Akira Sekiguchi

Rapamycin Promotes Autophagy and Reduces Neural Tissue Damage and Locomotor Impairment after Spinal Cord Injury in Mice

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that negatively regulates autophagy. Rapamycin, an inhibitor of mTOR signaling, can promote autophagy and exert neuroprotective effects in several diseases of the central nervous system (CNS). In the present study, we examined whether rapamycin treatment promotes autophagy and reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin significantly decreased the phosphorylation of the p70S6K protein and led to higher expression levels of LC3 and Beclin 1 in the injured spinal cord. In addition, neuronal loss and cell death in the injured spinal cord were significantly reduced in the rapamycin-treated mice compared to the vehicle-treated mice. Furthermore, the rapamycin-treated mice showed significantly higher locomotor function in Basso Mouse Scale (BMS) scores than did the vehicle-treated mice. These results indicate that rapamycin promoted autophagy by inhibiting the mTOR signaling pathway, and reduced neural tissue damage and locomotor impairment after SCI. The administration of rapamycin produced a neuroprotective function at the lesion site following SCI. Rapamycin treatment may represent a novel therapeutic strategy after SCI.

Spinal cord injury induces upregulation of Beclin 1 and promotes autophagic cell death

Autophagy is a degradation of the cytoplasm and it induces autophagic cell death in several neurodegenerative conditions. Beclin 1, a Bcl-2-interacting protein, is known to be a promoter of autophagy. We investigated the alterations in the Beclin 1 protein expression and the involvement of autophagy and autophagic cell death after spinal cord injury using a spinal cord hemisection model in mice. In the present study, the Beclin 1 expression dramatically increased at the lesion site after hemisection. The increased expression of Beclin 1 started from 4 h, peaked at 3 d, and lasted for at least 21 d after hemisection. The Beclin 1 expression was observed in neurons, astrocytes, and oligodendrocytes. The nuclei in the Beclin 1 expressing cells were round, which should normally be observed in autophagic cell death, and they were not either shrunken or fragmented as is observed in apoptotic nuclei. The results of the present study suggested that autophagy is activated in the injured spinal cord. Furthermore, autophagic cell death is considered to clearly contribute to neural tissue damage after spinal cord injury.

Induction of autophagy and autophagic cell death in damaged neural tissue after acute spinal cord injury in mice.

Study Design. Expression of light chain 3 (LC3), a characteristic marker of autophagy, was examined by immunohistochemistry and Western blot using a spinal cord injury (SCI) model in mice. Electron microscopic analysis was also performed to examine the anatomic formation of autophagy and autophagic cell death in the injured spinal cord. Objective. To examine both biochemically and anatomically the activity of autophagy in the damaged neural tissue after SCI. Summary of Background Data. Autophagy is the bulk degradation of intracellular proteins and organelles, and it is involved in a number of diseases. Autophagy can lead to nonapoptotic programmed cell death, which is called autophagic cell death. Recent researches have revealed the increased expression of LC3 and the anatomic formation of autophagy and autophagic cell death in damaged tissues of various disease models. However, previous studies have focused on apoptotic process but not autophagic activity as mechanism of neural tissue damage after SCI. To date, there has been no study to examine the expression of LC3 and the anatomic formation of autophagy after SCI. Methods. The spinal cord was hemitransected at T10 in adult female C57BL/6J mice. The LC3 expression was examined by immunohistochemistry and Western blot. The anatomic formation of autophagic activity was investigated using electron microscopy. Results. Immunohistochemistry showed that the number of the LC3-positive cells significantly increased at the lesion site after hemisection. The increase of LC3-positive cells was observed from 4 hours and peaked at 3 days, and it lasted for at least 21 days after hemisection. The LC3-positive cells were observed in neurons, astrocytes, and oligodendrocytes. Western blot analysis demonstrated that the level of LC3-II protein expression significantly increased in the injured spinal cord. Electron microscopy showed the formation of autophagic vacuoles to increase in the damaged cells. Furthermore, the nuclei in the transferase-mediated dUTP nick end labeling–positive cells expressed LC3 were round, which is consistent with autophagic cell death, and they were neither shrunken nor fragmented as is observed in apoptotic nuclei. Conclusion. This study suggested both biochemically and anatomically that autophagy was clearly activated and autophagic cell death was induced in the damaged neural tissue after SCI.

