Haruo Kanno

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.

Combination of engineered Schwann cell grafts to secrete neurotrophin and chondroitinase promotes axonal regeneration and locomotion after spinal cord injury.

Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function.

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.

Dynamic Change of Dural Sac Cross-Sectional Area in Axial Loaded Magnetic Resonance Imaging Correlates With the Severity of Clinical Symptoms in Patients With Lumbar Spinal Canal Stenosis

Study Design. Cross-sectional registry and imaging cohort study. Objective. To examine whether the dural sac cross-sectional area (DCSA) in axial loaded magnetic resonance imaging (MRI) correlates with the severity of clinical symptoms in patients with lumbar spinal canal stenosis (LSCS). Summary of Background Data. Many studies have analyzed the relationship between DCSA on conventional MRI and the severity of symptoms in LSCS, but the link is still uncertain. Recently, axial loaded MRI, which can stimulate the spinal canal of patients in the upright position, has been developed. Axial loaded MRI demonstrates significant reduction of DCSA and provides valuable radiologic findings in the assessment of LSCS. However, there has been no study of the correlation between DCSA in axial loaded MRI and the severity of symptoms in LSCS. Methods. In 88 patients with LSCS, DCSA in conventional MRI, axial loaded MRI, and changes in the DCSA were determined at the single most constricted intervertebral level. The severity of symptoms was evaluated on the basis of the duration of symptoms, walking distance, visual analogue scale of leg pain/numbness, and Japanese Orthopaedic Association score. Spearman correlations of the DCSA in conventional MRI, axial loaded MRI, and changes in the DCSA with the severity of symptoms were analyzed. In addition, the severity of symptoms and DCSA in conventional and axial loaded MRI were compared, respectively, between patients with and without significant (>15 mm2) changes in the DCSA. Results. The DCSA in axial loaded MRI had good correlations with walking distance and Japanese Orthopaedic Association score (rs = 0.46 and 0.45, respectively; P < 0.001). In addition, the change in the DCSA significantly correlated to walking distance, visual analogue scale of leg numbness, and Japanese Orthopaedic Association score (rs = 0.59, 0.44, and 0.54, respectively; P < 0.001). Furthermore, the symptoms were significantly worse in patients with more than 15 mm2 change in the DCSA (P < 0.001). Axial loaded MRI, but not conventional MRI, showed a significantly smaller DCSA in patients with more than 15 mm2 change in the DCSA (P < 0.05). Conclusion. DCSA in axial loaded MRI significantly correlated with the severity of symptoms. Axial loaded MRI demonstrated that changes in the DCSA significantly correlated with the severity of symptoms, which conventional MRI could not detect. Thus, MRI with axial loading provides more valuable information than the conventional MRI for assessing patients with LSCS.

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.

Axial loading during magnetic resonance imaging in patients with lumbar spinal canal stenosis: does it reproduce the positional change of the dural sac detected by upright myelography?

Study Design. We compared the sizes of the dural sac among conventional magnetic resonance imaging (MRI), axial loaded MRI, and upright myelography in patients with lumbar spinal canal stenosis (LSCS). Objective. To determine whether axial loaded MRI can demonstrate similar positional changes of the dural sac size as were detected by upright myelography in LSCS. Summary of Background Data. In patients with LSCS, constriction of the dural sac is worsened and symptoms are aggravated during standing or walking. To disclose such positional changes, upright myelography has been widely used. Recently, axial loaded MRI, which can simulate a standing position, has been developed. However, there has been no study to compare the dural sac size between axial loaded MRI and upright myelography. Methods. Forty-four patients underwent conventional MRI, axial loaded MRI, and myelography. Transverse and anteroposterior diameters and the cross-sectional areas of the dural sac from L2–L3 to L5–S1 were compared. Pearson correlations of the diameters between the MRIs and the myelograms were analyzed. On the basis of the myelograms, all disc levels were divided into severe and nonsevere constriction groups. In each group, the diameters and the cross-sectional areas were compared. Sensitivity and specificity to detect severe constriction were calculated for the conventional and axial loaded MRI. Results. Transverse and anteroposterior diameters at L4–L5 in the axial loaded MRI and myelogram were significantly smaller than those observed in the conventional MRI (P < 0.001). Cross-sectional areas in the axial loaded MRI were significantly smaller than those in the conventional MRI at L2–L3, L3–L4, and L4–L5 (P < 0.001). Between the axial loaded MRI and the myelography, Pearson correlation coefficients of the transverse and anteroposterior diameters were 0.85 and 0.87, respectively (P < 0.001), which were higher than those for conventional MRI. Reductions of the dural sac sizes in the axial loaded MRI were more evident in the severe constriction group. The axial loaded MRI detected severe constriction with a higher sensitivity (96.4%) and specificity (98.2%) than the conventional MRI. Conclusion. The axial loaded MRI demonstrated a significant reduction in the dural sac size and significant correlations of the dural sac diameters with the upright myelogram. Furthermore, the axial loaded MRI had higher sensitivity and specificity than the conventional MRI for detecting the severe constriction observed in the myelogram. Therefore, the axial loaded MRI can be used to represent positional changes of the dural sac size detected by the upright myelography in patients with LSCS.

Diagram specific to sacroiliac joint pain site indicated by one-finger test

BackgroundThe sacroiliac joint (SIJ) can be a source of low back and lower limb pain. The SIJ pain can originate not only from the joint space but also from the ligaments supporting the joint. Its diagnosis has been difficult because the physical and radiological examinations have proved less than satisfactory. Thus, to know the specific sites of SIJ pain, if these exist, could be very useful for making the diagnosis. The purpose of the present study was to identify the main site of SIJ pain according to the patient’s pointing with one finger and to confirm the site by a pain-provocation test and periarticular lidocaine injection.MethodsForty-six of 247 consecutive patients with low back pain at our outpatient clinic, who could indicate with one finger the main site of the pain, which presented at only one site and was reproducible, were the subjects of this study. The main site of pain was anatomically confirmed by fluoroscopy. Then, a periarticular SIJ injection was performed. The patients were blindly assessed and a diagram of the main site of the SIJ pain was made.ResultsThere were 19 males and 27 females and the age averaged 50 years. Eight patients showed a positive placebo response and were excluded from this study. Twenty-five of the remaining 38 patients indicated the main site of pain at the posterior-superior iliac spine (PSIS) or within 2 cm of the PSIS, and 18 of these patients showed a positive effect with periarticular SIJ block. The other 13 patients, including 2 patients with a positive response to the periarticular block, did not show the PSIS as the main site of pain.ConclusionsOur study clearly indicated that when patients point to the PSIS or within 2 cm of it as the main site of low back pain, using one finger, the SIJ should be considered as the origin of their low back pain.