Weakened rate-dependent depression of Hoffmann's reflex and increased motoneuron hyperactivity after motor cortical infarction in mice

Abstract

Abnormal reflexes associated with spasticity are considered a major determinant of motor impairments occurring after stroke; however, the mechanisms underlying post-stroke spasticity remain unclear. This may be because of the lack of suitable rodent models for studying spasticity after cortical injuries. Thus, the purpose of the present study was to establish an appropriate post-stroke spasticity mouse model. We induced photothrombotic injury in the rostral and caudal forelimb motor areas of mice and used the rate-dependent depression (RDD) of Hoffmann's reflex (H-reflex) as an indicator of spastic symptoms. To detect motoneuron excitability, we examined c-fos mRNA levels and c-Fos immunoreactivity in affected motoneurons using quantitative real-time reverse transcription PCR and immunohistochemical analysis, respectively. To confirm the validity of our model, we confirmed the effect of the anti-spasticity drug baclofen on H-reflex RDDs 1 week post stroke. We found that 3 days after stroke, the RDD was significantly weakened in the affected muscles of stroke mice compared with sham-operated mice, and this was observed for 8 weeks. The c-fos mRNA levels in affected motoneurons were significantly increased in stroke mice compared with sham-operated mice. Immunohistochemical analysis revealed a significant increase in the number of c-Fos-positive motoneurons in stroke mice compared with sham-operated mice at 1, 2, 4, and 8 weeks after stroke; however, the number of c-Fos-positive motoneurons on both sides of the brain gradually decreased over time. Baclofen treatment resulted in recovery of the weakened RDD at 1 week post stroke. Our findings suggest that this is a viable animal model of post-stroke spasticity.

OPEN

Citation: Cell Death and Disease (2014) 5, e1007; doi:10.1038/cddis.2013.544

& 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14 http://www.nature.com/cddis

Web End =www.nature.com/cddis

S Lee*,1, T Toda1, H Kiyama2 and T Yamashita3

Abnormal reexes associated with spasticity are considered a major determinant of motor impairments occurring after stroke; however, the mechanisms underlying post-stroke spasticity remain unclear. This may be because of the lack of suitable rodent models for studying spasticity after cortical injuries. Thus, the purpose of the present study was to establish an appropriate post-stroke spasticity mouse model. We induced photothrombotic injury in the rostral and caudal forelimb motor areas of mice and used the rate-dependent depression (RDD) of Hoffmanns reex (H-reex) as an indicator of spastic symptoms. To detect motoneuron excitability, we examined c-fos mRNA levels and c-Fos immunoreactivity in affected motoneurons using quantitative real-time reverse transcription PCR and immunohistochemical analysis, respectively. To conrm the validity of our model, we conrmed the effect of the anti-spasticity drug baclofen on H-reex RDDs 1 week post stroke. We found that 3 days after stroke, the RDD was signicantly weakened in the affected muscles of stroke mice compared with sham-operated mice, and this was observed for 8 weeks. The c-fos mRNA levels in affected motoneurons were signicantly increased in stroke mice compared with sham-operated mice. Immunohistochemical analysis revealed a signicant increase in the number of c-Fos-positive motoneurons in stroke mice compared with sham-operated mice at 1, 2, 4, and 8 weeks after stroke; however, the number of c-Fos-positive motoneurons on both sides of the brain gradually decreased over time. Baclofen treatment resulted in recovery of the weakened RDD at 1 week post stroke. Our ndings suggest that this is a viable animal model of post-stroke spasticity.

