Effects of photobiomodulation combined with MSCs transplantation on the repair of spinal cord injury in rat

Stem cell transplantation has shown promising regenerative effects against neural injury, and photobiomodulation (PBM) can aid tissue recovery. This study aims to evaluate the therapeutic effect of human umbilical cord mesenchymal stem cells (hUCMSCs) and laser alone or combined on spinal cord injury (SCI). The animals were divided into SCI, hUCMSCs, laser treatment (LASER) and combination treatment (hUCMSCs + LASER) groups. Cell‐enriched grafts of hUCMSCs (1 × 10⁶ cells/ml) were injected at the site of antecedent trauma in SCI model rats. A 2 cm² damaged area was irradiated with 630 nm laser at 100 mW/cm² power for 20 min. Locomotion was evaluated using Basso–Beattie–Bresnahan (BBB) scores, and neurofilament repair were monitored by histological staining and diffusion tensor imaging (DTI). First, after SCI, the motor function of each group was restored with different degrees, the combination treatment significantly increased the BBB scores compared to either monotherapy. In addition, Nissl bodies were more numerous, and the nerve fibers were longer and thicker in the combination treatment group. Consistent with this, the in situ expression of NF‐200 and glial fibrillary acidic protein in the damaged area was the highest in the combination treatment group. Finally, DTI showed that the combination therapy optimally improved neurofilament structure and arrangement. These results may show that the combination of PBM and hUCMSCs transplantation is a feasible strategy for reducing secondary damage and promoting functional recovery following SCI.     

Photobiomodulation therapy in knee osteoarthritis reduces oxidative stress and inflammatory cytokines in rats

Knee osteoarthritis (OA) is a chronic disease that causes pain and gradual degeneration of the articular cartilage. In this study, MIA‐induced OA knee model was used in rats to test the effects of the photobiomodulation therapy (PBM). We analyzed the inflammatory process (pain and cytokine levels), and its influence on the oxidative stress and antioxidant capacity. Knee OA was induced by monosodium iodoacetate (MIA) intra‐articular injection (1.5 mg/50 μL) and the rats were treated with eight sessions of PBM 3 days/week (904 nm, 6 or 18 J/cm²). For each animal, mechanical and cold hyperalgesia and spontaneous pain were evaluated; biological analyses were performed in blood serum, intra‐articular lavage, knee structures, spinal cord and brainstem. Cytokine assays were performed in knee, spinal cord and brainstem samples. The effects of the 18 J/cm² dose of PBM were promising in reducing pain and neutrophil activity in knee samples, together with reducing oxidative stress damage in blood serum and spinal cord samples. PBM improved the antioxidant capacity in blood serum and brainstem, and decreased the knee pro‐inflammatory cytokine levels. Our study demonstrated that PBM decreased oxidative damage, inflammation and pain. Therefore, this therapy could be an important tool in the treatment of knee OA.     

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.     

大鼠脊髓损伤的弱激光治疗参数选择

弱激光在周围神经损伤治疗中取得了理想效果,但在脊髓损伤修复方面的研究很少.本实验应用Allen＇s造模法构建大鼠急性脊髓损伤模型,通过测量穿透功率及组织温度变化筛选出用于脊髓损伤治疗的弱激光照射参数,照射组按照筛选出的参数连续治疗14 d,于术后1、3、7、14、21 d应用BBB评分法评价大鼠后肢运动功能的恢复情况,苏木精-伊红染色（HE染色）观察脊髓病理变化并测量空洞面积.结果显示应用筛选出的照射参数（810 nm、光斑面积0.2 cm2、500 mW/cm2、510 J/cm2）连续照射14 d,术后21 d照射组的运动功能评分显著高于未照射组（P＜0.05）,术后7、14、21d照射组脊髓空洞面积显著小于未照射组（P＜0.05）.结果表明810 nm、光斑面积0.2 cm2、500 mW/cm2、510 J/cm2的弱激光照射能促进急性脊髓损伤大鼠后期运动功能的恢复.     

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti‐inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV‐2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS‐induced M1 BV‐2 microglia, also the inflammatory secretion phenotype of M1 BV‐2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK‐/mTOR‐mediated autophagic flux stimulation.     

