sirolimus and Spinal-Cord-Injuries

Heterotopic ossification (HO) is associated with inflammation. The goal of this review is to examine recent findings on the roles of inflammation and the immune system in HO. We examine how inflammation changes in fibrodysplasia ossificans progressiva, in traumatic HO, and in other clinical conditions of HO. We also discuss how inflammation may be a target for treating HO.. Both genetic and acquired forms of HO show similarities in their inflammatory cell types and signaling pathways. These include macrophages, mast cells, and adaptive immune cells, along with hypoxia signaling pathways, mesenchymal stem cell differentiation signaling pathways, vascular signaling pathways, and inflammatory cytokines. Because there are common inflammatory mediators across various types of HO, these mediators may serve as common targets for blocking HO. Future research may focus on identifying new inflammatory targets and testing combinatorial therapies based on these results.

Topics: Adaptive Immunity; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthroplasty, Replacement, Hip; Blast Injuries; Brain Injuries, Traumatic; Burns; Cell Differentiation; Cytokines; Humans; Hypoxia; Immunosuppressive Agents; Inflammation; Janus Kinase Inhibitors; Macrophages; Mast Cells; Mesenchymal Stem Cells; Myositis Ossificans; Ossification, Heterotopic; Postoperative Complications; Pyrazoles; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Signal Transduction; Sirolimus; Spinal Cord Injuries; Stilbenes; Wounds and Injuries

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.

Topics: Animals; Cellular Senescence; Mice; Models, Biological; Nerve Regeneration; Signal Transduction; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Traumatic spinal cord injury (SCI) is still devastating. It was suggested that the inhibition of mTOR may alleviate neuronal inflammatory injury but its underlying mechanism remained to be elucidated. AIM2 (absent in melanoma 2) recruits ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 to form the AIM2 inflammasome, activate caspase-1, and elicit inflammatory responses. We designed this study to elucidate whether pre-treatments of rapamycin could suppress SCI induced neuronal inflammatory injury via AIM2 signaling pathway in vitro and in vivo.. We performed oxygen and glucose deprivation / re-oxygenation (OGD) treatment and rats clipping model to mimic neuronal injury after SCI in vitro and in vivo. Morphologic changes of injured spinal cord were detected by hematoxylin and eosin staining. The expression of mTOR, p-mTOR, AIM2, ASC, Caspase-1 and et al were analyzed by fluorescent staining, western blotting or qPCR. The polarization phenotype of microglia was identified by flow cytometry or fluorescent staining.. We found BV-2 microglia without any pre-treatment cannot alleviate primary cultured neuronal OGD injury. However, pre-treated rapamycin in BV-2 cells could transform microglia to M2 phenotype and protects against neuronal OGD injury via AIM2 signaling pathway. Similarly, pre-treatment of rapamycin could improve the outcome of cervical SCI rats through AIM2 signaling pathway.. It was suggested that resting state microglia pre-treated by rapamycin could protect against neuronal injury via AIM2 signaling pathway in vitro and in vivo. Pre-inhibition of mTOR pathway may improve neuronal protection after SCI.

Topics: Animals; Caspase 1; Cervical Cord; DNA-Binding Proteins; Microglia; Rats; Signal Transduction; Sirolimus; Spinal Cord; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Severe injury occurs in the lung after acute spinal cord injury (ASCI) and autophagy is inhibited. However, rapamycin-activated autophagy's role and mechanism in lung injury development after ASCI is unknown. Preventing lung injury after ASCI by regulating autophagy is currently a valuable and unknown area. Herein, we aimed to investigate the effect and possible mechanism of rapamycin-activated autophagy on lung damage post-ASCI. An experimental animal study of rapamycin's effect and mechanism on lung damage after ASCI. We randomly divided 144 female wild-type Sprague-Dawley rats into a vehicle sham group (n = 36), a vehicle injury group (n = 36), a rapamycin sham group (n = 36), and a rapamycin injury group (n = 36). The spine was injured at the tenth thoracic vertebra using Allen's method. At 12, 24, 48, and 72 h after surgery, the rats were killed humanely. Lung damage was evaluated via pulmonary gross anatomy, lung pathology, and apoptosis assessment. Autophagy induction was assessed according to LC3, RAB7, and Beclin 1 levels. ULK-1, ULK-1 Ser555, ULK-1 Ser757, AMPK α and AMPK β1/2 were used to investigate the potential mechanism. After rapamycin pretreatment, the lung showed no obvious damage (e.g., cell death, inflammatory exudation, hemorrhage, and pulmonary congestion) at 12 h and 48 h after injury and Beclin1, LC3 and RAB7 levels increased. After rapamycin pretreatment, ULK-1, ULK-1 Ser555, and ULK-1 Ser757 levels increased at 12 h and 48 h after injury compared with the vehicle group, but they decreased at 12 h after injury compared with the rapamycin sham group. After rapamycin pretreatment, AMPKα levels did not change significantly before and after injury; however, at 48 h after injury, its level was elevated significantly compared with that in the vehicle group. Rapamycin can prevent lung injury after ASCI, possibly via upregulation of autophagy through the AMPK-mTORC1-ULK1 regulatory axis.

