RESUMEN
The study aimed to design a reliable and straightforward PBM method by implanting a medical scattering fiber above surgically exposed spinal cord in SCI patients. Moreover, the safety of this method was examined. Twelve patients with acute SCI (ASIA B) requiring posterior decompression were recruited. The medical scattering fiber was implanted above the spinal cord, and was continuously irradiated at 810 nm, 300 mW, 30 min/day, once per day for 7 days. The vital signs (temperature, blood pressure, respiratory rate, heart rate, and oxygen saturation), infection indicators (WBC, NEUT, hs-CRP, and PCT), photo-allergic reaction indicators (Eosinophil and Basophil), coagulation function indicators (PT, APTT, TT) and neurological stability indicators (ASIA sensory and motor scores) were recorded to evaluate the safety of PBM. Three months after surgery, 12 patients completed follow-up. In our study, direct PBM on SCI site did not cause clinically pathologic changes in vital signs of the patients. All patients had higher WBC, NEUT, and hs-CRP at day 3 during irradiation than those before surgery, and returned to normal at day 7. The changes in Eosinophil and Basophil that were closely associated with allergic reactions were within normal limits throughout the course of irradiation. The coagulation function (PT, APTT, and TT) of patients were also in the normal range. The ASIA sensory and motor scores of all patients had no changes throughout the irradiation process. However, in the follow-up, both ASIA sensory and motor scores of all patients had minor improvement than those in pre-irradiation, and 7 patients had adverse events, but they were not considered to be related to PBM. Our study might firstly employ direct PBM in the SCI by using scattered optical fibers. In a limited sample size, our study concluded that direct PBM at the site of SCI would not produce adverse effects within the appropriate irradiation parameters. The method is safe, feasible, and does not add additional trauma to the patient. Our preliminary study might provide a new methodology for the clinical PBM treatment of acute SCI.
Asunto(s)
Proteína C-Reactiva , Terapia por Luz de Baja Intensidad , Traumatismos de la Médula Espinal , Humanos , Recuperación de la Función , Médula Espinal/patología , Traumatismos de la Médula Espinal/radioterapia , Traumatismos de la Médula Espinal/patologíaRESUMEN
BACKGROUND: Neurotoxic microglia and astrocytes begin to activate and participate in pathological processes after spinal cord injury (SCI), subsequently causing severe secondary damage and affecting tissue repair. We have previously reported that photobiomodulation (PBM) can promote functional recovery by reducing neuroinflammation after SCI, but little is known about the underlying mechanism. Therefore, we aimed to investigate whether PBM ameliorates neuroinflammation by modulating the activation of microglia and astrocytes after SCI. METHODS: Male Sprague-Dawley rats were randomly divided into three groups: a sham control group, an SCI + vehicle group and an SCI + PBM group. PBM was performed for two consecutive weeks after clip-compression SCI models were established. The activation of neurotoxic microglia and astrocytes, the level of tissue apoptosis, the number of motor neurons and the recovery of motor function were evaluated at different days post-injury (1, 3, 7, 14, and 28 days post-injury, dpi). Lipocalin 2 (Lcn2) and Janus kinase-2 (JAK2)-signal transducer and activator of transcription-3 (STAT3) signaling were regarded as potential targets by which PBM affected neurotoxic microglia and astrocytes. In in vitro experiments, primary microglia and astrocytes were irradiated with PBM and cotreated with cucurbitacin I (a JAK2-STAT3 pathway inhibitor), an adenovirus (shRNA-Lcn2) and recombinant Lcn2 protein. RESULTS: PBM promoted the recovery of motor function, inhibited the activation of neurotoxic microglia and astrocytes, alleviated neuroinflammation and tissue apoptosis, and increased the number of neurons retained after SCI. The upregulation of Lcn2 and the activation of the JAK2-STAT3 pathway after SCI were suppressed by PBM. In vitro experiments also showed that Lcn2 and JAK2-STAT3 were mutually promoted and that PBM interfered with this interaction, inhibiting the activation of microglia and astrocytes. CONCLUSION: Lcn2/JAK2-STAT3 crosstalk is involved in the activation of neurotoxic microglia and astrocytes after SCI, and this process can be suppressed by PBM.
