Down-regulation of Nogo receptor promotes functional recovery by enhancing axonal connectivity after experimental stroke in rats

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Down-regulation of Nogo receptor promotes functional recovery by enhancing axonal connectivity after experimental stroke in rats Tianzhu Wang a , Jing Wang a , Cheng Yin a , Ruen Liu b , John H. Zhang c , Xinyue Qin a,⁎ a

Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of Neurosurgery, China-Japan Friendship Hospital, Beijing100029, China c Department of Neurosurgery, Loma Linda University School of Medicine, Loma Linda, CA, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

The inability of axons in central nervous system (CNS) to regenerate after injury is related

Accepted 30 August 2010

partly to multiple endogenous axon growth inhibitors including Nogo receptor (NgR). This

Available online 7 September 2010

study tested the hypothesis that silencing NgR expression by adenovirus-mediated RNA interference (RNAi) (AD-NgR) may permit axonal connectivity after focal cerebral ischemia

Keywords:

in rats. Male Sprague–Dawley rats (250–280 g, n = 97) were assigned into seven groups: sham,

Adenovirus-mediated RNAi

MCAO (24 h and 2 weeks), MCAO plus AD-NgR (24 h and 2 weeks), and MCAO plus AD-HK

MCAO

(control oligonucleotides) (24 h and 2 weeks). After cerebral ischemia, NgR mRNA and

NgR

protein in the cortex and hippocampus were significantly increased at 24 h and 2 weeks.

Axonal connectivity

However, in AD-NgR treated rats, NgR mRNA and protein were reduced by 40–60% in the cortex and hippocampus at both time points as compared to controls. Although there was no significant difference in the infarct volume between the two groups, the number of midline-crossing fibers projecting to the contralateral red nucleus and corticostriatal fibers in the dorsolateral striatum were increased in AD-NgR injected rats, accompanied by improved behavioral outcomes. Taken together, these results suggest that NgR knockdown may promote CNS axonal regeneration and functional recovery after ischemic cerebral injury. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

The inability of neuronal axons in CNS to regenerate after injury results severe limitations in the functional recovery after injury (Donoghue, 1997. Regenerative failure has been

attributed in part to proteins associated with CNS myelin (Filbin, 2003) and with the glial scar that forms at the injury site (Silver and Miller, 2004). Several myelin inhibitors of axon growth, including Nogo (GrandPre et al., 2000), myelinassociated glycoprotein (MAG) (McKerracher et al., 1994) and

⁎ Corresponding author. Fax: + 86 23 89012478. E-mail address: [email protected] (X. Qin). Abbreviations: AAV, adenovirus-associated virus; BDA, biotinylated dextran amine; BSA, bovine serum albumin; CNS, central nervous system; CST, corticospinal tract; TTC, 2,3,5-triphenyltetrazolium chloride; MAG, myelin-associated glycoprotein; MCAO, middle cerebral artery occlusion; NEP1–40, nogo extracellular peptide 1–40; NgR, nogo receptor; OMgp, oligodendrocyte myelin glycoprotein; RNAi, RNA interference; RT-PCR, reverse transcriptase-polymerase chain reaction 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.08.101

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oligodendrocyte myelin glycoprotein (OMgp) (Wang et al., 2002a,b), exert their effects via the Nogo receptor (NgR) (Fournier et al., 2001) and other co-receptors, including p75NTR, LINGO-1 and TROY (Wang et al., 2002a,b). Signaling through these receptors activate intracellular Rho GTPase signal pathways that results in the collapse of the growth cone (Niederost et al., 2002). Consequently, NgR is believed to play a pivotal role in the suppression of axonal growth pathways (McGee and Strittmatter, 2003). These pathways have been studied most intensively in traumatic spinal cord injury (Wang et al., 2006). The IgG fusion protein, NgR (310) Ecto-Fc, has been found to be more effective than Nogo extracellular peptide 1–40 (NEP1–40) in promoting axonal growth and recovery after spinal cord injury (Li et al., 2004). In behavioral assays in rats, NgR antagonism promotes functional recovery by enhancing axonal plasticity after stroke (Lee et al., 2004). RNAi-induced gene knockdown is attractive for its faster speed, more usefulness, and lower cost, compared with the time-consuming conventional strategies such as gene targeting by homologous recombination (Kobayashi et al., 2004). In the present study, we examined whether adenovirus-mediated RNAi reduced the expression of NgR mRNA and protein in the cortex and hippocampus after cerebral ischemia/reperfusion. Furthermore, we determined whether knockdown of NgR promoted axon connectivity and functional recovery in rats after experimental stroke.