Topics: AMP-Activated Protein Kinases; Animals; Beclin-1; Excipients; Female; Lung Injury; Rats; Rats, Sprague-Dawley; Sirolimus; Spinal Cord Injuries; Thoracic Vertebrae

Spinal cord injury (SCI) is an intractable condition with complex pathological processes and poor prognosis. Reactive oxygen species (ROS) generation induced by the mammalian target of the rapamycin (mTOR) protein is one of the causes of secondary inflammation of SCI. Rapamycin (Rapa) is a pharmacological inhibitor of mTOR, which can inhibit ROS overproduction mediated by abnormal activation of the mTOR protein. Polydopamine, as a nanocarrier with excellent biological safety, has been reported to possess satisfactory ROS scavenging ability. Therefore, we designed a mesoporous polydopamine nanoparticle loaded with Rapa (mPDA@Rapa) for combination therapy, which simultaneously inhibited abnormally activated mTOR-mediated ROS production and eliminated already generated ROS. The synthesized mPDA nanoparticles could realize the effective encapsulation and sustained release of Rapa due to their mesoporous cavities and a hydrophobic benzene ring structure.

Topics: Animals; Delayed-Action Preparations; Indoles; Nanoparticles; Neurogenesis; Polymers; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases

The role of autophagy in the transplantation of induced pluripotent stem cells (iPSCs)-derived neural stem cells (NSCs) to treat spinal cord injury (SCI) and neurogenic bladder was investigated in this study. NSCs derived from human iPSCs were identified by and immunofluorescence assay. To clarify the role of autophagy, iPSCs were treated with either an autophagy inducer (rapamycin), or an autophagy inhibitor (chloroquine). Cell Counting kit-8 (CCK-8), western blot and flow cytometry were used to detect the effect of autophagy on the viability and differentiation of iPSCs. Sixty Wistar rats were selected to establish the SCI model and treated with iPSCs-derived NSCs transplantation. The effect of autophagy on the bladder function of rats with different treatments was evaluated by Basso, Beattie, and Bresnahan (BBB) score, bladder function score, bladder weight measurement, Hematoxylin & Eosin (H&E) staining, and Masson staining. The results of in vitro experiment showed that rapamycin enhanced the cell activity of iPSCs, increased the number of nestin positive cells, up-regulated Beclin-1 and LC3BI/II expressions, and down-regulated p62 expression. And the results of in vivo experiment showed that rapamycin improved exercise ability and bladder function, partially restored bladder weight, and significantly reduced bladder tissue damage in SCI rats. However, chloroquine showed the opposite results. The differentiation of iPSCs into NSCs could be promoted by induced autophagy, while neurogenic bladder of SCI was restored by autophagy activation.

Topics: Animals; Autophagy; Cell Differentiation; Heterografts; Humans; Immunosuppressive Agents; Induced Pluripotent Stem Cells; Neural Stem Cells; Rats; Rats, Wistar; Recovery of Function; Sirolimus; Spinal Cord Injuries; Stem Cell Transplantation; Urinary Bladder, Neurogenic

Spinal cord injury (SCI) is a serious medical problem, leading to lifelong disability and increasing the health burden worldwide. Traditional treatments have limited effects on neuronal function recovery. Previous studies showed that neurotrophin-3 (NT-3) promoted oligodendrocyte survival and improved neuronal functional recovery after SCI. However, the mechanism by which NT-3 promotes oligodendrocyte survival after SCI remains unclear, which limits its application.. A total of 75 female Sprague-Dawley rats were randomly divided into three groups: the NS group, NT-3 group, and NT-3 + rapamycin group. After successful modeling, the spinal cord specimens were taken at the corresponding time points. Western blot was used to detect autophagy-related proteins and Olig1 protein expression and combined with pathology, immunohistochemistry, flow cytometry, and other methods to detect the proliferation of oligodendrocytes after NT-3 application.. NT-3 was found to significantly promote the recovery of motor function by Basso-Beattie-Bresnahan scores analysis in the rat SCI model. Furthermore, intraspinal administration of NT-3 could downregulate the expression of Beclin-1 in oligodendrocytes, indicating that NT-3 could inhibit excessive autophagy of oligodendrocytes after SCI. The effects of NT-3 on oligodendrocyte survival could be blocked by an autophagy activator rapamycin.. This study found that NT-3 could promote the recovery of motor function after SCI in rats. The underlying reason may be that NT-3 inhibits the expression of autophagy proteins in oligodendrocytes and promotes oligodendrocyte proliferation. This study provided evidence for the future clinical application of NT-3 in SCI patients.