The role of mTOR signaling pathway in spinal cord injury

The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in multiple cellular functions, such as cell metabolism, proliferation and survival. Many previous studies have shown that mTOR regulates both neuroprotective and neuroregenerative functions in trauma and various diseases in the central nervous system (CNS). Recently, we reported that inhibition of mTOR using rapamycin reduces neural tissue damage and locomotor impairment after spinal cord injury (SCI) in mice. Our results demonstrated that the administration of rapamycin at four hours after injury significantly increases the activity of autophagy and reduces neuronal loss and cell death in the injured spinal cord. Furthermore, rapamycin-treated mice show significantly better locomotor function in the hindlimbs following SCI than vehicle-treated mice. These findings indicate that the inhibition of mTOR signaling using rapamycin during the acute phase of SCI produces neuroprotective effects and reduces secondary damage at lesion sites. However, the role of mTOR signaling in injured spinal cords has not yet been fully elucidated. Various functions are regulated by mTOR signaling in the CNS, and multiple pathophysiological processes occur following SCI. Here, we discuss several unresolved issues and review the evidence from related articles regarding the role and mechanisms of the mTOR signaling pathway in neuroprotection and neuroregeneration after SCI.

The role of autophagy in spinal cord injury

Previous studies have indicated that autophagy has an important function, not only in many neurodegenerative diseases, but also in traumatic and ischemic brain injury. However, no study has previously shown the contribution of autophagy to neural tissue damage after spinal cord injury. We recently investigated that the alterations in Beclin 1 expression and the involvement of autophagy and autophagic cell death after spinal cord injury using a spinal cord hemisection model in mice. The results showed that the expression of Beclin 1 dramatically increased in the damaged neural tissue and induced autophagic cell death after a spinal cord injury. These observations suggested that the increased expression of Beclin1 activates autophagy, while mediating a novel cell death mechanism at the lesion site in response to spinal cord injury. Here we discuss several unsolved issues and review the evidence in related articles regarding the role of autophagy and its contribution to the mechanism of cell death in spinal cord injury.

Genetic ablation of transcription repressor Bach1 reduces neural tissue damage and improves locomotor function after spinal cord injury in mice.

Heme oxygenase (HO)-1 is an inducible cytoprotective enzyme that degrades heme to iron, carbon monoxide (CO), and biliverdin, the latter two of which are thought to mediate the anti-inflammatory and antioxidant actions of HO-1. Bach1 is a transcriptional repressor of the HO-1 gene (Hmox-1). Previous reports have demonstrated that the genetic ablation of Bach1 engenders an increased HO-1 expression and a marked reduction in the degree of oxidative tissue damage in vivo. However, the function of Bach1 in spinal cord injury is still not understood. In the present study, we examined whether Bach1 deficiency increases HO-1 expression and reduces neural tissue damage in a spinal cord injury model using Bach1 knock-out (KO) mice and wild-type (WT) mice. The expression of HO-1 protein in the spinal cord was significantly higher in the Bach1 KO mice than in the WT mice before and after injury. The KO mice also had significantly higher Basso mouse scale scores for locomotor function and larger areas of spared white matter than the WT mice at 6 weeks after injury. Neuronal loss and apoptotic cell death in the injured spinal cord was significantly reduced in the KO mice in comparison to the WT mice. These results suggest that Bach1 deficiency engenders a constitutively higher expression of HO-1 and a dramatic increase in cytoprotection against spinal cord injury.

Low-energy extracorporeal shock wave therapy promotes vascular endothelial growth factor expression and improves locomotor recovery after spinal cord injury