2.

Results

2.1. Adenoiviral gene delivery effect on gene expression in the peri-infarct cortex and hippocampus GFP-adenovirus was injected as control indicator to measure how far adenovirus could reach from injection sites. There were no GFP-positive cells detected in contralateral cortex or hippocampus 24 h after viral transfection (Fig. 1, A1 and A2). GFP-positive neurons were observed among injected periinfarct cortex (Fig 1, C1 and C2), hippocampal regions CA1–3 and dentate gyrus (Fig. 1, B1 and B2).

2.2.

Infarct volume analysis

Infarct volume (white colored areas) was evaluated using TTC staining at 24 h and 2 weeks after MCAO. The infarct volumes at 24 h (Fig. 2, A1, B1 and C1) were not significantly different among MCAO, MCAO plus AD-NgR and MCAO plus AD-HK groups (P > 0.05). Even though TTC values were lower at 2 weeks when compared with those at 24 h in each group respectively (P < 0.01), no significance was observed among the three groups at 2 weeks (P > 0.05) (Fig. 2, A2, B2 and C2).

2.3. Expression of NgR mRNA in infarcted cortex and hippocampus At 24 h (Fig. 3, A1 and B1) and at 2 weeks (Fig. 3, A2 and B2) after MCAO, a marked increase (P < 0.01) in the expression of NgR mRNA in the infarcted cortex and ipsilateral hippocampus was observed when compared to the sham group. AD-NgR but

not AD-HK treatment significantly (P < 0.01) suppressed the level of NgR mRNA (Fig. 3, A3 and B3).

2.4.

Protein expression of NgR

A significant elevation (P < 0.01) of NgR protein was observed at 24 h and 2 weeks after MCAO (Fig. 4). Only AD-NgR treatment abolished the elevation of NgR in the ischemic cortex (Fig. 4, A1 and A2) and ipsilateral hippocampus (Fig. 4, B1 and B2).

2.5.

Sensorimotor function assessment

Rats were trained daily for 2 weeks to establish limb preference and baseline measurements prior to MCAO (Fig. 5). No significant difference among the groups was recorded before MCAO (P > 0.05). Animals that received MCAO surgery showed marked deficits in successfully obtaining pellets with the stroke-affected limb after 1 week. In AD-NgR treatment groups, a 50% recovery (3.2 ± 0.44) of the baseline behavioral performance (6.2 ± 0.45) was demonstrated after 3 weeks, which was significantly different from the values obtained from MCAO and MCAO plus AD-HK treatment groups. From 5 to 9 weeks, the behavioral performance of the rats which received AD-NgR treatment (4.8 ± 0.89) was able to recover to 75% of the baseline behavioral performance, significantly better than MCAO and MCAO plus AD-HK treatment groups (Fig. 5).

2.6.

Tracing corticorubral and corticospinal tracts

At the level of the midbrain, the lateral branches between the red nucleus and the primary motor cortex, was examined. A distinct characteristic of this unilateral projection to the red nucleus was that only a small number of fibers crossed the midline to project to the contralateral nucleus. In MCAO and AD-HK treatment groups, the corticorubral tract was ipsilateral dominant, with little evidence of fibers crossing to the contralateral red nucleus (Fig. 6, A2 and C2). In contrast, animals that underwent treatment with AD-NgR showed much more BDA-positive fibers crossing the midline and terminating in the contralateral red nucleus at appropriate target areas (Fig. 6, B2). Quantitative analysis (Fig. 6, D) demonstrated a significant (P < 0.01) increase in the fibers crossing the midline treated with AD-NgR (520 ± 56) as compared to animals in MCAO (220 ± 33) and AD-HK treatment groups (158 ± 67).

2.7.

