Effects Of Nano-sized Carbon Black On Lumbar Spinal Cord Injury And Inflammatory Factors Expression In Rats

Carbon black is a loose,light and extremely fine black powder,which is very stable and inert,and is often used in the manufacture of ink.The particle size of carbon black is small and the specific surface area is large.It is also widely used in scientific research.Carbon black particles are also the core of fine particulate matter in the atmosphere.A large number of previous experiments have proved that nano-level particles in polluted air will cause damage to the body.Besides respiratory disease that is the most common and the most obvious disease,nano-level particles can also affect other major systems and cause cancer.Recent studies have shown that the central nervous system is a potential target organ for the toxic effects of nanoparticles.Objective:To study the effects of nano-sized carbon black on lumbar spinal cord injury and changes of the expression of IL-1β、IL-6、TNF-ɑand IL-10 protein in rats.Methods:Thirty-two healthy male SD rats were used for carbon black inhalation experiment.They were randomly divided into four groups（n=8）:（1）Control（C）group:exposure to filtered air for 90 d.（2）Carbon black（CB）inhalation group:exposure to nano-sized carbon black particles for 90 d.（3）Recovery（R）group:exposure to nano-sized carbon black particles for 90 d and then filtered air for another 14 d.（4）Recovery control（RC）group:exposure to filtered air for 104 d.Rats in carbon black inhalation group and recovery group inhaled nano-sized carbon black particles for 6 h/day at a concentration of 30mg/m³for 90 d.The weight of the rats was recorded every 4 days during the exposure.At the end of the exposure,the rats were anesthetized and then blood samples were collected.Serum was harvested by centrifugation at 700×g for 10 min.The lumbar spinal cord tissue was harvested and stored in liquid nitrogen for Western blot（n=5 in each group）.The lumbar spinal cord tissue was separated and fixed in 4%paraformaldehyde,then embedded with paraffin（n=3 in each group）.Paraffin sections were prepared with routine methods for HE staining and immunohistochemistry.Results:1 The general condition and body weight of rats exposed to nano-sized carbon blackDuring the whole exposure of the experiment,all of the rats were survived.The body weight of rats in CB group was significantly lower than that of control group from the 21^th day（P<0.05）,and the body weight of rats in Rec group was significantly lower than that of RC group from the 21^th day（P<0.05）.2 White blood cell count and classification2.1 Total white blood cells（WBC）:Compared with control group,WBC of CB group increased significantly（P<0.05）.Compared with RC group,WBC of Rec group increased significantly（P<0.05）.2.2 Leukocyte classification:Compared with control group,the percentage of lymphocytes and basophils count decreased significantly in rats of CB group（P<0.05）;the percentage of monocytes and neutrophils,monocytes count,and neutrophils count increased significantly in rats of CB group（P<0.05）.Compared with CB group,the percentage of lymphocytes increased significantly in rats of Rec group（P<0.05）;the percentage of monocytes,neutrophils count decreased significantly in rats of Rec group（P<0.05）.Compared with RC group,the percentage of monocytes,neutrophils count decreased significantly in rats of Rec group（P<0.05）.3 HE stainingIn the gray matter of anterior horn of lumbar spinal cord,compared with control group,the number of neurons in CB group was no significantly different from control group,the number of glial cells in CB group was increased significantly,and satellite phenomenon was observed.Compared with CB group,the number of glial cells in Rec group was increased significantly,the nissl bodies of neurons dissipated.In the gray matter of posterior horn of lumbar spinal cord,compared with control group,the neurons in CB group was shrunken,the number of neurons in CB group was no significantly different from control group;however,the number of glial cells in CB group was increased significantly（P<0.05）.Compared with CB group,the number of glial cells in Rec group was increased significantly（P<0.05）.4 Western blot assay4.1 IL-1β:Compared with control group,the expression of IL-1βprotein in CB group was increased significantly（P<0.05）.Compared with CB group,the expression of IL-1βprotein in Rec group was further increased significantly（P<0.05）.Compared with control group,no significant difference of IL-1βprotein expression was observed in RC group（P>0.05）.4.2 The change trends of the expression of IL-6,TNF-ɑand IL-10 protein are