Cell Death and Disease (2014) 5, e1007; doi:http://dx.doi.org/10.1038/cddis.2013.544

Web End =10.1038/cddis.2013.544 ; published online 16 January 2014

Subject Category: Neuroscience

Spasticity is common in patients with brain and spinal cord injuries.1,2 Approximately 2040% of patients with stroke exhibit spasticity,35 whereby neurological damage disrupts voluntary movement control. Consequently, these patients quality of life is signicantly lower compared with that of patients without post-stroke spasticity.6

A velocity-dependent increase in muscle tone resulting from stretch reex hyperexcitability has been proposed as a cardinal feature of spasticity.7,8 In addition, spinal reex spasticity is known to be accompanied by weakened rate-dependent depression (RDD) of Hoffmanns reex (H-reex), which is the electrical analog of the tendon jerk reex and is mediated through monosynaptic pathways in the spinal cord. Electromyograms recorded duringH-reexes typically display two responses: an initial M wave, which is the result of direct activation of motor axons, and anH-reex, which is elicited by the synaptic activation of motoneurons. The RDD of the H-reex refers to the phenomenon by which the magnitude of the H-reex is temporarily attenuated by repetitive stimulation. Given that

Keywords: Stroke; spasticity; mouse

Abbreviations: RDD, rate-dependent depression; H-reex, Hoffmanns reex; PFA, paraformaldehyde; PBS, phosphate-buffered saline; LMD, laser microdissection; RT-PCR, reverse transcription-PCR; ChAT, choline acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ANOVA, analysis of variance; GABA, gamma-aminobutyric acid

Weakened rate-dependent depression of Hoffmanns reex and increased motoneuron hyperactivity after motor cortical infarction in mice

spasticity develops in conjunction with weakening RDD of the H-reex, which is gradually abolished following brain and spinal cord injury, RDD is considered a reliable measurement of spasticity in stroke patients and in animal models of spinal cord injury.911

It is clear that stroke can cause inhibitory and excitatory impulse imbalance, leading to upper motoneuron symptoms, and it is known that lesion location and extent can result in differing symptoms and degrees of spasticity severity; however, the pathophysiological basis of spasticity remains poorly understood.12 It is accepted that spasticity involves over-reactivity of alpha motoneurons and alterations in primary Ia reciprocal inhibition and Renshaw recurrent inhibition,1317 but further understanding of the underlying mechanisms is hindered by the lack of animal models. In one study, Fulton and Kennard18 used primates to demonstrate that lesions in both primary and premotor areas can induce spasticity; however, a rodent model is needed to analyze the precise mechanisms underlying spasticity after cortical injuries, such as those in stroke.

1Department of Rehabilitation Sciences, Graduate School of Medicine, Nagoya University, 1-1-20 Daiko-minami Higashi-ku, Nagoya-shi, Aichi, Japan; 2Department of Functional Anatomy and Neuroscience, Graduate School of Medicine, Nagoya University, 65 Tsurumai-tyou Shouwa-ku, Nagoya-shi, Aichi, Japan and 3Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita-shi, Osaka, Japan*Corresponding author: S Lee, Department of Rehabilitation Sciences, Graduate School of Medicine, Nagoya University, 1-1-20 Daiko-minami Higashi-ku, Nagoya-shi, Nagoya 461-8673, Japan. Tel: [phone]; Fax: mailto:+81 52 719 1365.3d

Web End =81 52 719 1365 ; E-mail: mailto:[email protected]

Web End [email protected]

Received 26.8.13; revised 04.12.13; accepted 09.12.13; Edited by A Verkhratsky

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Here, we investigated whether spasticity is induced after photothrombotic injury to the rostral and caudal forelimb motor areas of mice, which are considered the premotor and forelimb primary motor cortices in rodents, respectively.19 Given that enhanced excitability of affected motoneurons has frequently been reported in studies of spasticity after stroke and spinal cord injury, we further examined whether a similar phenomenon occurred in our mouse model of post-stroke spasticity.

Results

Location and volume of cortical infarct lesions. We unilaterally induced focal thrombotic stroke to the rostral and caudal forelimb motor areas of mice using the Rose Bengal photothrombotic method.2022 Lesions in the rostral and caudal forelimb motor areas were reproducible (Figures 1a and b). Nissl-stained coronal sections from the lesioned areas obtained 8 weeks after stroke demonstrated neuronal loss in the infarcted area, and volume quantication demonstrated that the lesion was 2.560.28 mm3 (n 5) in size.22