Tracing corticostriatal tract

At the level of striatum, there were large numbers of labeled fibers entering the ipsilateral striatum, most of which were located in the dorsolateral quadrant. There was no apparent difference in the intensity of labeling fibers in the ipsilateral striatum between these three groups (Fig. 7, A4, B4 and C4). In contrast with MCAO and AD-HK treatment groups, more BDApositive fibers were seen coursing through the corpus callosum into the contralateral dorsolateral striatum in ADNgR group (Fig. 7, A2, B2 and C2). Following quantitative analysis, the ratio of contralateral striatum BDA-positive fibers divided by ipsilateral striatum was significantly higher

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Fig. 1 – Adenoviral gene delivery effect on gene expression in the peri-infarct cortex and hippocampus. There was no GFP-positive cell detected in the region opposite to the injected site 24 h after viral transfection (A1, A2). GFP-positive neurons were observed among injected peri-infarct cortex (C1 and C2), hippocampal regions CA1–3 and dentate gyrus (B1 and B2). Scale bars: A1–C1, 200 μm, A2–C2,100 μm.

in AD-NgR treatment group than in the other two groups, respectively (P < 0.05) (Fig. 7, D).

3.

Discussion

Previously, we reported that after ischemic injury NgR mRNA and protein expression in ischemic cortex and hippocampus gradually increased during first 24 h and peaked at 2 weeks, and returned to normal levels by 3 weeks (Zhang et al., 2008). Therefore, in this study, we selected 24 h and 2 weeks as two relevant time points to observe gene silencing effects. This study demonstrated that early intracerebral infusion of ADNgR suppressed the elevation of NgR expression which was induced by MCAO and resulted in functional recovery in skilled forelimb movements. Furthermore, AD-NgR treatment showed an increase of axon connectivity in corticorubral and corticostriatal pathways from the non-lesioned hemisphere, resulting in bilateral innervations. In the early stages of focal cerebral ischemia, Nogo-A/NgR pathway may interfere with neural plasticity and impede subsequent neural recovery (Zhou et al., 2003). At advanced stages of focal cerebral ischemia, the expression of Nogo-A,

rather than MAG and OMgp, lasted for 4 weeks with concomitant oligodendrocyte proliferation (Wang et al., 2007). A number of studies have demonstrated that blocking NgR can promote central nervous axonal regeneration. Administration of NEP1–40, an antagonist of NgR, promoted axonal regeneration and also improved motor functions in mice which underwent transverse spinal cord injury (Cao et al., 2008). After spinal cord and ischemic brain injury, NgR (310) Ecto-Fc treatment improved axonal regeneration and motor functions in rats (Li et al., 2004). However, drug-intervention approaches have inherent shortcomings that would hinder the clinical usage especially when long-term treatment is required to achieve a certain blood concentration. Therefore we utilized NgR as the target for intervention and employed gene therapy to reduce the expression of NgR after cerebral ischemia. Now, two methods are used to reduce endogenous gene expression. The one is to knock out the gene of interest to suppress gene expression completely. The other one is to knock down gene expression by RNAi. For clinical relevance and acute treatment approach, using RNAi to suppress NgR overexpression after cerebral ischemia seems an ideal approach (Grimm and Kay, 2007). Furthermore, adenovirus-mediated RNAi can be highly effective in inhibiting endogenous gene expression

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Fig. 2 – TTC staining was used to evaluate infarct volume at 24 h and 2 weeks after middle cerebral artery occlusion–reperfusion from MCAO groups (A1 and A2), AD-HK groups (B1, B2), AD-NgR groups(C1, C2). White regions represented areas of infarct; it was clear that the infarct sustained among these three groups was no significant difference at 24 h and 2 weeks after ischemia–reperfusion .Whereas the infarct volume was significantly reduced at 2 weeks after middle cerebral artery occlusion-reperfusion compared to at 24 h after ischemia–reperfusion (*P < 0.01vs 24 h after ischemia–reperfusion). Scale bars: 7.5 mm.