Topics: Animals; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Beclin-1; Cell Proliferation; Cell Survival; Disease Models, Animal; Female; Humans; Motor Activity; Nerve Tissue Proteins; Neurotrophin 3; Oligodendroglia; Rats; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; Sirolimus; Spinal Cord Injuries; Spinal Nerves

Surgical decompression after spinal cord injury (SCI) is a conventional treatment. Although it has been proven to have clinical effects, there are certain limitations, such as the surgical conditions that must be met and the invasive nature of the treatment. Therefore, there is an urgent need to develop a simple and maneuverable therapy for the emergency treatment of patients with SCI before surgery. Rapamycin (RAPA) has been reported to have potential as a therapeutic agent for SCI. In this study, we observed the therapeutic effects of rapamycin and surgical decompression, in combination or separately, on the histopathology in rabbits with SCI. After combination therapy, intramedullary pressure (IMP) decreased significantly, autophagic flux increased, and apoptosis and demyelination were significantly reduced. Compared with RAPA/surgical decompression alone, the combination therapy had a significantly better effect. In addition, we evaluated the effects of mechanical pressure on autophagy after SCI by assessing changes in autophagic initiation, degradation, and flux. Increased IMP after SCI inhibited autophagic degradation and impaired autophagic flux. Decompression improved autophagic flux after SCI. Our findings provide novel evidence of a promising strategy for the treatment of SCI in the future. The combination therapy may effectively improve emergency treatment after SCI and promote the therapeutic effect of decompression. This study also contributes to a better understanding of the effects of mechanical pressure on autophagy after neurotrauma.

Topics: Animals; Apoptosis; Autophagy; Cell Count; Decompression, Surgical; Demyelinating Diseases; Disease Models, Animal; Female; Neurons; Rabbits; Sirolimus; Spinal Cord; Spinal Cord Injuries

Spinal cord ischemia and reperfusion (SCIR) injury can induce autophagy, which is involved in the survival of neurons. However, whether autophagy plays a neuroprotective or a detrimental role in SCIR injury remains controversial. Angiopoietin-1 (Ang-1), an endothelial growth factor, has been shown to have neuroprotective effects. The present study aimed to explore the neuroprotective mechanisms of Ang-1 in neuronal cells in a rat model of SCIR injury in vivo. Ang-1 protein and rapamycin were injected intrathecally. Basso Beattie Bresnahan (BBB) scoring and hematoxylin and eosin staining were used to assess the degree of SCIR injury. Proteins that reflected the level of autophagy expression, such as Beclin-1 and LC3, were evaluated by western blotting. The results indicated that SCIR injury resulted in loss in lower limb motor function. Ang-1 protein inhibited the expression of Beclin-1 and LC3, which improved the BBB score and alleviated spinal cord injury. In contrast, rapamycin, an autophagy activator, caused the opposite effect. This study provides evidence that Ang-1 plays a neuroprotective role by inhibiting of autophagy expression in SCIR injury. Overall, findings could be useful for the treatment of SCIR injury.

Topics: Angiopoietin-1; Animals; Autophagy; Male; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Reperfusion Injury; Sirolimus; Spinal Cord; Spinal Cord Injuries; Spinal Cord Ischemia

Hydrogel scaffolds provide a beneficial microenvironment in transected rat spinal cord. A combinatorial biomaterials-based strategy provided a microenvironment that facilitated regeneration while reducing foreign body reaction to the three-dimensional spinal cord construct. We used poly lactic-co-glycolic acid microspheres to provide sustained release of rapamycin from Schwann cell (SC)-loaded, positively charged oligo-polyethylene glycol fumarate scaffolds. The biological activity and dose-release characteristics of rapamycin from microspheres alone and from microspheres embedded in the scaffold were determined in vitro. Three dose formulations of rapamycin were compared with controls in 53 rats. We observed a dose-dependent reduction in the fibrotic reaction to the scaffold and improved functional recovery over 6 weeks. Recovery was replicated in a second cohort of 28 animals that included retransection injury. Immunohistochemical and stereological analysis demonstrated that blood vessel number, surface area, vessel diameter, basement membrane collagen, and microvessel phenotype within the regenerated tissue was dependent on the presence of SCs and rapamycin. TRITC-dextran injection demonstrated enhanced perfusion into scaffold channels. Rapamycin also increased the number of descending regenerated axons, as assessed by Fast Blue retrograde axonal tracing. These results demonstrate that normalization of the neovasculature was associated with enhanced axonal regeneration and improved function after spinal cord transection.