Reduction of H-reex RDD after stroke. To test whether an affected muscle exhibited spastic symptoms after stroke, we measured H-reex RDDs in the affected and unaffected abductor digiti minimi muscles 3 days and 1, 2, 3, 4, 6, and 8 weeks after stroke. The H-reex magnitudes were attenuated by repeated activations at frequencies 40.1 Hz. In particular, we observed a reduction of 480% of the H-reex magnitude at 5 Hz in sham-operated mice (Figure 2a, upper panel). In contrast, the H-reex magnitude in 4-week stroke mice was not strongly attenuated by repeated activation (Figure 2a, lower panel). Moreover, we found that RDDs for the affected muscles were signicantly weakened in stroke mice (n 6) compared with sham-operated mice (n 7) 3

days after stroke, and this was still observed on week 8 after stroke (Po0.01, Figure 2b); however, the RDDs were not

signicantly weakened at week 3. In addition, the RDDs of stroke mice were signicantly smaller at week 6 than at weeks 4 and 8 (Po0.01). Four weeks after stroke, the RDDs induced at all frequencies 40.1 Hz were signicantly smaller in stroke mice than in sham-operated mice (Figure 2c). This indicates that the affected muscles exhibited spastic post-stroke symptoms in our model.

In addition, we compared H-reex RDDs elicited by 5 Hz in the unaffected muscles of the sham-operated and stroke mice (Figure 2d). We found that at weeks 1 and 8, the RDDs in the unaffected muscles were signicantly smaller in stroke mice compared with sham-operated mice (Po0.01 for week 1 and

Po0.05 for week 8); this suggests that the RDDs in the unaffected muscles were also inuenced by cortical injuries.

However, it should be noted that the RDDs of sham-operated and stroke mice were not signicantly different at any other time point in the unaffected muscles (Figure 2d).

Similarly, we compared the RDDs at 5 Hz for the affected and unaffected muscles of stroke and sham-operated mice separately (Figures 3a and b). In stroke mice, the RDDs for the affected side were signicantly smaller than those for the unaffected side at 1, 2, 3, and 6 weeks after stroke (Po0.01 for 1, 2, and 6 weeks and Po0.05 for 3 weeks; Figure 3a);

however, the weakened depressions at 4 and 8 weeks were not signicantly different between the two sides. In contrast, there were no differences in the RDDs between the affected and unaffected sides of sham-operated mice (Figure 3b).

We also examined RDDs in the abductor pollicis brevis muscles after stroke and found that at 1 week after stroke they were signicantly smaller in stroke mice compared with sham-operated mice (Po0.01, data not shown). These results indicate that some muscles developed spastic symptoms after cortical injury in this model system.

Motoneuron hyperactivation after stroke. It has been reported that one of the mechanisms underlying spasticity in stroke patients is increased neuronal excitability of the

1 mm

Figure 1 (a) Schematic representation showing the lesion areas (n 4). These hemicortical lesions were reproducible. (b) Coronal sections of Nissl-stained injured brain;

specically, the rostral and caudal forelimb areas were injured (Nissl staining)

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Figure 2 RDD of H-reex in sham-operated and stroke mice. (a) Repeated stimulation at 5 Hz weakened H-reex amplitude in normal mice (upper). In stroke mice, H-reex was not weakened upon repeated stimulation at 5 Hz (lower). (b) Changes in H-reex depression (%) with 5-Hz stimulation on the affected side in stoke and sham-operated mice. (c) H-reex RDD 4 weeks after stroke. Response data are expressed as percentages relative to the mean responses at 0.1 Hz in the same series of measurements. (d) Changes in H-reex depression (%) with stimulation at 5 Hz on the unaffected side in stroke and in sham-operated mice. (sham-operated group, dotted line; stroke group, solid line) Data are shown as the meanS.E.M. *Po0.05, **Po0.01 (n 7 for sham-operated mice and n 6 for stroke mice, one-way ANOVA followed

by TukeyKramer tests)

propriospinal neurons and motoneurons.14,17,23 To determine whether motoneuron excitability was increased after stroke, we performed quantitative real-time reverse transcription PCR (RT-PCR) to examine the gene expression of c-fos, an activity-dependent immediate early gene, in spinal motoneurons.24,25 A retrograde tracer and laser microdissection (LMD) technique was used to obtain affected motoneurons (Figure 4A). Expression of c-fos mRNA in the affected neurons was signicantly increased for stroke mice (n 3)

compared with sham-operated mice (n 4) (B50 neurons

from each mouse were analyzed; MannWhitney U test, Po0.01, Figure 4B).