Baicalin effects on rats with spinal cord injury by anti‐inflammatory and regulating the serum metabolic disorder

Baicalin had neuroprotective effects on inhibiting neuronal cell apoptosis induced by spinal cord ischemic injury. This study aimed to explore the protective effects of Baicalin on rats with spinal cord injury (SCI) and its mechanism of action. The recovery of spinal cord nerve function in rats was evaluated by the Basso, Beattie, and Bresnahan (BBB) score and the combine behavioral score (CBS). The expressions of cytokines tumor necrosis factor α (TNF‐α), interleukin‐1β (IL‐1β), and IL‐6 were detected by the enzyme‐linked immunosorbent assay method. Expressions of inflammation‐related proteins were detected by Western blot. Multivariate statistical analysis was performed for serum metabolites. The BBB and CBS score results showed that Baicalin had a certain improvement on rats with SCI. SCI symptoms were significantly improved in low‐dose and high‐dose groups. The levels of TNF‐α, IL‐1β, and IL‐6 in the SCI group were significantly increased. The expressions of NF‐κB p65, NF‐κB p50, p‐IκBα, and IKKα in the SCI group showed the opposite trend compared with the low‐dose and high‐dose groups. Compared with the sham group, glutamine, levels of 3‐OH‐butyrate, N‐acetylaspartate, and glutathione were significantly reduced, and the levels of glutamate and betaine were significantly increased in the SCI group. When Baicalin was administered, the contents of glutamine synthase (GS) and glutaminase (GLS) were significantly reduced, indicating that Baicalin had the effect of improving GS and GLS. Baicalin has protective effects on improving SCI and lower extremity motor function, has a significant anti‐inflammatory effect, and regulates the serum metabolic disorder caused by SCI in rats.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.     

Dual regulation of microglia and neurons by Astragaloside IV‐mediated mTORC1 suppression promotes functional recovery after acute spinal cord injury

Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS‐IV), in vitro and in vivo. Our results showed that AS‐IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS‐IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS‐IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS‐IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS‐IV may become a novel therapeutic medicine for SCI.     

Superficial photothermal laser ablation of ex vivo sheep esophagus using a cone‐shaped optical fiber tip

Superficial photothermal laser ablation (SPLA) may be useful as a therapeutic approach producing a depth of injury that is sufficient to eliminate mucosal lesion but not deep enough to induce thermal effects in deeper tissue layers. The purpose of this preliminary study is twofold: (a) to describe design steps of a fiber probe capable of delivering a tightly focused laser beam, including Monte‐Carlo‐based simulations, and (b) to complete the initial testing of the probe in a sheep esophagus model, ex vivo. The cone‐shaped (tapered) fiber tip was obtained by chemical etching of the optical fiber. A 1505 nm diode laser providing power up to 500 mW was operated in continuous wave. The successful SPLA of the sheep mucosa layer was demonstrated for various speed‐power combinations, including 300 mW laser power at a surface scanning rate of 0.5 mm/s and 450 mW laser power at a surface scanning rate of 2.0 mm/s. Upon further development, this probe may be useful for endoscopic photothermal laser ablation of the mucosa layer using relatively low laser power.     

Cellular transformations in near‐infrared light‐induced apoptosis in cancer cells revealed by label‐free CARS imaging

Photobiomodulation (PBM) involves light to activate cellular signaling pathways leading to cell proliferation or death. In this work, fluorescence and Coherent anti‐Stokes Raman Scattering (CARS) imaging techniques were applied to assess apoptosis in human cervical cancer cells (HeLa) induced by near infrared (NIR) laser light (808 nm). Using the Caspase 3/7 fluorescent probe to identify apoptotic cells, we found that the pro‐apoptotic effect is significantly dependent of irradiation dose. The highest apoptosis rate was noted for the lower irradiation doses, that is, 0.3 J/cm² (~58%) and 3 J/cm² (~28%). The impact of light doses on proteins/lipids intracellular metabolism and distribution was evaluated using CARS imaging, which revealed apoptosis‐associated reorganization of nuclear proteins and cytoplasmic lipids after irradiation with 0.3 J/cm². Doses of NIR light causing apoptosis (0.3, 3 and 30 J/cm²) induced a gradual increase in the nuclear protein level over time, in contrast to proteins in cells non‐irradiated and irradiated with 10 J/cm². Furthermore, irradiation of the cells with the 0.3 J/cm² dose resulted in lipid droplets (LDs) accumulation, which was apparently caused by an increase in reactive oxygen species (ROS) generation. We suggest that PBM induced apoptosis could be caused by the ability of NIR light to trigger excessive LDs formation which, in turn, induces cellular cytotoxicity.      

p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser³⁹². Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.     