Topics: Animals; Cell Line; Cells, Immobilized; Delayed-Action Preparations; Female; Microspheres; Polyethylene Glycols; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Inbred F344; Schwann Cells; Sirolimus; Spinal Cord Injuries; Spinal Cord Regeneration; Tissue Engineering; Tissue Scaffolds

Autonomic dysreflexia (AD) is a potentially life-threatening syndrome in individuals with spinal cord injury (SCI) above the T6 spinal level that is characterized by episodic hypertension in response to noxious stimuli below the lesion. Maladaptive intraspinal plasticity is thought to contribute to the temporal development of AD, and experimental approaches that reduce such plasticity mitigate the severity of AD. The mammalian target of rapamycin (mTOR) has gained interest as a mediator of plasticity, regeneration, and nociceptor hypersensitivity in the injured spinal cord. Based on our preliminary data that prolonged rapamycin (RAP) treatment markedly reduces mTOR activity in the cord weeks after high-thoracic (T4) spinal transection, we sought to determine whether RAP could modulate AD development by impeding intraspinal plasticity. Naïve and injured rats were administered RAP or vehicle every other day, beginning immediately after injury for four weeks, and hemodynamic monitoring was conducted to analyze the frequency of spontaneously occurring AD, as well as the severity of colorectal distention (CRD) induced AD. Results showed that after SCI, RAP significantly exacerbated sustained body weight loss and caused a marked elevation in resting blood pressure, with average daily blood pressure rising above even normal naïve levels within one week after injury. Moreover, RAP significantly increased the frequency of daily spontaneous AD and increased the absolute blood pressure induced by CRD at three weeks post-injury. These dynamic cardiovascular effects were not, however, correlated with changes in the density of nociceptive c-fibers or c-Fos+ neurons throughout the spinal cord, indicating that intraspinal plasticity associated with AD was not altered by treatment. These findings caution against the use of RAP as a therapeutic intervention for SCI because it evokes toxic weight loss and exacerbates cardiovascular dysfunction perhaps mediated by increased peripheral nociceptor sensitivity and/or vascular resistance.

Topics: Animals; Autonomic Dysreflexia; Blood Pressure; Female; Immunosuppressive Agents; Male; Neuronal Plasticity; Rats; Sirolimus; Spinal Cord Injuries; Thoracic Vertebrae; Weight Loss

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal-lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.

Topics: Animals; Apoptosis; Astrocytes; Cell Survival; Cyclooxygenase 2; Cytokines; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Morpholines; Motor Activity; Multiprotein Complexes; Nerve Tissue; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitrites; Pyrimidines; Signal Transduction; Sirolimus; Spinal Cord; Spinal Cord Injuries; TOR Serine-Threonine Kinases

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. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:93-103, 2017.

Topics: Animals; Calcium-Binding Proteins; Drug Evaluation, Preclinical; Female; Glial Fibrillary Acidic Protein; Hyperalgesia; Locomotion; Mice, Inbred C57BL; Microfilament Proteins; Neuralgia; Neuroglia; p38 Mitogen-Activated Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Several studies suggest that glial scars pose as physical and chemical barriers that limit neurite regeneration after spinal cord injury (SCI). Evidences suggest that the activation of the PI3K/Akt/mTOR signaling pathway is involved in glial scar formation. Therefore, inhibition of the PI3K/Akt/mTOR pathway may beneficially attenuate glial scar formation after SCI. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulates the PI3K/Akt/mTOR pathway. Therefore, we hypothesized that the overexpression of PTEN in the spinal cord will have beneficial effects after SCI. In the present study, we intrathecally injected a recombinant adenovirus carrying the pten gene (Ad-PTEN) to cause overexpression of PTEN in rats with contusion injured spinal cords. The results suggest overexpression of PTEN in spinal cord attenuated glial scar formation and led to improved locomotor function after SCI. Overexpression of PTEN following SCI attenuated gliosis, affected chondroitin sulfate proteoglycan expression, and improved axon regeneration into the lesion site. Furthermore, we suggest that the activation of the PI3K/Akt/mTOR pathway in astrocytes at 3 days after SCI may be involved in glial scar formation. Because delayed treatment with Ad-PTEN enhanced motor function recovery more significantly than immediate treatment with Ad-PTEN after SCI, the results suggest that the best strategy to attenuate glial scar formation could be to introduce 3 days after SCI. This study's findings thus have positive implications for patients who are unable to receive immediate medical attention after SCI.

Topics: Animals; Cicatrix; Disease Models, Animal; Female; Gliosis; Green Fluorescent Proteins; Humans; Immunosuppressive Agents; Locomotion; Nerve Tissue Proteins; Neuroglia; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; PTEN Phosphohydrolase; Rats; Rats, Wistar; Signal Transduction; Sirolimus; Spinal Cord Injuries; Time Factors

Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons.