Furthermore, we measured c-Fos immunoreactivity in motoneurons labeled for choline acetyltransferase (ChAT) using spinal sections from the cervical to the thoracic level (C4T1) obtained 1, 2, 4, and 8 weeks after stroke. At 1 week after stroke, c-Fos induction in the affected and unaffected neurons was signicantly increased in stroke mice compared with sham-operated mice at most spinal levels (n 3 for

groups; **Po0.01, in C4T1, compared between sham-operated and stroke mice on the affected side; #Po0.05 in

C4, ##Po0.01, in C6T1, compared between sham-operated and stroke mice on the unaffected side; Figures 4c and d).

Moreover, at 2 weeks after stroke, the number of c-Fos-positive motoneurons at C4T1 of stroke mice signicantly increased on the affected side compared with the affected side of the sham-operated mice (n 3 for groups; *Po0.05,

Figure 4E). Similarly, at 4 weeks after stroke, the number of c-Fos-positive motoneurons in the affected side of stroke mice signicantly increased compared with the affected side of the sham-operated mice at C4C7 (n 3 for groups; **Po0.01 in

C4C7; Figure 4F). At 8 weeks after stroke, the number of c-Fos-positive neurons at C7 and T1 on the affected side was signicantly higher in stroke mice compared with sham-operated mice (n 3 for groups; *Po0.05; 8 weeks in C7 and

T1; Figure 4G); however, stroke mice showed a gradual decrease in this number during the eighth week after stroke (C4; r 0.33, Po0.01, C5; r 0.31, Po0.01,

C6; r 0.39, Po0.01, C7; r 0.32, Po0.01, T1;

r 0.48, Po0.01; Figure 5a). In the unaffected side in

stroke mice, we observed a similar increase in the number of c-Fos-positive neurons (C6; r 0.41, Po0.01, C7;

r 0.46, Po0.01, T1; r 0.30, Po0.01; Figure 5b).

In addition, compared with the unaffected side of stroke mice, their affected side showed signicantly increased number of c-Fos-positive motoneurons at C4 and C5 on week 1 and at C6 on week 4 after stroke (wwPo0.01 at 1 week;

wPo0.05 at 4 weeks; Figures 4d and f). These results suggest that the motoneurons, which innervate both the affected and unaffected muscles, were excited.

Effective spasticity suppression with baclofen. Many clinical and experimental animal pharmacology studies have used the anti-spasticity agent baclofen, which is a gamma-aminobutyric acid (GABA)B receptor agonist.26,27 To conrm whether spastic symptoms, such as weakened RDD, were inhibited following administration of an anti-spasticity agent, we measured H-reex RDD in mice intraperitoneally injected with baclofen at 1 week after stroke. The mice, which exhibited weakened RDD at 3 days after stroke (Figure 6a), were injected with baclofen or 0.9% NaCl as vehicle before H-reex RDD measurement. We found that weakened RDDs

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Figure 3 RDDs of H-reexes of the affected and unaffected sides in (a) stroke and (b) sham-operated mice. Data are represented as the meanS.E.M. *Po0.05, **Po0.01 (n 7 for sham-operated mice and n 6 for stroke mice,

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were signicantly recovered in baclofen-injected stroke mice (n 5) compared with vehicle-injected stroke mice (n 4,

Po0.01; Figure 6b).

We also examined RDDs in baclofen-injected intact mice and found that RDDs were not signicantly different from those in vehicle-injected intact mice (data not shown).

Discussion

Spasticity is a common complication after stroke; however, the underlying pathophysiology remains unclear, and research into this area is hampered by the lack of appropriate animal models. The present study reveals that lesions in the rostral and caudal forelimb motor areas of the mouse brain induce spasticity, as indicated by weakened H-reex RDDs, increased motoneuron excitability of affected muscles and recovered RDDs in stroke mice following baclofen treatment. Our ndings raise the possibility that this protocol may yield a viable animal model of post-stroke spasticity.