(Hommel et al., 2003). In previous studies, adenovirus-mediated RNAi were developed for the treatment of CNS neurodegenerative disease, including spinocerebellar ataxia (Xia et al., 2004) and Huntington Disease (Franich et al., 2008). These studies employed AAV as the vector since AAV can integrate into the host genome and as a result silence genes for prolonged time periods (Kobayashi et al., 2004). Our previous study indicated that the expression of NgR persisted for approximately 3 weeks (Zhang et al., 2008). Therefore adenovirus-mediated RNAi suits our needs for a short term suppression of endogenous gene expression (Kitagawa et al., 1999). In the study we found that adenovirus could transfect ischemic brain tissue efficiently, and neurons in the hippocampus had higher infection efficiency (Fig. 1). We demonstrated that after 24 h of treatment with ADNgR, the level of NgR mRNA and protein in cortex and hippocampus dropped 60% when compared to MCAO animals. After 2 weeks, the inhibitory effect on NgR mRNA expression maintained at 40% level. An interesting observation in this study was that the infarct volume was not changed by RNAi. At first, the infarct volume was not different among groups at 24 h, which seemed that suppression of Nogo-A/NgR signaling pathway did not translated into early morphological neuroprotection in this animal model of cerebral ischemia. Then the infarct volume was decreased till 2 weeks in each group, but the difference among groups were still not found. Even the TTC stain result was not much reliable for measuring infarction at 2 weeks, for

36 h after ischemia macrophages with intact mitochondria would replace injured neurons and be stained, it could be still concluded that RNAi failed to reduce the infarct volume at the two points after MCAO. This observation is somehow in agreement with another study using antagonist to block NogoA/NgR signaling pathway after experimental stroke in rats (Lee et al., 2004). Even though acute neuroprotection was not observed, acute application of RNAi to reduce NgR mRNA and protein expression seemed to lead to functional recovery up to 9 weeks after ischemic stroke. The underlying mechanisms might be related to axonal connectivity especially the corticorubral and corticostriatal pathways, which may constitute the anatomical substrate responsible for recovery of motor function (Kolb et al., 1992; Z'Graggen et al., 1998; Kartje et al., 1999; Papadopoulos et al., 2002). In the AD-NgR treatment animals, we found large number of BDA-positive fibers crossing the midline from the red nucleus of uninjured side in the midbrain. The similar phenomenon was also observed in the level of striatum. These results suggested that the increased density of corticorubral fibers and corticostriatal fibers originated in part from the contralateral site, uninjured cortex. Experimental studies of motor corticofugal pathways had shown that after unilateral sensorimotor cortical lesions in newborn rats, the spared, contralateral hemisphere formed new, bilateral connections with the striatum (Kartje et al., 1999), thalamus (Yu et al., 1995), red nucleus (Naus et al., 1985), tectum (Leong and Lund, 1973), basilar pontine gray (Castro

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and Mihailoff, 1983), and spinal cord (Castro, 1975). In our study, at least corticorubral and corticostriatal pathways may constitute the anatomical substrate responsible for recovery of motor function. To sum up, inhibiting the Nogo-A/NgR pathway can enhance the functional recovery and axonal connectivity in a rat model of cerebral ischemic injury. Gene silencing of NgR may block inhibitory axonal growth signals and thus remove the constraint of axonal regeneration, indicating RNAi could be a potential treatment strategy for ischemic stroke.

4.

Experimental procedures

4.1.

Adenoviral vector

In our previous study (Peng et al., 2010) we constructed three short hairpin segments against NgR (numbered as NgR1, 2, 3 shRNA) based on type-5 adenovirus that was essentially the same as that in previous reports (Luo et al., 2007). AD-NgR was amplified in HEK293 cells and purified, whose titer was measured as previously described (Ogorelkova et al., 2004). After RT-PCR and western blotting analyses, NgR2 shRNA had the highest efficiency of gene silencing among the three adenoviruses and was selected for this study (AD-NgR).

4.2.

Animals

Seventy seven adult male Sprague–Dawley rats (250–280 g) were randomly divided into 7 groups (n = 11 in each group): sham, MCAO (24 h and 2 weeks), MCAO plus AD-NgR (24 h and 2 weeks), and MCAO plus AD-HK (negative control groups, 24 h and 2 weeks). In addition, another 20 rats were randomly assigned into sham, MCAO, MCAO plus AD-NgR, and MCAO plus AD-HK (n = 5 in each group) for labelling corticorubral tract, corticospinal tract and corticostriatal tract. All protocols for animal experiments were approved by the Administrative Panel on Laboratory Animal Care of Chongqing Medical University.

