Intravital Imaging Reveal Microglia Function In Melanoma Brain Metastasis And Neuropathic Pain

Microglia/macrophages(M/Ms)are the main immune cells with the property of high plasticity in activation state and function in the central nervous system(CNS).They not only play important roles in sustaining the homeostasis of CNS,but also participate in the occrruence and development of many neurological diseases.Many studies demonstrate that the morphology and motility of M/Ms is the direct manifestation of their activation state in different physiological and pathological conditions.Depicting the rule of alteration in morphology and motility of M/Ms via intravital microscopic imaging technology will make it possible to uncover their function in different diseases directely.Nowadays,cancer is a huge threat to the health of human beings.However,metastasis is the reason that makes cancer difficulty to be radical cure.Because of the complex in brain structure and function,metastasis to brain possess the characteristics with difficulty in cures,high risk and poor prognosis.Neuropathic pain is a chronic pain caused by injury of nervous system and happens accompanioned with diseases such as bone shrink in cancer,infection,autoimmune diseases,trauma,diabetes,which affect the life quality of patients very deeply.However,how does the activation state of M/Ms change and what is their function during the chronic development of the two diseases haven' t been elucidated well to date.Taking advantages of longitudinal in vivo microscopic imagin,this study focused on longitudinal monitoring the morphology and motility behaviors of M/Ms during the development of brain metastasis and neuropathic pain to reveal the function they played in diseases.The main results were shown as below:(1)In the first place,we realized the monitoring of the melanoma brain metastasis and normal brain tissues in mice at the same time with dual-lateral cranial windows.Having imaged the mice for as long as 3 weeks,we observed that the morphology of M/Ms changed from typical ramified shape to activated amboied shape during the development of melanoma brain metastasis.The quantitation of imaging data shown that,compared with the normal brain tissues,the M/Ms in the meatstasis changed their soma diameter whic increased 1.56 folds,decreased 1.68 folds of their processes.Except the alteration in morphology,the cell numbers and soma velocity of M/Ms were 1.57 folds and 2.36 folds of that in their control groups separately.Additionally,we sumerized the information of soma diameter and processes of M/Ms and proposed a parameter named ‘branching parameter' for manisfetion the activate degree of them more directly and comprehensively.These results indicated that plenty of M/Ms had been activated on day 5 after melanoma cell inoculation,and they kept the activation state during melanoma brain metastasis and achieved to the strongest activation state on day 21 after melanoma cell inoculation.(2)Through depletion of M/Ms,we testified that they participated in the occurrence of melanoma brain metastasis.Meanwhile,they upregulated the MMP3 expression during the development of melanoma brain metastasis.Up to 94% M/Ms expressed MMP3,and the expression of MMP3 from M/Ms was 114.2 folds of that in melanoma cells.The correlation analysis between the MMP3 expressed by M/Ms and the tight junction protein expression or the activate parameter of M/Ms showed that,the autocrine MMP3 promote their activation as a stimulation and meanwhile participated in disruption of BBB to realize the facilitation of melanoma brain metastasis.(3)Having established the mice model with different pain grade with CCI injury on sciatic nerve and implanted the spinal cord window on lumbar 5 of spinal,we observed the dynamic change of M/Ms during the development of neuropathic pain after 2weeks imaging.Comparing the M/Ms in mice with different pain grade and both lateral spinal cord,we found that,they were activated on day 2 after CCI injury,and their morphology turned to be amboid shape.To day 14 after CCI injury,they kept the activation still,and decreased their velocity in processes,and increased the volume density to 1.75 folds of that in control groups.These results indicated that,after CCI injury,M/Ms were quickly activated,and the longlasting activation state may closely relate with the property of persistence and lasting in the development of neuropathic pain.(4)Last,for studying the modulation of S1 HL cortex in neuropathic pain,we detected the oxygen satuation in normal and pain mice with unlabled,high resolution photoacoustic microscopic system.The results showed that the proportion of oxyhemoglobin was higher in pain mice.The phenomenon of higher oxygen satuation indicated that in pain mice,the active exchange of material and energy was taking place,in which more oxygen were needed,and the M/Ms in SIHL cortex may be in the activation state as well to participate in the modulation of neuropathic pain.In summary,we monitored that the M/Ms were quickly activated and alterated significantly in the morphology,motility behaviors and distribution,along with the development of melanoma brain metastasis and neuropathic pain,with the use of intravital microscopic imaging techniques.Furthermore,we uncovered their function in both of the diseases with traditional biological methods and photoacoustic microscopic imaging to offer new proof fore related mechanism research and new insights for the therapy of the diseases.