Asunto(s)
Astrocitos/efectos de la radiación , Terapia por Luz de Baja Intensidad , Microglía/efectos de la radiación , Recuperación de la Función/efectos de la radiación , Traumatismos de la Médula Espinal/patología , Animales , Astrocitos/metabolismo , Janus Quinasa 2/metabolismo , Janus Quinasa 2/efectos de la radiación , Lipocalina 2/metabolismo , Lipocalina 2/efectos de la radiación , Masculino , Microglía/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/efectos de la radiación , Transducción de Señal/efectos de la radiación , Traumatismos de la Médula Espinal/metabolismo , Regulación hacia ArribaRESUMEN
To study the effect of photobiomodulation (PBM) on axon regeneration and secretion change of dorsal root ganglion (DRG) under oxidative stress after spinal cord injury (SCI), and further explore the effect of changes in DRG secretion caused by PBM on the polarization of macrophages. The PBM-DRG model was constructed to perform PBM on neurons under oxidative stress simulated in vitro. And the irradiation conditions were as follows: wavelength, 810 nm; power density, 2 mW/cm2; irradiation area, 4.5 cm2; and irradiation time, 440 s. Then resulted in an energy of 4 J (2 mW/cm2 × 4.5 cm2 × 440 s). About 100 µM H202 was added to the culture medium to simulate oxidative stress after SCI. An ROS (reactive oxygen species) assay kit was used to measure ROS contend in the DRG. The survival level of the neurons was measured using the CCK-8 method, and the axon regeneration of neurons was observed by using immunofluorescence. The secretion level of CCL2 from DRG was determined by RT-qPCR and ELISA. Further culturing macrophages of DRG-conditioned medium culture, the expression level of iNOS and Arg-1 in macrophages was assessed using Western blot analysis. The expression level of TNF-α and IL-1ß was determined by ELISA. After adding the neutralizing antibody of CCL2 to the DRG neuron-conditioned medium following PBM irradiation to culture macrophages to observe the effects on macrophage polarization and secretion. PBM could reduce ROS levels in neurons, increase neuronal survival under oxidative stress, and promote neuronal axon regeneration. In addition, PBM could also promote CCL2 secretion by DRG under oxidative stress. By constructing a DRG supernatant-M1 macrophage adoptive culture model, we found that the supernatant of DRG after PBM intervention could reduce the expression level of iNOS and the secretion of TNF-α and IL-1ß in M1 macrophages; at the same time, it could also up-regulate the expression of Arg-1, one of the markers of M2 macrophages. Furthermore, these effects could be prevented by the addition of neutralizing antibodies of CCL2. PBM could promote survival and axonal regeneration of DRG under SCI oxidative stress, increase the secretion level of CCL2 by DRG, and this change can reduce the polarization of macrophages to M1, further indicating that PBM could promote spinal cord injury repair.
Asunto(s)
Axones/metabolismo , Quimiocina CCL2/metabolismo , Macrófagos/citología , Estrés Oxidativo , Fototerapia/métodos , Traumatismos de la Médula Espinal/terapia , Regeneración de la Medula Espinal , Animales , Axones/efectos de la radiación , Diferenciación Celular , Células Cultivadas , Quimiocina CCL2/genética , Femenino , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiología , Interleucina-1beta/metabolismo , Luz , Macrófagos/inmunología , Macrófagos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos BALB C , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Spinal cord injury (SCI) stimulates reactive astrogliosis and the infiltration of macrophages, which interact with each other at the injured area. We previously found Photobiomodulation (PBM) significantly decreases the number of M1 macrophages at the injured area of SCI. But the exact nature of the astrocyte response following PBM and relationship with the macrophage have not been explored in detail. In this study, a BALB/c mice model with standardized bilateral spinal cord compression and a macrophage-astrocyte co-culture model were applied to study effects of PBM on astrocytes. Results showed that PBM inhibit the expression of the astrocyte markers glial fibrillary acidic protein (GFAP) and the secretion of chondroitin sulfate proteoglycans (CSPG) in the para-epicenter area, decrease the number of M1 macrophage in vivo. The in vitro experiments indicated M1 macrophages promote the cell viability of astrocytes and the expression of CSPG. However, PBM significantly inhibited the expression of GFAP, decreased activation of astrocyte, and downregulated the expression of CSPG by regulating M1 macrophages. These results demonstrate that PBM may regulate the interaction between macrophages and astrocytes after spinal cord injury, which inhibited the formation of glial scar.
Asunto(s)
Astrocitos/efectos de la radiación , Polaridad Celular/efectos de la radiación , Terapia por Luz de Baja Intensidad , Macrófagos/efectos de la radiación , Animales , Astrocitos/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Medios de Cultivo Condicionados/farmacología , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos BALB C , Actividad Motora/efectos de los fármacos , Actividad Motora/efectos de la radiación , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Recuperación de la Función/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/radioterapiaRESUMEN
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.