Clinically, increased muscle tone (i.e., an increase in resistance to passive stretch) occurs because of increased reex activity and intrinsic changes in the muscles.3 Reex-mediated increases in patient muscle tone reach a maximum between 1 and 3 months after stroke,3,28 and it has been proposed that after 3 months, the increased resistance to passive stretch is attributable to intrinsic changes in the muscles.28 We found that the weakened H-reex RDDs exhibited two phases of spasticity (Figures 2b and 3a). In the rst phase (04 weeks after stroke), we observed a relatively small, yet signicant, difference in the RDDs of sham-operated and stroke mice. However, in the second phase (48 weeks after stroke), the H-reex in stroke mice was not highly depressed by 5-Hz stimulation. Moreover, the number

of c-Fos-positive cells was elevated in the period from 0 to 4 weeks after stroke but gradually decreased during 48 weeks after stroke (Figures 5a and b). It can thus be hypothesized that plasticity at the spinal and neuromuscular levels may occur, for instance, the number of motoneuron synapses may increase29 or the development of motoneuron axonal arbors may be enhanced.30 These changes could account for the signicant difference in RDDs that we observed during the second phase compared with the rst phase.

Our results demonstrated that the number of c-Fos-positive motoneurons in for both affected and unaffected motoneurons was signicantly elevated in stroke mice compared with sham-operated mice (Figures 4dg). Previous studies have also reported neuronal plasticity in unaffected neurons and contra-lesioned corticospinal axons after cortical injury.31,32 One reason for plasticity in the unaffected side may be injury of neurons in the rostral and caudal forelimb motor areas that disynaptically project to unaffected spinal cord motoneurons. In other words, motor and supplemental motor cortical areas project to both sides of the brainstem but especially to the ipsilateral brainstem.33,34 As brainstem neurons project to the spinal cord, bilateral spinal cord motoneurons are indirectly inuenced. However, H-reex RDDs in unaffected motoneurons were not signicantly weakened in stroke mice compared with sham-operated mice except at 1 and 8 weeks post stroke (Figure 2d). The reason why H-reex RDDs in unaffected side post-stroke mice were not signicantly weakened compared with sham-operated mice is unclear.

With regard to spinal cord injury, it was previously hypothesized that one of the main mechanisms of spasticity was increased motoneuron excitability.11,35,36 In our mouse spasticity model, we examined motoneuron excitability by assessing c-fos mRNA expression and c-Fos immuno-reactivity in motoneurons, and we found that levels of both were signicantly increased in the affected spinal cord after stroke. Boulenguez et al.11 recently reported that one of the reasons for motoneuron excitability after spinal cord injury is down-regulation of the potassium chloride cotransporter KCC2; when this occurs, inhibitory GABAA and glycine receptors switch from depolarizing to hyperpolarizing actions of GABA and glycine, respectively, resulting in low intracellular Cl concentrations.11,37 Moreover, a previous study reports as other spasticity mechanism that the serotonergic 5-HT2C receptor changes constitutive active after spinal cord injury.38 Furthermore, patients with multiple sclerosis, which is an autoimmune disease, also exhibit spasticity. These lines of evidence indicate that several other mechanisms may contribute to post-stroke spasticity.

We used H-reex RDD, which is believed to cause both presynaptic and motoneuron excitability, as a measure of spasticity.14,17,39,40 In previous studies, H-reex RDD was used as a measure of spasticity, we also observed signicance weakened H-reex RDDs at 4 weeks after spinal cord injury (data not shown).1,9,11 It is known that repetitive ring of synapses leads to a temporary decrease in synaptic strength,41 possibly due to a decrease in presynaptic Ca2 current,42 vesicle depletion,41 postsynaptic receptor desensitization,43,44 activity-dependent decrease in neurotransmitter release probability45,46 and action potential conduction failure in the postsynaptic neuron.

