Spasticity obstructs motor function recovery post-stroke, and has been reported to occur in spinal cord injury and electrophysiological studies. The purpose of the present study was to assess spinal cord circuit spasticity in post-stroke mice. At 3, 7, 21, and 42 d after photothrombotic ischemic cortical injury in C57BL/6J mice, we observed decreased rate-dependent depression (RDD) of the Hoffmann reflex (H reflex) in the affected forelimb of mice compared with the limbs of sham mice and the non-affected forelimb. This finding suggests a hyper-excitable stretch reflex in the affected forelimb. We then performed immunohistochemical and western blot analyses to examine the expression of the potassium-chloride cotransporter 2 (KCC2) and phosphorylation of the KCC2 serine residue, 940 (S940), since this is the main chloride extruder that affects neuronal excitability. We also performed immunohistochemical analyses on the number of vesicular glutamate transporter 1 (vGluT1)-positive boutons to count the number of Ia afferent fibers that connect to motoneurons. Western bolts revealed that, compared with sham mice, experimental mice had significantly reduced KCC2 expression at 7 d post-stroke, and dephosphorylated S940 at 3 and 7 d post-stroke in motoneuron plasma membranes. We also observed a lower density of KCC2-positive areas in the plasma membrane of motoneurons at 3 and 7 d post-stroke. However, western blot and immunohistochemical analyses revealed that there were no differences between groups 21 and 42 d post-stroke, respectively. In addition, at 7 and 42 d post-stroke, experimental mice exhibited a significant increase in vGluT1 boutons compared with sham mice. Our findings suggest that both the down-regulation of KCC2 and increases in Ia afferent fibers are involved in post-stroke spasticity.

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.

Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1–8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson’s disease.

Previous epidemiological studies have revealed that occupational exposure to extremely low frequency weak magnetic fields (ELF‐WMF) is associated with the development of neurodegenerative diseases including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). The molecular mechanism of these detrimental effects, however, remain mostly unelucidated. We examined the effects of extremely low frequency fluctuation of weak magnetic fields (ELF‐WMF) on cultured mammalian cells. While applying ELF‐WMF, we examined the temporal profiles of expression levels of VDAC1, a mitochondrial outer membrane protein, and seven electron transport complex (ETC) proteins of NDUFS1 (ETC I), NDUFB8 (ETC I), SDHB (ETC II), UQCRFS1 (ETC III), UQCRC2 (ETC III), MTCOI (ETC IV), and ATP5A (ETC V). ELF‐WMF downregulated the amounts of these proteins at 3 h and recovered them at 12 h. Quantification of the amount of mitochondria with MitoTracker Green showed that the mitochondrial mass was decreased to 70% at 3 h and recovered at 12 h. In addition, mitophagy‐related proteins of PINK1 and LC3‐II were increased by ELF‐WMF at 1.5–2.5 h. Quantification of ETC enzymatic activities in protein homogenates revealed that ELF‐WMF exclusively suppressed the activity of ETC II in 10 min. We report that ELF‐WMF much weaker than geomagnetic fields suppresses the enzymatic activity of ETC II, and provokes mitophagy. Mitophagy is a cellular quality‐assurance system to eliminate damaged mitochondria. Mitophagy induced by ELF‐WMF may be causally associated with the development and progression of AD and ALS. Contrarily, ELF‐WMF finely tuned to preferentially eliminate damaged mitochondria may be able to ameliorate Parkinson’s disease, in which compromised mitophagy is a cause of the disease. Support or Funding Information This research was supported by grants‐in‐aids from MEXT, MHLW, and AMED of Japan.

Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have not been reported. We found that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1–8 Hz, which was three or more times weaker than the geomagnetic field, reduced mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullified the effect of ELF-WMF. Suppression of ETC complex II subsequently induced mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induced PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negated the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson’s disease.