Topics: Animals; Animals, Newborn; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Female; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelin-Associated Glycoprotein; Nerve Regeneration; Neurotrophin 3; Nogo Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Somatosensory Disorders; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Whether rapamycin has neuroprotective effects in spinal cord injury remains controversial. The present study shows that rapamycin protects neurons from death after spinal cord injury by inhibiting the secondary inflammatory response. The effects of rapamycin were tested using a myeloperoxidase assay, Western blotting, immunohistochemistry, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The experimental results showed that after spinal cord injury, rapamycin reduced the numbers of activated microglia and neutrophils in the damage zone, lowered the expression levels of TNF-α and IL-1β, reduced the apoptotic cells, and increased the survival of neurons. The above data proved that rapamycin diminishes inflammatory cell activation and proliferation, downregulates the expression of inflammatory factors, reduces the microenvironmental damage effects on neurons in the acute injury phase, and thus promotes the survival of neurons. Therefore, we believe that rapamycin has neuroprotective effects in spinal cord injury.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Cell Survival; Down-Regulation; Female; Immunosuppressive Agents; Interleukin-1beta; Microglia; Neurons; Neuroprotective Agents; Rats; Rats, Wistar; Sirolimus; Spinal Cord Injuries; Tumor Necrosis Factor-alpha

Spinal cord injury (SCI) frequently leads to a permanent functional impairment as a result of the initial injury followed by secondary injury mechanism, which is characterised by increased inflammation, glial scarring and neuronal cell death. Finding drugs that may reduce inflammatory cell invasion and activation to reduce glial scarring and increase neuronal survival is of major importance for improving the outcome after SCI. In the present study, we examined the effect of rapamycin, an mTORC1 inhibitor and an inducer of autophagy, on recovery from spinal cord injury. Autophagy, a process that facilitates the degradation of cytoplasmic proteins, is also important for maintenance of neuronal homeostasis and plays a major role in neurodegeneration after neurotrauma. We examined rapamycin effects on the inflammatory response, glial scar formation, neuronal survival and regeneration in vivo using spinal cord hemisection model in mice, and in vitro using primary cortical neurons and human astrocytes. We show that a single injection of rapamycin, inhibited p62/SQSTM1, a marker of autophagy, inhibited mTORC1 downstream effector p70S6K, reduced macrophage/neutrophil infiltration into the lesion site, microglia activation and secretion of TNFα. Rapamycin inhibited astrocyte proliferation and reduced the number of GFAP expressing cells at the lesion site. Finally, it increased neuronal survival and axonogenesis towards the lesion site. Our study shows that rapamycin treatment increased significantly p-Akt levels at the lesion site following SCI. Similarly, rapamycin treatment of neurons and astrocytes induced p-Akt elevation under stress conditions. Together, these findings indicate that rapamycin is a promising candidate for treatment of acute SCI condition and may be a useful therapeutic agent.

Topics: Animals; Astrocytes; CD11b Antigen; Cell Count; Cell Survival; Cells, Cultured; Disease Models, Animal; ELAV-Like Protein 3; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Humans; Immunosuppressive Agents; Inflammation; Ki-67 Antigen; Male; Mice; Mice, Inbred C57BL; Neurons; Rats; Sirolimus; Spinal Cord Injuries; Time Factors

Chronic spinal cord injury (SCI) is a formidable hurdle that prevents a large number of injured axons from crossing the lesion, particularly the corticospinal tract (CST). This study shows that Pten deletion in the adult mouse cortex enhances compensatory sprouting of uninjured CST axons. Furthermore, forced upregulation of mammalian target of rapamycin (mTOR) initiated either 1 month or 1 year after injury promoted regeneration of CST axons. Our results indicate that both developmental and injury-induced mTOR downregulation in corticospinal motor neurons can be reversed in adults. Modulating neuronal mTOR activity is a potential strategy for axon regeneration after chronic SCI.. As one of the long descending tracts controlling voluntary movement, the corticospinal tract (CST) plays an important role for functional recovery after spinal cord injury. The regeneration of CST has been a major challenge in the field, especially after chronic injuries. Here we developed a strategy to modulate Pten/mammalian target of rapamycin signaling in adult corticospinal motor neurons in the postinjury paradigm. It not only promoted the sprouting of uninjured CST axons, but also enabled the regeneration of injured axons past the lesion in a mouse model of spinal cord injury, even when treatment was delayed up to 1 year after the original injury. The results considerably extend the window of opportunity for regenerating CST axons severed in spinal cord injuries.