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Figure 4 Increased c-fos mRNA levels in affected motoneurons after stroke. (A) Cross-sections of the spinal cord at C7. The motoneurons were labeled with the retrograde tracer True Blue to isolate the motoneurons with LMD. Bar 200 mm. (a) Magnied view of the square in panel (A). The arrowheads indicate motoneurons labeled

with True Blue. (b) Labeled motoneurons were isolated by LMD. (a and b): Bar 100 mm. (B) Quantication of c-fos mRNA expression in the affected motoneurons 1 week

after stroke (by RT-PCR). The fold change in c-fos mRNA expression in the stroke group relative to the sham-operated group is presented as the meanS.E.M. (n 4 for

sham-operated mice and n 3 for stroke mice, approximately 50 motoneurons for each animal; **Po0.01 by MannWhitney U-test). (C) At 1 week after stroke, c-Fos-

positive motoneurons in C7 increased in both the affected and unaffected sides of stroke mice compared with the respective side of sham-operated mice. Bars 100 mm.

(DG) Quantitative analyses of the number of c-Fos-ChAT double-positive motoneurons (arrowhead) in affected and unaffected spinal cord levels (C4T1) in sham-operated and stroke mice at 1, 2, 4, and 8 weeks after stroke. *Po0.05, **Po0.01 between sham-operated and stroke mice on the affected side; #Po0.05, ##Po0.01 between sham-operated and stroke mice on the unaffected side; wPo0.05, wwPo0.01 between unaffected and affected sides in stroke mice (one-way ANOVAs followed by TukeyKramer tests)

H-reex RDD is useful for determining changes in spinal cord function after injury.10,11,4749 In our model, motoneuron excitability, an important spastic symptom, was conrmed

by changes in H-reex RDD and c-Fos expression, which highlights the potential of using this protocol to model post-stroke spasticity.

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Figure 6 H-reex RDDs 1 week post stroke in mice treated with baclofen. H-reex RDD in (a) baclofen- or vehicle-injected intact mice or (b) 1-week stroke mice. Response data are expressed as percentages relative to the mean responses at 0.1 Hz in the same series of measurements. Data are represented as meanS.E.M. (**Po0.01, one-way ANOVA followed by TukeyKramer tests)

Although there are other parameters for assessing spasticity in patients with spinal cord injury and stroke, such as the modied Ashworth scale scored by the degree of muscle tone, these measurements cannot be used in animal models.5053 Apparent spastic behavior was not detected in our model, which may be due to the lack of an appropriate parameter for assessing spastic behavior in rodents. Given that we did not clearly observe spastic behavior, we are currently trying to detect abnormal muscle contractions or neuromuscular junction changes using morphological analyses. These parameters could be used as spasticity markers in the future. Our model of spasticity after stroke can potentially help elucidate spasticity pathophysiology, which could aid the development of pharmacological and rehabilitation treatments.

Materials and MethodsAnimals. Adult male C57BL/6J mice (2030 g, 810-weeks old, Japan SLC, Inc., Shizuoka, Japan) were housed in standard cages with food and water ad libitum and maintained under a 12-h light-dark cycle. All procedures were approved by the Nagoya University Guidelines for the Care and Use of Laboratory Animals.

Photothrombotic cortical lesion. Focal cortical ischemia was induced by cortical microvessel photothrombosis, as described previously.21,22 Mice were

anesthetized with intraperitoneal sodium pentobarbital (Somnopentyl, 50 mg/kg body weight; Kyouritu Seiyaku, Tokyo, Japan) and then individually placed on a stereotaxic frame for surgery. Rose Bengal (30 mg/kg body weight; Sigma-Aldrich, St. Louis, MO, USA) was injected into the tail vein 5 min before illumination, and the skull was exposed via a midline incision of the skin. Focal illumination of the rostral and caudal forelimb motor areas ( 3.0-mm to 1.5-mm anteroposterior,

3.00.5-mm lateral to bregma) (Figures 7a and b) was carried out with a cold light source (KL1500LCD, Zeiss, Oberkochen, Germany), with a 4.5-mm aperture54

through the intact skull; the brain was illuminated for 15 min.22 The scalp was then sutured, and the mice were allowed to recover. Control mice either received the same injection of Rose Bengal without illumination (n 10) or were

photostimulated after an intravenous injection of 0.9% NaCl solution (n 4).