consistent with that of IL-1β.5 Immunohistochemistry analysis5.1 IL-1βIn the gray matter of anterior horn of lumbar spinal cord,compared with control group,the positive staining of IL-1βin neurons,glial cells and nerve fibers was significantly increased in CB group,and the IOD of IL-1βexpression was significantly increased（P<0.05）.Compared with CB group,the positive staining of IL-1βin neurons,glial cells and nerve fibers was further increased in Rec group,especially in cytoplasma of neurons,and the IOD of IL-1βexpression was significantly increased（P<0.05）.In the gray matter of posterior horn of lumbar spinal cord,compared with control group,the positive staining of IL-1βin nerve fibers was significantly increased in CB group,the staining of neurons and glial cells was slightly increased,and the IOD of IL-1βexpression was significantly increased in CB group（P<0.05）.Compared with CB group,the positive staining of IL-1βin neurons and glial cells was significantly increased in Rec group,the positive staining of IL-1βinnerve fibers was also significantly increased,and the IOD of IL-1βexpression was significantly increased in Rec group（P<0.05）.5.2 IL-6In the gray matter of anterior horn of lumbar spinal cord,compared with control group,the positive staining of IL-6 in neurons,glial cells and nerve fibers was significantly up-regulated in CB group,and the IOD of IL-6expression was significantly increased（P<0.05）.Compared with CB group,the positive staining of IL-6 in neurons,glial cells and nerve fibers was significantly up-regulated in Rec group,especially in cytoplasma of neurons,and the IOD of IL-6 expression was significantly increased（P<0.05）.In the gray matter of posterior horn of lumbar spinal cord,compared with control group,the positive staining of IL-6 in neurons,glial cells and nerve fibers was significantly raised in CB group,and the IOD of IL-6 expression was significantly increased（P<0.05）.Compared with CB group,the staining of neurons and glial cells in Rec group was significantly deepened,the staining of nerve fibers was also significantly deepened,and the IOD of Rec group was increased significantly（P<0.05）.5.3 TNF-ɑIn the gray matter of anterior horn of lumbar spinal cord,compared with control group,the positive staining of TNF-ɑin neurons,glial cells and nerve fibers was significantly elevated in CB group,and the IOD of TNF-ɑexpression was significantly increased（P<0.05）.Compared with CB group,the positive staining of TNF-ɑin neurons,glial cells and nerve fibers was significantly elevated in Rec group,and the IOD of TNF-ɑexpression was significantly increased（P<0.05）.In the gray matter ofposterior horn of lumbar spinal cord,compared with control group,the positive staining of TNF-ɑin neurons,glial cells and nerve fibers was significantly deepened in CB group,and the IOD of TNF-ɑexpression was significantly increased（P<0.05）.Compared with CB group,the positive staining of TNF-ɑin neurons,glial cells andnerve fibers was significantly deepened in Rec group,especially in cytoplasma of neurons,and the IOD of TNF-ɑexpression was significantly increased（P<0.05）.5.4 IL-10In the gray matter of anterior horn of lumbar spinal cord,compared with control group,the positive staining of IL-10 in neurons,glial cells and nerve fibers was significantly deepened in CB group,and the IOD of IL-10expression was significantly increased（P<0.05）.Compared with CB group,the positive staining of IL-10 in neurons,glial cells and nerve fibers was significantly deepened in Rec group and the IOD of IL-10 expression was significantly increased（P<0.05）.The posterior horn of lumbar spinal cord:Compared with control group,the positive staining of IL-10 in neurons,glial cells and nerve fibers was significantly deepened in CB group,and the IOD of IL-10 expression was significantly increased（P<0.05）.Compared with CB group,the positive staining of IL-10 in neurons,glial cells and nerve fibers was significantly deepened in Rec group,,especially in cytoplasma of neurons,and the IOD of IL-10 expression was significantly increased（P<0.05）.Conclusion:After the CB inhalation,the body weight of rats significantly decreased from 21 days compared with control group.The CB inhalation induced systemic inflammation;the expression of proinflammatory factor IL-1β,IL-6 and TNF-ɑwas significantly increased in gray matter of anterior and posterior horn of lumbar spinal cord,and the anti-inflammatory factor IL-10 was also significantly increased.Without treatment,the lasting effect of carbon black on the inflammation injury was observed in the lumbar spinal cord in rats after being separated from the CB environment.