Asunto(s)
Axones/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Terapia por Luz de Baja Intensidad , Macrófagos/metabolismo , Macrófagos/efectos de la radiación , Factores de Crecimiento Nervioso/metabolismo , Regeneración Nerviosa , Animales , Axones/efectos de los fármacos , Axones/efectos de la radiación , Medios de Cultivo Condicionados/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Isoquinolinas/farmacología , Macrófagos/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Factores de Crecimiento Nervioso/genética , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/efectos de la radiación , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sulfonamidas/farmacologíaRESUMEN
A large number of animal experiments and clinical trials have confirmed that electrical stimulation can accelerate the growth of axons and recovery of motor function, all of which are inseparable from the formation of myelin. Therefore, establishment of a suitable electrical stimulation platform to study the effects of electrical stimulation on the myelin process of dorsal root ganglia and Schwann cells is of great significance for understanding the recovery of electrical stimulation. We designed a simple conductive glass cell culture system to overcome the shortcomings of direct contact of the electrode with the culture solution, and the number of culture chambers can be selected based on the purpose of the experiment in order to reduce experimental time and cost.
Asunto(s)
Técnicas de Cocultivo/instrumentación , Estimulación Eléctrica/instrumentación , Ganglios Espinales/citología , Vaina de Mielina/metabolismo , Células de Schwann/citología , Animales , Células Cultivadas , Técnicas de Cocultivo/métodos , Estimulación Eléctrica/métodos , Terapia por Estimulación Eléctrica , Diseño de Equipo , Ganglios Espinales/metabolismo , Ratas Sprague-Dawley , Células de Schwann/metabolismoRESUMEN
BACKGROUND/AIMS: Low-level laser therapy (LLLT) leads to complex photochemical responses during the healing process of spinal cord injury (SCI). Confocal Raman Microspectral Imaging (in combination with multivariate analysis) was adopted to illustrate the underlying biochemical mechanisms of LLLT treatment on a SCI rat model. METHODS: Using transversal tissue sections, the Raman spectra can identify areas neighboring the injury site, glial scar, cavity, and unharmed white matter, as well as their correlated cellular alterations, such as demyelination and up-regulation of chondroitin sulfate proteoglycans (CSPGs). Multivariate data analysis methods are used to depict the underlying therapeutic effects by highlighting the detailed content and distribution variations of the biochemical constituents. RESULTS: It is confirmed that photon-tissue interactions might lead to a decay of the inhibitory response to remyelination by suppressing CSPG expression, as also morphologically demonstrated by reduced glial scar and cavity areas. An inter-group comparison semi-quantitatively confirms changes in lipids, phosphatidic acid, CSPGs, and cholesterol during SCI and its LLLT treatment, paving the way for in vitro and in vivo understanding of the biochemical changes accompanying pathobiological SCI events. CONCLUSION: The achieved results in this work not only have once again proved the well-known cellular mechanisms of SCI, but further illustrate the underlying biochemical variability during LLLT treatment, which provide a sound basis for developing real-time Raman methodologies to monitor the efficacy of the SCI LLLT treatment.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Láseres de Semiconductores/uso terapéutico , Terapia por Luz de Baja Intensidad , Traumatismos de la Médula Espinal/radioterapia , Animales , Colesterol/metabolismo , Análisis por Conglomerados , Modelos Animales de Enfermedad , Femenino , Metabolismo de los Lípidos , Microscopía Confocal , Ácidos Fosfatidicos/metabolismo , Análisis de Componente Principal , Ratas , Ratas Sprague-Dawley , Espectrometría Raman , Médula Espinal/patología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patologíaRESUMEN
Electromagnetic fields (EMF) was considered as a non-invasive modality for treatment of osteoporosis while the effects were diverse with EMF parameters in time domain. In present study, we extended analysis of EMF characteristics from time domain to frequency domain, aiming to investigate effects of four kinds of EMF (LP (1-100 Hz), BP (100-3,000 Hz), HP (3,000-50,000 Hz) and AP (1-50,000 Hz)) on ovariectomized (OVX) osteoporosis (OP) in mice. Forty-eight 3-month-old female BALB/c mice were equally assigned to Sham, OVX, OVX + LP, OVX + BP, OVX + HP and OVX + AP groups (n = 8). After 8-week exposure (3 h/day), LP and BP significantly increased serum bone formation markers and osteogenesis-related gene expressions compared with OVX. Bedsides, LP and BP also slightly increased bone resorption activity compared with OVX, evidenced by increased RANKL/OPG ratio. HP sharply decreased serum bone formation and resporption markers and osteogenesis and osteoclastogenesis related gene expressions compared with OVX. AP had accumulative effects of LP, BP and HP, which significantly increased bone formation and decreased bone resporption activity compared with OVX. As a result, LP, BP and HP exposure did not later deterioration of bone mass, microarchitecture and mechanical strength in OVX mice with OP. However, AP stimulation attenuated OVX-induced bone loss.