Electrophysiological recordings. We modied a previously described method to use the RDD of the H-reex as a measurement of spastic symptoms11,55 Mice were anesthetized with ketamine (200 mg/kg, CS Pharmacology Co., Ltd., Aichi, Japan), which is widely used during H-reex recordings (Figure 8a). If required, an additional 25% dose of anesthesia was given every 30 min to suppress whisker tremors or voluntary movements. The forelimbs and hindlimbs of the mice were xed on a plate with a plastic tape, and their positions were controlled to avoid unnecessary pressure and stretch that could affect the electrophysiological responses of the muscles and nerves (Figure 8b). To maintain the body temperature, the plate was placed on a warm pad maintained at approximately 37 1C. We transcutaneously inserted a pair of stainless needle electrodes (EKA2-1508, Bioresearch Center Corporation, Aichi, Japan) xed with micromanipulator (SM-15, NARISHIGE, Tokyo, Japan) into nerve bundles, including the ulnar nerve and stimulated with a stimulator (13 mA in 0.1 mA increments, SEN7103, Nihon Kohden Corporation, Aichi, Japan). For recording, a pair of stainless needle electrodes xed with manipulator (YOU-2, NARISHIGE) was transcutaneously placed into the abductor digiti minimi muscles and obtained recordings with an amplier (High pass: 0.1, SS-201J, Nihon Kohden Corporation) and an A/D converter (Low pass: 1 kHz, PowerLab, ADInstruments Japan Inc., Aichi, Japan). We initially stimulated the nerves for 0.2 ms at 0.1 Hz and then gradually increased the current intensity; in this way, we were able to determine the intensity necessary to obtain a maximal H-reex. Following this, we used an intensity of 23 stimulation trains at 0.1, 0.5, 1, 2, and 5 Hz, with 2-min intervals

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Light - exposured Area

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Figure 7 Stroke lesion areas in the left rostral and caudal forelimb motor areas. (a) Image of a photochemically injured cortex. (b) The light beam was exposed to the brain at the following coordinates: 3-mm to 1.5-mm antero-posterior and from 0.5-mm to 3.0-mm lateral to the left of bregma in the rostral and caudal forelimb areas

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H reflex

(-) stimulation

(+) stimulation

reference

(-) record

(+) record

Figure 8 (a) Illustration of the M wave and H-reex and (b) the method used to measure the H-reex in the abductor digiti minimi muscle of the mouse

between each train, to measure the RDD. To determine the RDD at different frequencies, we discarded the responses to the rst three stimulations that were necessary for depression to occur and expressed all of the remaining

responses as percentages relative to the mean response at 0.1 Hz in the same

series of measurements.

LMD and real time RT-PCR preparation. To obtain samples of the affected motoneurons in the abductor muscles of the little nger, we labeled the motoneurons with the retrograde tracer 1% True Blue (T5891, Sigma-Aldrich); the tracer was diluted with water and injected into the right abductor digiti minimi muscle. The spinal cords (C6 to Th1) were removed from both sham-operated and stroke mice, and the collected samples were cut into 30-mm sections with a cryostat. The labeled motoneurons were collected with an LMD system (LMD 6500, Leica Microsystems K. K., Tokyo, Japan; Figure 3). After total RNA was isolated from the obtained neurons, the RNAs were transcripted to cDNA and amplied with a Prelude and Ovation WTA system (PicoSL WTA system V2, NuGEN Technologies, Inc., San Carlos, CA, USA).

Quantitative analysis of real-time RT-PCR. Quantitative analysis for real-time RT-PCR was performed using an oligonucleotide primer sets that corresponded to the cDNA sequences of c-fos (forward: 50-GGGAGGACCTTACC

TGTTCGT-30, reverse: 50-GGATGCTTGCAAGTCCTTGAG-30), ChAT (forward: 50-ACATATGATGACAGGCAACAAGAAG-30, reverse: 50-GGGAGCAGGGAGTTC ACTCA-30), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, forward: 50-GGGCTGGCATTGCTCTCA-30, reverse: 50-TGTAGCCGTATTCATTGTCATA CCA-30). We used the ABI StepOne Plus system (Applied Biosystems, Life Technologies Corporation, Grand Island, NY, USA). The reaction mixture (10 ml), containing 5 ml SYBR Green real-time PCR master mix (THUNDERBIRD SYBR q

