briefly re-anesthetized to open the wound. Both carotidarteries were temporarily occluded for 10min, without anesthesia,with aneurysm clips. Silk sutures through the neck were tightenedto prevent any collateral blood flow. Core temperature wasregulated at 37.5 ± 0.5 °C, by an infrared lamp during surgery,until rats became fully ambulatory. Rats that became unrespon-sive during ischemia, showed initial running behavior, loss ofrighting reflexes and dilation of pupils were included. However,rats that ceased to remain in coma, developed righting reflexesduring ischemia or displayed seizure activity during or afterischemia were excluded. In the sham ischemia group, thevertebral arteries were not electrocauterized during the prepara-tory surgery and the carotids were manipulated but not occluded.

4.3. Assessment of hippocampal injury

Rats were sacrificed at 7 days after ischemia. They wereanesthetized and perfused with phosphate-buffered saline (PBS)and fixed with 10% (w/v) formaldehyde. The brains were left insitu overnight at 4 °C. Coronal sections (10 μm) of paraffin-embedded brains were cut at 3.3 mm posterior to bregma. Afterde-paraffinization in xylene and clearing in 100% (v/v) ethanol, thesections were gradually rehydrated by passing through ethanolseries (95–30%) and were then stained with hematoxylin andeosin. Healthy, viable cells were counted in the entire CA1 band ofthe non-ischemic animals, as well as in ischemic CA1 of deaf-ferented and non-deafferented hemisphere. The count obtainedfrom CA1 of non-ischemic animals was considered as 100%. Thenumber of viable cells counted in ischemic CA1 of deafferentedand non-deafferented sides was normalized to those obtainedfrom CA1 of non-ischemic animals. Fifteen Wistar rats were usedfor this part of study.

4.4. Immunohistochemistry

Coronal hippocampal paraffin sections (10 μm thick) wereincubated overnight at 4 °C with rabbit anti-GluR2 (1:200; Upstate,NY; immunogen: synthetic peptide corresponding to amino acids864–883 of rat GluR2), and mouse anti-microtubule-associatedprotein subunit 2 (MAP2; 1:200; Sigma; immunogen: bovine brainmicrotubule protein) primary antibodies and washed with in PBS(3 × 5 min). The sections were subsequently incubated for 2 h withgoat anti rabbit-Fluorescein Isothiocyanate (FITC) (Zymed) and

goat anti mouse-Cy3 (Zymed) fluorescent-tagged secondary anti-bodies. Sections were washed in PBS and mounted on speciallycoated slides with a mounting medium containing 4′, 6-Diami-dino-2-phenylindole (DAPI) as a counter stain. Fluorescence wasdetected using Olympus BX51 microscope. Optical intensities forGluR2 fluorescence were analyzed by ImagePro Plus software.

GluR2 and MAP2 immunohistochemistry was also performedusing diamino-benzidine (DAB) as a substrate. Briefly, coronalhippocampal sections, incubated overnight at 4 °C with rabbitanti-GluR2 (1:200), and mouse anti-MAP2 (1:200), were washedwith PBS and incubated for 2 h with biotinylated goat anti-rabbitand goat anti-mouse secondary antibodies (Zymed), respectively.After incubation with avidin–biotin solution (Zymed), sectionswere developed with DAB. Brown color formation indicated thelocalization of protein of interest. This part of the experimentalstudy utilized 15 male Wistar rats.

4.5. Western blotting

Immunoblot analysis of GluR2 protein levels was performed onprotein extracts of CA1 harvested from the dorsolateral hippo-campus of animals sacrificed at 7 days post-ischemia. We usedfifteen rats for this study.