4.3.

Transient middle cerebral artery occlusion in rats

Right middle cerebral artery occlusion (MCAO) was induced with an intraluminal filament (Zhang et al., 2008). Briefly, animals were anesthetized by 3.5% Chloral Hydrate (350 mg/ kg). Under an operating microscope, the right common carotid artery (CCA) was exposed through a midline neck incision and dissected from its bifurcation to the base of the skull. After coagulation of the occipital artery branches of the external carotid artery (ECA), the right ECA was coagulated along with the terminal lingual and maxillary artery branches. The right internal carotid artery (ICA) was isolated, and the pterygopalatine artery was ligated close to its origin with a silk suture. A microaneurysm clip was placed across both CCA and them ICA to prevent bleeding during the insertion of the suture. After the silk suture was tied loosely around the mobilized ECA stump, a small incision was made on the ECA stump, and a 40-mm-length monofilament nylon suture, heat blunted at the tip and coated with melting paraffin wax, was inserted into the lumen of the ICA. The temporary clip on the ICA was

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removed, and the nylon suture was advanced 18–20 mm from the bifurcation of the CCA until mild resistance was felt. The silk suture around the ECA stump was tightened on the intraluminal nylon suture. After removal of the microaneurysm clip, the neck incision was closed. After 2 h of occlusion, the animals were re-anesthetized and the filament was withdrawn. Animals subjected to sham surgery were treated similarly, except that the filament was not advanced to the origin of the MCA. Rats that failed to exhibit neurological abnormalities at 2 h after stroke were excluded from this study. Physiological parameters were monitored before and during MCAO. Rats' rectal temperatures were maintained at 37 ± 0.5 °C with a heating pad. Regional cerebral blood flow (rCBF) was measured during the surgery by a laser-Doppler flow (LDF) (Periflux system 5000; Perimed) to confirm the successful occlusion of MCA.

4.4.

Stereotactic surgery

Immediately after MCAO, rats were anesthetized again with 3.5% Chloral Hydrate (350 mg/kg) and placed in a stereotaxic apparatus. The bregma was used as stereotaxic zero. Two sites in the cortex surrounding the infarction (Zhao et al., 2002) were targeted for injection at the following coordinates: 1.0 mm rostral to the bregma, 2.0 mm lateral to the midline, 1.2 mm ventral to the dura; 3.0 mm caudal to the bregma, 1.5 mm lateral to the midline, 1.2 mm ventral to the dura. One site in the hippocampus was targeted for injection coordinate: 3.5 mm caudal to the bregma, 2.5 mm lateral to the midline, 3.5 mm ventral to the dura. AD-NgR (1.58 × 108 pfu/10 μl) or ADHK (1.42 × 108 pfu/10 μl) was injected into the three sites independently. The injection rate was 0.3 μl/min and the total volume was 2 μl in each site. At the end of the injection, the microinjector was kept in its position for 5 min before withdrawal of the needle. The animals were kept warm by a heating pad.

4.5.

Quantification of infarct volume

Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) stained sections (+4.7 to −5.5 mm from the bregma (Paxinos and Watson, 1986). Using a rat brain slicer (Activational Systems, Warren, MI, USA), 2 mm coronal sections were dissected. After sectioning, slices were immediately immersed in 2%TTC in 0.9% NaCl at 37 °C for 10 min for vital staining, photographed, and transferred in 4% PFA for immersion fixation for 24 h. The infarct size was measured as described by Kawamata et al. (1997) (the total area of the intact contralateral hemisphere minus the total area of the intact ipsilateral hemisphere, multiplied by the total distance between sections).

4.6.

Detection of virus delivery

Slices from the anterior and posterior sections (previously stained in TTC) were dehydrated in 30% sucrose. After that, the sections were cut into coronal slices of 30 μm on a microtome (Leica CM3050S, Germany) and mounted onto slides. The GFP fluorescence was detected by fluorescence microscope directly.