PCR Mix, Toyobo Co., Ltd., Osaka, Japan), 0.2 ml of both the sense and antisense primers (10 mM), and 1 ml cDNA sample (10 ng), was preheated at 95 1C for 3 min and then treated with 40 cycles of amplication (denaturation at 95 1C for 15 s and annealing and extension at 60 1C for 1 min). The relative intensity against GAPDH and the fold-change relative to the controls were calculated with DDCT analyses. mRNA levels of ChAT, a motoneuron differentiation marker, were also measured.

c-Fos immunohistochemical analysis. At 1, 2, 4, and 8 weeks after stroke, some mice (n 3 each) were deeply anesthetized and then transcardially

perfused with 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer. The spinal cord from the cervical to the upper thoracic region was removed and xed in 4% PFA at 4 1C overnight and then cryoprotected in 30% sucrose for 3 days. The tissues were embedded in optimal cutting temperature compound (Tissue-Tek, SAKURA Finetek, Tokyo, Japan) and stored at 80 1C.

Coronal sections of the spine (2030-mm thick) were prepared with a cryostat.

Cell Death and Disease

Spasticity after stroke S Lee et al

8

Sections were then stored in the cryoprotectant ethylene glycol at 20 1C until

the time of use. Briey, we removed tissue sections from the cryoprotectant and washed them in phosphate-buffered saline (PBS). We then pre-incubated the tissue in blocking buffer (5% normal bovine serum and 0.25% Triton X-100 in PBS) for 1 h at 25 1C, followed by incubation with a c-Fos antibody (sc-52, 1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ChAT antibody (AB144P, 1 : 500, Millipore, Billerica, MA, USA) in blocking buffer for 24 h at room temperature. After washing in PBS, the tissue was incubated for 1 h in a solution containing Alexa Fluor 488 and 568 conjugated to donkey anti-rabbit (1 : 1000) and donkey anti-goat (1 : 500) immunoglobulin G, respectively (Molecular Probes, Invitrogen, Carlsbad, CA, USA). The sections were counterstained with DAPI (40,6-diamidino-2-phenylindole). All the sections were examined by uorescence microscopy (magnication, 100, BZ-9000 BIOREVO, KEYENCE, Osaka, Japan)

or confocal laser scanning microscopy (magnication, 100, A1Rsi, Nikon,

Tokyo, Japan). For quantication, c-Fos-positive motoneurons, stained with ChAT, were counted in 10 cross-sections per cervical and upper thoracic level (C4T1).

Baclofen treatment. This blind experiment of intraperitoneal injection of baclofen was performed using a modied procedure as previously described.27 We

conrmed the weakened RDDs in the abductor digiti minimi muscle at 3 days after stroke. The stroke mice were intraperitoneally injected with baclofen (n 5, 9 mg/kg

weight, diluted with 0.9% NaCl, Sigma-Aldrich) or 0.9% NaCl (n 4) as vehicle at

7 days post stroke. H-reex RDDs of injected mice were measured 90 min after the injection. To assess the effect of baclofen in intact mice, we also measured RDDs in baclofen-injected intact mice.

Statistical analyses. Group measurements are expressed as mean S.E.M. H-reex RDDs were determined as a measurement of spasticity and analyzed with one-way analysis of variance (ANOVA) and TukeyKramer Tests. For real-time RT-PCR analysis, we used MannWhitney U-tests and Students t-tests to compare data obtained from the sham-operated and stroke groups. For c-Fos immunohistochemical analysis, we used ANOVA, TukeyKramer tests, and Spearmans correlation coefcient by rank test. P-values o0.05 were considered signicant.

Conict of InterestThe authors declare no conict of interest.

Acknowledgements. This work was supported by a grant from the JSPS KAKENHI (Grant Number 23700598) and a grant from the Hayashi Memorial Foundation for Female Natural Scientists. We thank Fumitaka Kimura for his helpful support and experiment suggestions.

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