The protein concentration of extracts was determined usingBioRad Dc Assay that utilizes the method described by Lowry et al.(1951). Bovine serum albumin was used as standard. Electropho-resis of the total proteins (50 μg of protein/lane) was performed ona denaturing SDS-PAGE [12% (w/v) acrylamide]. The proteins weretransferred overnight at 4 °C onto a nitrocellulose membraneusing BioRad mini protean II wet transfer unit. The membranewas washed with tris-buffered saline containing Tween-20 [TBS-Tween-20; 20 mM Tris–HCl, 100 mM NaCl, 0.25% (v/v) Tween 20;3 × 20min], and incubated for 1 h in blocking buffer [containing 7%(w/v) dry skim milk]. Successively, the blots were incubated for 2h with polyclonal rabbit anti-GluR2 (1:5000; Upstate, Lake Placid,NY) and mouse anti-actin (1:5000; Santa Cruz Biotechnologies,Santa Cruz, CA). After washing (3 × 15min), the blots were blockedand incubated for 2 h in with horseradish peroxidase (HRP)conjugated goat anti-rabbit and goat anti-mouse secondaryantibodies (1:10,000; Upstate, NY). The same antibody was utilizedfor labeling GluR2 in immunocytochemistry and immunoblottingexperiments. Protein bands were visualized using enhancedchemiluminescence under FlourS-Max Multimager (BioRad, Her-cules, CA, USA). Densitometric data of bands was determinedusing QuantityOne software. Band intensities of GluR2 obtainedfrom CA1 of normal, non-ischemic CA1 were considered as 100%.The densitometric values of GluR2 protein in ischemic CA1 ofdeafferented and non-deafferented hemispheres were normal-ized to those obtained from non-ischemic CA1.

4.6. Real-time quantitative polymerase chainreaction (PCR)

Isolation of total RNA from CA1: Rats subjected to either deaf-ferentation and ischemia, ischemia alone, or normal control weresacrificed 7 days post-ischemia. Total RNA was extracted fromCA1 microdissected from the dorsolateral hippocampus usingTrizol reagent (Invitrogen, Burlington, ON) according to manufac-turer's instructions.

qPCR primers: The following primers for GluR2 were selectedfrom published sequences (Gorter et al., 1997): GluR2 (accessionno. M85035) forward primer: 5′-CGGGTAGGGATGGTTCAGTTT-3′and reverse primer: 5′-TGGCTACCTCCACCTTGTCGAT-3′ (Invitro-gen, Burlington, ON). The primers were checked for hairpinstructures on Primer3 Express (online version). The length of theamplicon was 62 bp and the melting temperatures were set at 60°C (Horii et al., 2002). 18 S RNA was used as an internal control andthe abundance of target mRNAs were expressed relative to thisreference gene.

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4.7. Reverse transcription and PCR amplification

cDNA synthesis from total RNA extracts was performed usingSuperscript II RT (Invitrogen, Burlington, ON) and dNTPs accordingto manufacturer's instructions. The PCRs were performed on anABI PRISM 7000 real-time thermal cycler using SYBR green, intriplicates in a final volume of 30 μl containing 5 μl cDNA sample, 4mM MgCl2, 200 nM each oligonucleotide primer, 200 μM dATP,dCTP, dGTP, 400 μMdUTP, 1× SYBR Green PCR buffer and 0.625 U oftaq-polymerase (Invitrogen, Burlington, ON). Samples were heat-ed for 10 min at 95 °C and subjected to 45 cycles of denaturation at95 °C for 15 s and annealing and elongation at 60 °C for 1 min. Anegative control without cDNA template was included in everyassay. All PCR products were checked by melting point analysis(Applied Biosystems, Foster City, CA, USA). Analysis of PCR resultswas done using the comparative Ctmethod. Relative expression ofgene of interest relative to reference gene was calculated using thefollowing formula (Horii et al., 2002):

RFE ¼ 2�DDCt

Where RFE is the Relative Fluorescence Expression, ΔΔCt = (Ct geneof interest) − (Ct reference gene), and Ct is the cycle threshold.

This method enables quantification of expression levels ofgene of interest relative to a reference control. Expression level ofGluR2mRNA obtained fromCA1 of normal, non-ischemic CA1wasconsidered as 100%. Values obtained for GluR2 expression inischemic CA1 of deafferented and non-deafferented hemisphereswere normalized to those of non-ischemic CA1.

4.8. Statistical analysis

Paired Student t test was used to determine statistical significancebetween groups. Correlation between groups was establishedusing Pearson's product moment correlation coefficient.

Acknowledgments

Alastair Buchan is supported by a Heart and Stroke Founda-tion Professor of Stroke Research award and a Heart andStroke Foundation of Alberta, NWT and Nunavut operatinggrant, and also by an Alberta Heritage Foundation for MedicalResearch Scientist award. Rose Geransar was supported by aCanadian Stroke Consortium Summer Studentship award.This research was also generously supported by the DavidAshford fund of the Calgary Foundation.

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