Topics: Animals; Axons; Cerebral Cortex; Dependovirus; Disease Models, Animal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Longitudinal Studies; Mice; Mice, Transgenic; Nerve Regeneration; PTEN Phosphohydrolase; Pyramidal Tracts; Recovery of Function; Sirolimus; Spinal Cord Injuries; Time Factors; Vesicular Glutamate Transport Protein 1

In this study we examined the relationship between autophagy and apoptosis in diabetic rats after spinal cord injury (SCI), also we determined the role of autophagy in diabetes-aggravated neurological injury in vivo and in vitro. Our results showed that diabetes decreased the survival of neurons, promoted astrocytes proliferation, increased inflammatory cells infiltration and inhibited functional recovery after SCI. Diabetes was shown to confer increased activation of apoptotic pathways, along with an increase in autophagy; similar effects were also observed in vitro in neuronal PC12 cells. Treatment with rapamycin, an autophagy activator, partially abolished the adverse effect of diabetes, suggesting that diabetes may enhance neurological damage and suppress locomotor recovery after SCI, in addition to its effects on apoptosis and autophagy. In contrast, further stimulation of autophagy improved neurological function via inhibition of apoptosis. These results explained how diabetes exacerbates SCI in cellular level and suggested autophagy stimulation to be a new therapeutic strategy for diabetic SCI.

Topics: Animals; Apoptosis; Astrocytes; Autophagy; Cell Proliferation; Cell Survival; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Locomotion; Neurons; PC12 Cells; Rats; Rats, Sprague-Dawley; Recovery of Function; Sirolimus; Spinal Cord Injuries

Autophagy is an intracellular catabolic mechanism that maintains the balance of proteins, lipids and aging organelles. 3-Methyladenine (3-MA) is a selective inhibitor of autophagy, whereas rapamycin, an antifungal agent, is a specific inducer of autophagy, inhibiting the protein mammalian target of rapamycin. In the present study, we examined the role of autophagy, inhibited by 3-MA and enhanced by rapamycin, in a model of acute spinal cord injury in rats. We found that rapamycin could significantly increase the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 at the injury site. At the same time, the number of neurons and astrocytes with LC3 positive in the spinal cord was upregulated with time. In addition, administration of rapamycin produced an increase in the Basso, Beattie and Bresnahan scores of injured rats, indicating high recovery of locomotor function. Furthermore, expression of the proteins Bcl-2 and Bax was upregulated and downregulated, respectively. By contrast, the results for rats treated with 3-MA, which inhibits autophagy, were the opposite of those seen with the rapamycin-treated rats. These results show that induction of autophagy can produce neuroprotective effects in acute spinal cord injury in rats via inhibition of apoptosis.

Topics: Animals; Apoptosis; Autophagy; Male; Motor Activity; Neurons; Rats; Rats, Sprague-Dawley; Recovery of Function; Sirolimus; Spinal Cord Injuries; Thoracic Vertebrae

Erlotinib and Rapamycin are both in clinical use and experimental inhibition of their respective molecular targets, EGFR and mTORC1, has improved recovery from spinal cord injury. Our aim was to determine if daily Erlotinib or Rapamycin treatment started directly after spinal contusion injury in rats improves locomotion function or recovery of bladder function.. Stockholm, Sweden.. Rats were subjected to contusion injuries and treated during the acute phase with either Erlotinib or Rapamycin. Recovery of bladder function was monitored by measuring residual urine volume and hindlimb locomotion assessed by open-field observations using the BBB rating scale as well as by automated registration of gait parameters. Body weights were monitored. To determine whether Erlotinib and Rapamycin inhibit the same signaling pathway, a cell culture system and western blots were used.. Erlotinib accelerated locomotor recovery and slightly improved bladder recovery; however, we found no long-term improvements of locomotor function. Rapamycin did neither improved locomotor function nor bladder recovery. In vitro studies confirmed that Erlotinib and Rapamycin both inhibit the EGFR-mTORC1 signaling pathway.. We conclude that none of these two drug regimes improved long-term functional outcome in our current model of spinal cord injury. Nevertheless, oral treatment with Erlotinib may offer modest temporary advantages, whereas treatment with Rapamycin does not.

Topics: Administration, Oral; Animals; Disease Models, Animal; Erlotinib Hydrochloride; Female; Hindlimb; Immunosuppressive Agents; Locomotion; Protein Kinase Inhibitors; Quinazolines; Rats, Sprague-Dawley; Recovery of Function; Sirolimus; Spinal Cord Injuries; Urinary Bladder

To explore the effects of rapamycin-induced autophagy on apoptosis in a rat model of acute spinal cord injury (SCI), and to explore the effect of rapamycin on apoptosis in primary spinal cord cell culture.. SCI was induced at T10 in female adult Sprague-Dawley rats. After injury was induced, the rats were injected with rapamycin and/or methylprednisolone and were sacrificed at various days after injury. Apoptosis and autophagy were examined with TUNEL staining and electron microscopy. Hind limb function was assessed by the Gale scale.. The expression of the apoptosis-related protein caspase-3 did not significantly increase until 21 days following injury, while increases in LC3II and LC3I began 10 days after injury, but then declined. TUNEL staining and electron microscopy confirmed that following injury autophagy occurred before apoptosis, but by 14 days after the injury, the level of autophagy had decreased significantly while the level of apoptosis showed a continued increase. Following treatment with rapamycin, apoptosis was significantly higher than in the vehicle control group, but significantly lower than in the sham-operated group, showing a protective effect of rapamycin. Gale scale grades in rats treated with rapamycin were significantly higher compared with the vehicle control group, suggesting a functional effect of rapamycin-induced inhibition of apoptosis.. The results indicate that rapamycin significantly improved the prognosis of acute SCI in rats by inhibiting cell apoptosis. Rapamycin might be useful as a therapeutic agent for acute SCI.