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Fig. 3 – RT-PCR analysis demonstrates the expressions of NgR mRNA in the infarct cortex and hippocampus from sham group and ischemic insult groups, AD-NgR groups, negative control groups (AD-HK groups), respectively. Representative bands for every group as well as corresponding β-actin are shown (A1, A2, B1, B2). The right panels show the change of NgR mRNA expressions in each group. Bars represent mean ± SD of n = 6 rats per group. The expression of NgR mRNA is significantly increased at 24 h and 2 weeks after cerebral ischemia–reperfusion. AD-NgR treatment significantly decreases the level of NgR mRNA expression (*P < 0.01 vs. control, #P < 0.01vs. ischemia–reperfusion). M: marker, 1 indicates sham group, 2 indicates cerebral ischemia–reperfusion groups, 3 indicates AD-HK groups, 4 indicates AD-NgR groups.

4.7.

Evaluation of NgR mRNA by RT-PCR

Animals were deep anesthetized and sacrificed by decapitation. Arachnoid was kicked off by equipment processed with DEPC (diethypyrocarbonate) firstly overnight, then put into high pressure cylinder for 2–3 h and dried in 60 °C oven. The brain tissue was placed on the ice, rapidly separated, weighed on analytical balance, quickly put into a marked frozen pipe, and finally stored in liquid nitrogen. The entire process was no more than 3 min. Primers were designed using GenBank sequences: AF462390 (Nogo receptor as template was optimized using Primer Premier 5.0 software (Molecular Biology Insights, Inc., Cascade, CO).The primer sequences were designed as following: sense-5'TGC TGGCAT GGG TGT TAT GG-3', anti-sense-5'CGG AAGGTG TTG TCG GGA AG-3', (NgR1, product size 493 bp); sense-5'CGT AAA GAC CTC TAT GCC AAC

A-3', antisense-5'CGG ACT CAT CGT ACT CCT GCT-3' (β-actin, product size 229 bp). Total RNA was extracted from isolated cerebral cortex and hippocampus by the acid guanidinium thiocyanate-phenol-chloroform method using TRIZOL reagent. The RNA for assay was dissolved in Rnase-free water. A260/A280 ratios of purified RNA were between 1.6 and 1.8. RNA samples were processed for cDNA synthesis using Random (dT) 12–18 primers and MMLV reverse transcriptase (Toyobo Bio-Technology, Japan). PCR was carried out in 50 μL of reaction mixture containing 10 μl 5 × PCR buffer, 0.25 μl TaKaRa Ex Taq HS (TaKaRa Bio-Technology, Japan), 10 μl cDNA, 28.75 μl ddH2O2, 1 μl forward and reverse primers. The amplification program consisted of a denaturing step at 94 °C for 2 min, an annealing step at 59.3 °C for 50 s and an extension step at 72 °C for 2 min, for 35 cycles. The 5 μL PCR products separated on a 1.5% agarose gel (E-Gel, Invitrogen,

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Fig. 4 – Western blot analysis shows the expression of NgR protein in the infarct cortex and hippocampus from sham group and MCAO groups, AD-NgR groups, negative control groups (AD-HK groups), respectively. Representative bands for every group as well as corresponding β-actin are shown (A1, A2, B1, B2). The right panels show the change of NgR protein expressions in each group. Bars represent mean ± SD of n = 6 rats per group. The expression of NgR protein is significantly increased at 24 h and 2 weeks after cerebral ischemia–reperfusion. AD-NgR treatment significantly decreases the level of NgR protein expression (*P < 0.01 vs. control, #P < 0.01vs. ischemia–reperfusion). M: marker, 1 indicates sham group, 2 indicates cerebral ischemia–reperfusion groups, 3 indicates AD-HK groups, 4 indicates AD-NgR groups.

Carlsbad, CA, USA). The gel was photographed under UV transillumination with the AlphaImager system (AlphaInnotech, San Leandro, CA, USA).

4.8.