Low‐level laser therapy 810‐nm up‐regulates macrophage secretion of neurotrophic factors via PKA‐CREB and promotes neuronal axon regeneration in vitro

Macrophages play key roles in the secondary injury stage of spinal cord injury (SCI). M1 macrophages occupy the lesion area and secrete high levels of inflammatory factors that hinder lesion repair, and M2 macrophages can secrete neurotrophic factors and promote axonal regeneration. The regulation of macrophage secretion after SCI is critical for injury repair. Low‐level laser therapy (810‐nm) (LLLT) can boost functional rehabilitation in rats after SCI; however, the mechanisms remain unclear. To explore this issue, we established an in vitro model of low‐level laser irradiation of M1 macrophages, and the effects of LLLT on M1 macrophage polarization and neurotrophic factor secretion and the related mechanisms were investigated. The results showed that LLLT irradiation decreased the expression of M1 macrophage‐specific markers, and increased the expression of M2 macrophage‐specific markers. Through forward and reverse experiments, we verified that LLLT can promote the secretion of various neurotrophic factors by activating the PKA‐CREB pathway in macrophages and finally promote the regeneration of axons. Accordingly, LLLT may be an effective therapeutic approach for SCI with clinical application prospects.     

Oral mucosa stem cells alleviates spinal cord injury-induced neurogenic bladder symptoms in rats

Background

Spinal cord injury (SCI) deteriorates various physical functions, in particular, bladder problems occur as a result of damage to the spinal cord. Stem cell therapy for SCI has been focused as the new strategy to treat the injuries and to restore the lost functions. The oral mucosa cells are considered as the stem cells-like progenitor cells. In the present study, we investigated the effects of oral mucosa stem cells on the SCI-induced neurogenic bladder in relation with apoptotic neuronal cell death and cell proliferation.

Results

The contraction pressure and the contraction time in the urinary bladder were increased after induction of SCI, in contrast, transplantation of the oral mucosa stem cells decreased the contraction pressure and the contraction time in the SCI-induced rats. Induction of SCI initiated apoptosis in the spinal cord tissues, whereas treatment with the oral mucosa stem cells suppressed the SCI-induced apoptosis. Disrupted spinal cord by SCI was improved by transplantation of the oral mucosa stem cells, and new tissues were increased around the damaged tissues. In addition, transplantation of the oral mucosa stem cells suppressed SCI-induced neuronal activation in the voiding centers.

Conclusions

Transplantation of oral mucosa stem cells ameliorates the SCI-induced neurogenic bladder symptoms by inhibiting apoptosis and by enhancing cell proliferation. As the results, SCI-induced neuronal activation in the neuronal voiding centers was suppressed, showing the normalization of voiding function.     

810nm弱激光照射对大鼠急性脊髓损伤后巨噬细胞极化的作用研究

本研究构建急性大鼠脊髓夹伤模型,并将大鼠随机分为单纯脊髓损伤对照组及脊髓损伤联合弱激光照射组。照射组应用810 nm波长,150 m W照射功率,照射光斑0.3 cm^2的弱激光对脊髓损伤区进行经皮照射,连续照射3天,7天或14天。应用免疫荧光、免疫印迹实验方法,测定脊髓损伤区巨噬细胞及小胶质细胞的极化表达。应用酶联免疫吸附法测定脊髓损伤区白细胞介素4的表达情况。应用坚牢蓝髓鞘染色测定两组损伤脊髓中髓鞘保留的差异。采用BBB评分对两组大鼠后肢运动功能的恢复进行评估。结果表明,810 nm弱激光对脊髓损伤区连续照射3天,7天后,可显著减少M1型巨噬细胞及其标志物诱导型一氧化氮合酶的表达,在7天时间增加M2型巨噬细胞及其标志物精氨酸酶1的表达。弱激光照射组白细胞介素4的表达明显增加。损伤后14天,弱激光照射组脊髓损伤区髓鞘保留面积比值明显提高。损伤后7天及14天时,弱激光照射组大鼠的BBB评分明显升高。该实验结果表明,810 nm弱激光经皮照射,可增加大鼠急性脊髓损伤区M2型巨噬细胞及小胶质细胞的表达,并减少脊髓损伤后的髓鞘脱失,促进脊髓损伤大鼠运动功能的恢复。     

