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.

Figure 1

(a) Schematic representation showing the lesion areas (n=4). These hemicortical lesions were reproducible. (b) Coronal sections of Nissl-stained injured brain; specifically, the rostral and caudal forelimb areas were injured (Nissl staining)

Figure 2

RDD of H-reflex in sham-operated and stroke mice. (a) Repeated stimulation at 5 Hz weakened H-reflex amplitude in normal mice (upper). In stroke mice, H-reflex was not weakened upon repeated stimulation at 5 Hz (lower). (b) Changes in H-reflex depression (%) with 5-Hz stimulation on the affected side in stoke and sham-operated mice. (c) H-reflex 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-reflex 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 mean±S.E.M. *P<0.05, **P<0.01 (n=7 for sham-operated mice and n=6 for stroke mice, one-way ANOVA followed by Tukey–Kramer tests)

Figure 3

RDDs of H-reflexes of the affected and unaffected sides in (a) stroke and (b) sham-operated mice. Data are represented as the mean±S.E.M. *P<0.05, **P<0.01 (n=7 for sham-operated mice and n=6 for stroke mice, one-way ANOVA followed by Tukey–Kramer tests)

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 μm. (a) Magnified 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 μm. (B) Quantification 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 mean±S.E.M. (n=4 for sham-operated mice and n=3 for stroke mice, approximately 50 motoneurons for each animal; **P<0.01 by Mann–Whitney 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 μm. (D–G) Quantitative analyses of the number of c-Fos-ChAT double-positive motoneurons (arrowhead) in affected and unaffected spinal cord levels (C4–T1) in sham-operated and stroke mice at 1, 2, 4, and 8 weeks after stroke. *P<0.05, **P<0.01 between sham-operated and stroke mice on the affected side; ^#P<0.05, ^##P<0.01 between sham-operated and stroke mice on the unaffected side; ^†P<0.05, ^††P<0.01 between unaffected and affected sides in stroke mice (one-way ANOVAs followed by Tukey–Kramer tests)

Figure 5

Changes in the number of c-Fos-positive motoneurons in (a) affected and (b) unaffected sides of the spinal cord from 1 to 8 weeks after stroke. Data are represented as mean±S.E.M. (Spearman's correlation coefficient by rank test)

Figure 6

H-reflex RDDs 1 week post stroke in mice treated with baclofen. H-reflex 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 mean±S.E.M. (**P<0.01, one-way ANOVA followed by Tukey–Kramer tests)

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

Figure 8

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