Topics: Animals; Apoptosis; Autophagy; Blotting, Western; Disease Models, Animal; Female; Flow Cytometry; In Situ Nick-End Labeling; Microscopy, Electron, Transmission; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Recovery of Function; Sirolimus; Spinal Cord Injuries

Apoptosis has been widely reported to be involved in the pathogenesis associated with spinal cord injury (SCI). Recently, autophagy has also been implicated in various neuronal damage models. However, the role of autophagy in SCI is still controversial and its interrelationship with apoptosis remains unclear. Here, we used an in vitro SCI model to observe a time-dependent induction of autophagy and apoptosis. Mechanical injury induced autophagy markers such as LC3 lipidation, LC3II/LC3I conversion, and Beclin-1 expression. Injured neurons showed decreased cell viability and increased apoptosis. To elucidate the effect of autophagy on apoptosis, the mechanically-injured neurons were treated with the mTOR inhibitor rapamycin and 3-methyl adenine (3-MA), which are known to regulate autophagy positively and negatively, respectively. Rapamycin-treated neurons showed the highest level of cell viability and lowest level of apoptosis among the injured neurons and those treated with 3-MA showed the reciprocal effect. Notably, rapamycin-treated neurons exhibited slightly reduced Bax expression and significantly increased Bcl-2 expression. Furthermore, by plasmid transfection, we showed that Beclin-1-overexpressing neuronal cells responded to mechanical injury with greater LC3II/LC3I conversion and cell viability, lower levels of apoptosis, higher Bcl-2 expression, and unaltered Bax expression as compared to vector control cells. Beclin-1-knockdown neurons showed almost the opposite effects. Taken together, our results suggest that autophagy may serve as a protection against apoptosis in mechanically-injured spinal cord neurons. Targeting mTOR and/or enhancing Beclin-1 expression might be alternative therapeutic strategies for SCI.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Survival; Cells, Cultured; Female; Male; Neurons; Rats; Rats, Wistar; Sirolimus; Spinal Cord; Spinal Cord Injuries; TOR Serine-Threonine Kinases

The role of autophagy in the recovery of spinal cord injury remains controversial; in particular, the mechanism of autophagy regulated degradation of ubiquitinated proteins has not been discussed to date. In this study, we investigated the protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the rat model of trauma. bFGF administration improved recovery and increased the survival of neurons in spinal cord lesions in the rat model. The protective effect of bFGF is related to the inhibition of autophagic protein LC3II levels; bFGF treatment also enhances clearance of ubiquitinated proteins by p62, which also increases the survival of neuronal PC-12 cells. The activation of the downstream signals of the PI3K/Akt/mTOR pathway by bFGF treatment was detected both in vivo and in vitro. Combination therapy including the autophagy activator rapamycin partially abolished the protective effect of bFGF. The present study illustrates that the role of bFGF in SCI recovery is related to the inhibition of excessive autophagy and enhancement of ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new trend for bFGF drug development for central nervous system injuries and sheds light on protein signaling involved in bFGF action.

Topics: Animals; Autophagy; Disease Models, Animal; Female; Fibroblast Growth Factor 2; Heat-Shock Proteins; Humans; Motor Neurons; Neuroprotective Agents; PC12 Cells; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Recovery of Function; Sequestosome-1 Protein; Signal Transduction; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases; Ubiquitinated Proteins

The mammalian target of rapamycin (mTOR) is known to regulate cell growth, and it also participates in pain transmission as has been recently verified in inflammatory and neuropathic pain models. The targeting of mTOR represents a new strategy for the control of chronic pain. In the present study, we investigated the effect of mTOR in the expression of PSD95 and NR2B-PSD95 or GluA2-PSD95 interaction ratio in a chronic constriction injury (CCI) mice model.. Paw mechanical withdrawal threshold (PMWT) and paw withdrawal thermal latency (PWTL) were respectively used to assess mechanical allodynia and thermal hyperalgesia after CCI operation and intrathecal injection of rapamycin. Western blot and co-immunoprecipitation were used to investigate the effects of rapamycin on the expression of PSD95 and interaction ratio of NR2B-PSD95 or GluA2-PSD95 in the spinal dorsal horn of mice.. Our study demonstrated that the inhibition of spinal mTOR with intrathecal injections of rapamycin (1 μg/5 μL) for days 1-6 after CCI surgery led to an obvious decrease in CCI-induced neuropathic pain. Rapamycin significantly reduced the PMWT of CCI mice, whereas there was no significant effect on PWTL. The active form of the mTOR signaling pathway (p-mTOR, p-4EBP1 and p-p70S6k) at the spinal level remarkably increased in CCI mice, and rapamycin could inhibit this up-regulation. The increased expression of PSD95 and the interaction ratio of GluA2-PSD95 or NR2B-PSD95 could also be inhibited by intrathecal injection of rapamycin.. These data suggest that the mTOR pathway is activated in the spinal dorsal horn in CCI-induced neuropathic pain, and the intrathecal injection of rapamycin can reduce mechanical allodynia. Our findings indicate that spinal mTOR is an important component of CCI-induced neuropathic pain, and mTOR may be a potential target for chronic pain therapy.

