| Objective: To establish a modified mice model of lumbar nerve root compression and observe the changes of lumbar lymphatic system after lumbar nerve root compression;to observe the effects of VEGFR3 inhibitor on nerve recovery,inflammatory injury and lymphatic function in the model of mice lumbar nerve root compression;to study the specific mechanism of acupuncture on inflammatory injury in the mice model of lumbar nerve root compression.Methods:1.Changes of lymphatic system in mice model of lumbar nerve root compression: 20 male mice were randomly divided into model group and sham operation group to establish mice model of lumbar nerve root compression.After 24 hours,the behavioral changes of mechanical pain threshold and thermal pain threshold were observed.The mice in the model group and sham operation group were sacrificed,and the compressed lumbar segments and lymph nodes were removed.The morphology of lumbar segments was observed by HE staining,and the area of lymphatic vessels and proliferation of lymphatic endothelial cells in the lymph nodes were observed by immunofluorescence staining.2.The role of VEGFR3 inhibitor in the mice model of lumbar nerve root compression: 60 male mice were randomly divided into sham operation group,model group and VEGFR3 inhibitor intervention group.The mice model of lumbar nerve root compression was established in the model group and VEGFR3 inhibitor group.After operation and 7 days,the behavioral changes of mechanical pain and thermal pain threshold in all groups were observed.The mice in each group were sacrificed after 7 days,and the compressed lumbar segments and lymph nodes were removed.Immunofluorescence staining was used to observe the distribution of GFAP and Neu N in spinal cord,the area of lymphatic vessels and proliferation of lymphatic endothelial cells in lymph nodes,the immune cells in lymph nodes and the innervation rate of nerve endings at the neuromuscular junction of tibialis anterior muscle.Quantitative imaging flow cytometry was used to observe the proportion of immune cells in lymph nodes.3.Mechanism of acupuncture in mice model of lumbar nerve root compression: 60 male mice were randomly divided into sham operation group,model group and acupuncture group.Lumbar nerve root compression model was established in model group and acupuncture group.The behavioral changes of mechanical pain and thermal pain threshold of model mice were observed after operation and 7 days after treatment.The compressed lumbar segment,anterior tibial muscle and lumbar aorta were taken out after 7days of treatment.Immunofluorescence staining was used to observe the morphology of spinal cord neurons and astrocytes,the neuromuscular junction of tibialis anterior muscle,the area of lymphatic vessels,the proliferation of lymphatic endothelial cells and the changes of M1 macrophages in lymph nodes.Flow cytometry was used to observe B cells,T cells,mononuclear neutrophils,monocytes,neutrophils in lumbar aortic lymph nodes.Results:1.Behavioral changes showed that the hind limb of the affected side of the model group had obvious lower pain threshold;Evans blue injection found that the lumbar aortic lymph nodes were lumbar backflow lymph nodes;HE staining showed obvious morphological changes of lumbar nerve root and DRG neuron in the model group;immunofluorescence staining showed that the area of lymphatic vessels and the number of lymphatic endothelial cells in the lumbar aortic lymph nodes of the model group increased significantly.2.Behavioral changes showed that the mechanical pain threshold of the affected hind limb in the VEGFR3 inhibitor group was higher than that in the model group;astrocytes and neurons in the VEGFR3 inhibitor group were more concentrated in the dorsal horn of the spinal cord than those in the model group,but arranged disorderly;the terminal nerve innervation of the neuromuscular junction in the VEGFR3 inhibitor group and the model group was significantly less than that in the sham operation group,but there was no significant difference between the VEGFR3 inhibitor group and the model group.Compared with the model group and sham operation group,the number of B cells in lymph nodes and the number of M1 and M2 macrophages in VEGFR3 inhibitor group was increased;the number of MSM in VEGFR3 inhibitor group was increased.3.Behavioral changes showed that the pain threshold of the affected hind limb in the model group was significantly decreased,while acupuncture treatment significantly increased the pain threshold.Immunofluorescence staining showed that acupuncture could significantly repair the astrocytes and neurons in the compressed lumbar spinal cord,increase the innervation rate of nerve endings at the neuromuscular junction of tibialis anterior muscle,and reduce the proliferation of lymphatic endothelial cells in lumbar aortic lymph nodes.Flow cytometry showed that the number of monocytes increased significantly in the model group,while acupuncture could significantly reduce the number of monocytic neutrophils.Conclusion:1.The behavioral changes of pain threshold and the deformation and rupture of DRG neurons in the compressed segment were observed in the nerve root compression model mice,which simulated the human lumbar disc herniation.At the same time,it was found that the lymph nodes of lumbar nerve root compression model were lumbar aortic lymph nodes,and the lymphatic system changed significantly after nerve root compression,which laid a foundation for studying the role of lymphatic system in nerve root model.2.The VEGFR3 inhibitor aggregated neural cells and astrocytes in the spinal dorsal horn of the mice model with nerve root compression,activated the immune function of lymphatic system,and increased the local inflammatory response.VEGFR3 inhibitor inhibited the Lymphangiogenesis in the lymph node and increased the inflammatory response.3.Acupuncture can attenuated the pain symptoms of hind limbs of mice model of lumbar nerve root compression,improve the nerve repair,inhibit the immune response in lumbar lymph nodes,and reduce the inflammatory response caused by nerve root compression model.This paper explains the mechanism of the Traditional Chinese Medicine rehabilitation theory of promoting qi and activating blood circulation,relaxing tendons and removing blood stasis. |
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