Western blot analysis for NgR protein

The brains were sampled as described above, with protein-free equipments. The remained isolated cerebral cortex and hippocampus were homogenized in ice-cold lysis buffer containing 1 ml Radio Immunoprecipitation Assay Lysis Buffer (Keygen biotechnology company, China) and 5 μl of 100 μg/ml Phenylmethyl SulphonylFluoride (PMSF), 5 μl phosphatase inhibitor, 1 μl proteinase inhibitor mixture for purifying proteins according to the manufacturers' instructions. Protein concentration was determined by Bradford method (Bio-Rad, Hercules CA, USA) using bovine serum albumin (BSA) as the standard. Equal amounts of protein per lane (50 μg) were loaded onto an 12% polyacrylamide gel and separated by electrophoresis at 80 V for 90 min. Proteins were then trans-

ferred to PVDF membrane at 250 mA for 70 min and the membrane was blocked with 5% nonfat dried milk/0.05% Tween 20 in Tris-buffered saline. The membrane was then incubated with different antibodies overnight at 4 °C: goat anti-NgR (Santa Cruz Biotechnology, 1:200, CA, USA) and mouse anti-β-actin (Abcam Ltd, 1:2000, England). Following washes, the membranes were incubated with peroxidaseconjugated rabbit anti-goat IgG and anti-mouse IgG (Santa Cruz Biotechnology, 1:1000, CA, USA) in blocking solution for 1 h at 37 °C. ECL Western blot detection reagents (Pierce Biotechnology, Rockford, IL, USA) detected antibody labeling. The Western blot result was quantified by densitometry. Relative optical density of protein bands was measured following subtraction of the background.

4.9.

Evaluation of sensorimotor performance

Montoya's staircase test was carried out to test skilled forepaw use as described in detail previously (Montoya et al., 1991).

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Fig. 5 – Staircase test. Quantitative measurements of animals reaching for food at baseline (Preolesion). One week after stroke, the numbers of food pellets retrieved with the contralateral forepaw. Significantly greater recovery during the postoperative period (3 weeks, 5 weeks, 7 weeks, 9 weeks) is observed in the AD-NgR-treated animals (*P < 0.01 vs. ischemia–reperfusion groups and AD-HK groups). There was no significant change of recovery during the postoperative period (3 weeks, 5 weeks, 7 weeks, 9 weeks) in AD-HK-injected animals and received no treatment animals.

Rats were placed in a Plexiglas box with a baited double staircase. Food pellets were placed on the staircase and presented bilaterally at seven graded stages of reaching difficulty to provide objective measures of maximum forelimb extension and grasping skill. Each step of the stairs was baited with two chow pellets of 45 mg. Food was restricted during the pre-stroke training period and 5 d after surgery to provide motivation for food rewards. All animals were food restricted to 85–90% of their free feeding weight. Each test session lasted 15 min. The number of pellets seized and eaten per side was used as a measure of forelimb reaching ability. Animals were received 2 weeks training before being subjected to surgery to establish the baseline performance, and animals that did not learn to seize four pellets from each side during the last training session was excluded from the study. The tests were made in weekly intervals on days 7, 21, 35, 49, and 63 postoperatively.

4.10.

Biotinylated Dextran Amine (BDA) tracing

Seven weeks after MCAO, the sensorimotor cortex opposite to the stroke lesion site was exposed, and 5 injections of 1 μl each of a 10% solution of BDA (MW10,000, Molecular Probes, CA, USA), in 0.01 mol/L phosphate buffer (pH 7.4) were injected to the left forelimb sensorimotor cortex following these five coordinates: 1.0 mm rostral to the bregma, 1.0 mm lateral to the midline, 1.5 mm ventral to the dura; 4.0 mm caudal to the bregma, 1.0 mm lateral to the midline, 1.5 mm ventral to the dura; 1.0 mm rostral to the bregma, 5.0 mm lateral to the midline, 1.5 mm ventral to the dura; 4.0 mm caudal to the bregma, 5.0 mm lateral to the midline, 1.5 mm ventral to the dura; 1.5 mm caudal to the bregma, 2.5 mm lateral to the midline, 1.5 mm ventral to the dura. Two weeks after BDA injection, animals were deeply anesthetized with 3.5% Chloral Hydrate (350 mg/kg) intraperitoneally and perfused cardially with 200 mL of PBS followed by 200 mL of 4% paraformaldehyde in 0.1 mol/L phosphate buffer. Brain was dissected, postfixed overnight, and embedded in tissue-freezing medium for cryostat sectioning. Forty micrometer coronal sections were