Tauroursodeoxycholic acid alleviates secondary injury in the spinal cord via up-regulation of CIBZ gene

Spinal cord injury (SCI) is generally divided into primary and secondary injuries, and apoptosis is an important event of the secondary injury. As an endogenous bile acid and recognized endoplasmic reticulum (ER) stress inhibitor, tauroursodeoxycholic acid (TUDCA) administration has been reported to have a potentially therapeutic effect on neurodegenerative diseases, but its real mechanism is still unclear. In this study, we evaluated whether TUDCA could alleviate traumatic damage of the spinal cord and improve locomotion function in a mouse model of SCI. Traumatic SCI mice were intraperitoneally injected with TUDCA, and the effects were evaluated based on motor function assessment, histopathology, apoptosis detection, qRT-PCR, and western blot at different time periods. TUDCA administration can improve motor function and reduce secondary injury and lesion area after SCI. Furthermore, the apoptotic ratios were significantly reduced; Grp78, Erdj4, and CHOP were attenuated by the treatment. Unexpectedly, the levels of CIBZ, a novel therapeutic target for SCI, were specifically up-regulated. Taken together, it is suggested that TUDCA effectively suppressed ER stress through targeted up-regulation of CIBZ. This study also provides a new strategy for relieving secondary damage by inhibiting apoptosis in the early treatment of spinal cord injury.     

The regenerative effects of electromagnetic field on spinal cord injury

Traumatic spinal cord injury (SCI) is typically the result of direct mechanical impact to the spine, leading to fracture and/or dislocation of the vertebrae along with damage to the surrounding soft tissues. Injury to the spinal cord results in disruption of axonal transmission of signals. This primary trauma causes secondary injuries that produce immunological responses such as neuroinflammation, which perpetuates neurodegeneration and cytotoxicity within the injured spinal cord. To date there is no FDA-approved pharmacological agent to prevent the development of secondary SCI and induce regenerative processes aimed at healing the spinal cord and restoring neurological function. An alternative method to electrically activate spinal circuits is the application of a noninvasive electromagnetic field (EMF) over intact vertebrae. The EMF method of modulating molecular signaling of inflammatory cells emitted in the extra-low frequency range of <100 Hz, and field strengths of <5 mT, has been reported to decrease inflammatory markers in macrophages, and increase endogenous mesenchymal stem cell (MSC) proliferation and differentiation rates. EMF has been reported to promote osteogenesis by improving the effects of osteogenic media, and increasing the proliferation of osteoblasts, while inhibiting osteoclast formation and increasing bone matrix in vitro. EMF has also been shown to increase chondrogenic markers and collagen and induce neural differentiation, while increasing cell viability by over 50%. As advances are made in stem cell technologies, stabilizing the cell line after differentiation is crucial to SCI repair. Once cell-seeded scaffolds are implanted, EMF may be applied outside the wound for potential continued adjunct treatment during recovery.     

Expression of neural cell adhesion molecule in spinal cords following a complete transection

Neural cell adhesion molecule (NCAM) regulates tissue organization during development and in the adult. NCAM upregulation occurs after an injury to brains and sciatic nerves. However, little is known about NCAM expression after spinal cord injury (SCI). By using a complete spinal cord transection with a 5 mm tissue removal, an increase in the NCAM level is detected in spinal cord stumps proximal and distal to the transection site at 1 d and 3 d post injury, while its expression at 8 d is declined to a lower level than that observed in sham-operated spinal cords. The strong NCAM expression is present in motor neurons at 3 d post transection whereas the intensive NCAM immunostaining is localized in dorsal sensory and corticospinal fiber tracts at 8 d following injury. Collectively, NCAM level is elevated and strongly expressed in dorsal fiber tracts after SCI, implying that the endogenous process for spinal cord regeneration may take place after SCI.