Topics: Animals; Behavior, Animal; Chronic Disease; Male; Mice; Mice, Inbred C57BL; Pain Threshold; Sirolimus; Spinal Cord Compression; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Spinal cord injuries (SCIs) are serious and debilitating health problems that lead to severe and permanent neurological deficits resulting from the primary mechanical impact followed by secondary tissue injury. During the acute stage after an SCI, the expression of autophagy and inflammatory responses contribute to the development of secondary injury. In the present study, we examined the multifaceted effects of rapamycin on outcomes of rats after an SCI.. We used 72 female Sprague-Dawley rats for this study. In the SCI group, we performed a laminectomy at T10, followed by impact-contusion of the spinal cord. In the control group, we performed only a laminectomy without contusion. We evaluated the effects of rapamycin using the Basso, Beattie, and Bresnahan scale for functional outcomes, Western blot analyses for analyzing LC3-II, tumor necrosis factor expression, and p70S6K phosphorylation, and an immunostaining technique for localization and enumeration of microglial and neuronal cells.. Basso, Beattie, and Bresnahan scores after injury significantly improved in the rapamycin-treated group compared with the vehicle group (on Day 28 after the SCI; P < .05). The Western blot analysis demonstrated that rapamycin enhanced LC3-II expression and decreased p70S6K phosphorylation compared with the vehicle (P < .01), which implies promotion of autophagy through mammalian target of rapamycin inhibition. Furthermore, rapamycin treatment significantly attenuated tumor necrosis factor production and microglial expression (P < .05). Immunohistochemistry of NeuN (antibodies specific to neurons) showed remarkable neuronal cell preservation in the rapamycin-treated group compared with the vehicle-treated group (P < .05), which suggests a neuroprotective effect of rapamycin.. Rapamycin is a novel neuroprotectant with multifaceted effects on the rat spinal cord after injury. Use of such a clinically established drug could facilitate early clinical trials in selected cases of human SCIs.

Topics: Animals; Anti-Inflammatory Agents; Autophagy; Female; Laminectomy; Microglia; Models, Animal; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Recovery of Function; Sirolimus; Spinal Cord Injuries; Treatment Outcome

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.

Topics: Animals; Autophagy; Blotting, Western; Disease Models, Animal; Female; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Motor Activity; Nerve Degeneration; Neuroprotective Agents; Sirolimus; Spinal Cord Injuries

We investigated microRNAs (miRs) associated with PTEN/mTOR signaling after spinal cord injury (SCI) and after hind limb exercise (Ex), a therapy implicated in promoting spinal cord plasticity. After spinalization, rats received cycling Ex 5 days/week. The expression of miRs, their target genes and downstream effectors were probed in spinal cord tissue at 10 and 31 days post injury. Ex elevated expression of miR21 and decreased expression of miR 199a-3p correlating with significant change in the expression of their respective target genes: PTEN mRNA decreased and mTOR mRNA increased. Western blotting confirmed comparable changes in protein levels. An increase in phosphorylated-S6 (a downstream effector of mTOR) within intermediate grey neurons in Ex rats was blocked by Rapamycin treatment. It thus appears possible that activity-dependent plasticity in the injured spinal cord is modulated in part through miRs that regulate PTEN and mTOR signaling and may indicate an increase in the regenerative potential of neurons affected by a SCI.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Exercise Therapy; Female; Gene Expression Regulation; MicroRNAs; Motor Activity; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases

Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberin's ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras-MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration.

Topics: Analysis of Variance; Animals; Astrocytes; Cells, Cultured; Chromones; Disease Models, Animal; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Excitatory Amino Acid Transporter 2; Flavonoids; Glial Fibrillary Acidic Protein; Immunosuppressive Agents; Male; Monomeric GTP-Binding Proteins; Morpholines; Neuropeptides; Protein Kinases; Ras Homolog Enriched in Brain Protein; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases; Transcription Factors; Transfection; Up-Regulation; Vimentin