OBJECT Extracorporeal shock wave therapy (ESWT) is widely used for the clinical treatment of various human diseases. Recent studies have demonstrated that low-energy ESWT upregulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis and functional recovery in myocardial infarction and peripheral artery disease. Many previous reports suggested that VEGF produces a neuroprotective effect to reduce secondary neural tissue damage after spinal cord injury (SCI). The purpose of the present study was to investigate whether low-energy ESWT promotes VEGF expression and neuroprotection and improves locomotor recovery after SCI. METHODS Sixty adult female Sprague-Dawley rats were randomly divided into 4 groups: sham group (laminectomy only), sham-SW group (low-energy ESWT applied after laminectomy), SCI group (SCI only), and SCI-SW group (low-energy ESWT applied after SCI). Thoracic spinal cord contusion injury was inflicted using an impactor. Low-energy ESWT was applied to the injured spinal cord 3 times a week for 3 weeks. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan (BBB) Scale (open field locomotor score) at different time points over 42 days after SCI. Hematoxylin and eosin staining was performed to assess neural tissue damage in the spinal cord. Neuronal loss was investigated by immunostaining for NeuN. The mRNA expressions of VEGF and its receptor, Flt-1, in the spinal cord were assessed using real-time polymerase chain reaction. Immunostaining for VEGF was performed to evaluate VEGF protein expression in the spinal cord. RESULTS In both the sham and sham-SW groups, no animals showed locomotor impairment on BBB scoring. Histological analysis of H & E and NeuN stainings in the sham-SW group confirmed that no neural tissue damage was induced by the low-energy ESWT. Importantly, animals in the SCI-SW group demonstrated significantly better locomotor improvement than those in the SCI group at 7, 35, and 42 days after injury (p < 0.05). The number of NeuN-positive cells in the SCI-SW group was significantly higher than that in the SCI group at 42 days after injury (p < 0.05). In addition, mRNA expressions of VEGF and Flt-1 were significantly increased in the SCI-SW group compared with the SCI group at 7 days after injury (p < 0.05). The expression of VEGF protein in the SCI-SW group was significantly higher than that in the SCI group at 7 days (p < 0.01). CONCLUSIONS The present study showed that low-energy ESWT significantly increased expressions of VEGF and Flt-1 in the spinal cord without any detrimental effect. Furthermore, it significantly reduced neuronal loss in damaged neural tissue and improved locomotor function after SCI. These results suggested that low-energy ESWT enhances the neuroprotective effect of VEGF in reducing secondary injury and leads to better locomotor recovery following SCI. This study provides the first evidence that low-energy ESWT can be a safe and promising therapeutic strategy for SCI.

4,8-Dichloroocta-t-butyltetracyclo[3.3.0.02,7.03,6]octagermane

A polyhedral germane, 4,8-dichloroocta-t-butyltetracyclo[3.3.0.02,7.03,6]octagermane, was prepared by reductive reactions of either 1,2-di-t-butyl-1,1,2,2-tetrachlorodigermane or t-butyltrichlorogermane with lithium naphthalenide. The structure of the new compound was determined by X-ray crystallographic analysis as well as by NMR spectra.

Cage Compounds of Si and Ge: Synthesis and Structures

Abstract Syntheses, characterization, and reactions of octasilacubane Si8R8 (R = 2,6-diethylphenyl), octagermacubane Ge8R8 (R = 2,6-diethylphenyl), hexasilaprismane Si6R6 (R = 2,6-diisopropylphenyl), and hexagermaprismane Ge6R6 (R = 2,6-diisopropylphenyl) are reported.

Rapamycin suppresses microglial activation and reduces the development of neuropathic pain after spinal cord injury.

Rapamycin is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, plays an important role in multiple cellular functions. Our previous study showed rapamycin treatment in acute phase reduced the neural tissue damage and locomotor impairment after spinal cord injury (SCI). However, there has been no study to investigate the therapeutic effect of rapamycin on neuropathic pain after SCI. In this study, we examined whether rapamycin reduces neuropathic pain following SCI in mice. We used a mouse model of thoracic spinal cord contusion injury, and divided the mice into the rapamycin‐treated and the vehicle‐treated groups. The rapamycin‐treated mice were intraperitoneally injected with rapamycin (1 mg/kg) 4 h after SCI. The rapamycin treatment suppressed phosphorylated‐p70S6K in the injured spinal cord that indicated inhibition of mTOR. The rapamycin treatment significantly improved not only locomotor function, but also mechanical and thermal hypersensitivity in the hindpaws after SCI. In an immunohistochemical analysis, Iba‐1‐stained microglia in the lumbar spinal cord was significantly decreased in the rapamycin‐treated mice. In addition, the activity of p38 MAPK in the lumbar spinal cord was significantly attenuated by rapamycin treatment. Furthermore, phosphorylated‐p38 MAPK‐positive microglia was relatively decreased in the rapamycin‐treated mice. These results indicated rapamycin administration in acute phase to reduce secondary neural tissue damage can contribute to the suppression of the microglial activation in the lumbar spinal cord and attenuate the development of neuropathic pain after SCI. The present study first demonstrated that rapamycin has significant therapeutic potential to reduce the development of neuropathic pain following SCI.