cut using a vibratome (Leica CM3050S，Germany). All sections were collected in 0.01 mmol/L PBS and incubated with avidin– peroxidase in 0.01 mmol/L PBS /0.3% Triton X-l00 for 4 h at 37 °C. Then the slides were washed three times for 30 min in PBS. The tissue was incubated with 0.05% 3,3-diaminobenzidine (DAB; Molecular Probes, CA, USA) for 5 min. The sections were air dried, dehydrated, coverslipped with mounting medium. Brain structures needed to be analyzed were identified with the atlas of Paxinos and Watson (Paxinos and Watson, 1986). Sections were analyzed by image analysis software (MCID/M2Analyzing Programe, Imaging Research, Ontario, Canada). The number of labeled corticospinal tract (CST) fibers in the pons ipsilateral to the injection site was counted and used for error correction in BDA labeling. Images of two consecutive sections at cerebral peduncle level were captured using × 10 objective. Then four squares, each measuring 0.45 × 0.67 mm, were centered on the cerebral peduncle, and the BDA-positive fibers within these squares were examined using × 40 objective. The total number of labeled CST fibers for each section was estimated by the means of the four values .The values obtained from the two consecutive sections were averaged. The corticorubral tract projecting to the red nucleus in infarct side was examined by counting all BDA-positive fibers crossing the midline on each section. To account for differences in tracing, the number of midline-crossing BDA-positive fibers was divided by the total number of CST fibers. The method described by Kartje was used to evaluate the cortical projections to the striatum (Kartje et al., 1999). The sections chosen for analysis began at the rostral level (AP + 1.20 mm) and extended through the entire dorsal striatum to the caudal level (AP −1.20 mm). Approximately every fourth section was counted in the following manner: using a rectangular counting frame, BDA-positive fibers that intersected a total line length of 1 mm were counted in each striatal quadrant, both ipsilateral and contralateral to the injection site. The number of intersections in each quadrant was averaged. As an internal control to take into account tracer variability between animals, the average fiber count from the contralateral side was divided by the ipsilateral count to provide a ratio.

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Fig. 6 – Cross-sections of the nucleus ruber. There is a few visible fibers crossing the midline to the contralateral red nucleus in animal received no treatment after ischemia–reperfusion (A1, A2). Many axons (arrow) crossing the midline and ending in the contralateral red nucleus are seen in an AD-NgR-treated animal (B1, B2). It is similar to the ischemia group; a few axons crossing the midline can be seen in an AD-HK -treated animal (C1, C2). Labeled corticospinal tract fibers are in the cerebral peduncle of the same section (A3–C3, A4–C4). Scale bars: A2–C2, A4–C4, 50 μm; A1–C1, 200 μm; A3–C3, 100 μm. (D) Midline fiber crossing index :Number of midline crossing fibers in the area of the red nucleus divided by the total number of labeled CST fibers, to correct for the differences in the tracing (*P < 0.01).

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Statistical analysis

All results were presented as means ± standard deviation (SD). Statistical differences between the control and

each group of ischemia (with or without adenovirus treatment) were compared by using one-way analysis of variance (ANOVA) followed by a post hoc Tukey test. The P value < 0.05 was considered statistically significant.

Fig. 7 – Cross sections of the striatum. There are a few visible fibers in the dorsolateral striatum, contralateral to BDA injection site in a animal received no treatment after ischemia–reperfusion (A1, A2). Many axons (arrow) are seen in the same region in an AD-NgR-treated animal (B1, B2). It is similar to the ischemia group, a few visible fibers in the dorsolateral striatum, contralateral to BDA injection site can be seen in an AD-HK-treated animal (C1, C2). Labeled BDA-positive fibers are in the striatum, ipsilateral to the biotinylated dextran amine (BDA) injection site (A3–C3, A4–C4). Scale bars: A2–C2, A4–C4, 50 μm; A1–C1, A3–C3, 200 μm. (D) The ratio of BDA-positive fibers in the dorsolateral striatum (contralateral to BDA injection site) divided by fibers in the same region (ipsilateral to the BDA injection site), to correct for the differences in the tracing (*P < 0.01).

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Statistical analysis was performed using SPSS 11.5 for windows.

Acknowledgment These studies were supported by the National Natural Science Foundation of China (no: 30470607) to Dr. Xinyue Qin.

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