The Pathological Correlation Study Between DLAV Formation Loss And Bone Damage Of Zebrafish Larvae Induced By Fluoride Exposure

Objective:The study intends to take wild-type and Tg(Flk1:EGFP)transgenic zebrafish as research object and observe the bone morphological development and pathological changes of endochondral bone mineralization and bone formation in zebrafish larvae induced by fluoride exposure,in order to establish a bone damage model of zebrafish larvae induced by fluoride exposure.Through observing the effects of fluoride exposure on DLAV formation of zebrafish larvae in order to explore the correlation between DLAV generation and bone damage.Methods:The control group(0mg/L NaF),the low fluoride group(25mg/L NaF)and the high fluoride group(100mg/L NaF)were established according to median lethal concentration of fluoride on nine days post fertilization zebrafish larvae and previous literature.Six hours post fertilization wild-type zebrafish embryos and Tg(Flk1:EGFP)transgenic zebrafish embryos were cultured to the nineth day according to the experimental groups.The fluorine ion selective electrode method was used to detect the body fluorine content of Tg(Flk1:EGFP)transgenic zebrafish larvaes in each group,the mortality and morphological development of Tg(Flk1:EGFP)transgenic zebrafish larvaes in each group were observed and statistically analyzed under the stereo microscope.Alizarin red was used to trace the mineralized bone area,integrated optical density and stained vertebrae of Tg(Flk1:EGFP)transgenic zebrafish larvaes in each group,alcin blue staining was used to trace the chondrogenesis area of Tg(Flk1:EGFP)transgenic zebrafish larvaes in each group.Real time fluorescence quantitative polymerase chain reaction was used to detect the bone developing-specific genes expression level(Sox9a,Sox9 b,OC,ALP,OPG,Runx2 b,RANKL)of Tg(Flk1:EGFP)transgenic zebrafish larvaes in each group.The DFCH-DA probe was used to detect the reactive oxygen species level of wild-type zebrafish larvaes in each group.The angiogenesis of Tg(Flk1:EGFP)transgenic zebrafish larvaes in each group were observed under the fluorescence microscope,so that to count the number of dorsal longitudinal anastomotic vessel loss and endothelial cell number of DLAV in the tail of the spine.Real time fluorescence quantitative polymerase chain reaction was used to detect the EGFP expression level of Tg(Flk1:EGFP)transgenic zebrafish larvaes in each group.The correlation between DLAV formation index and bone formation index was analyzed by Pearson correlation.Results:(1)Compared with the control group,the body fluorine content of zebrafish larvae in low fluoride group and high fluoride group were significantly increased(P < 0.05).There was no significant difference in mortality of zebrafish larvae among all groups(P > 0.05).Compared with the control group,the rate of swim bladder development loss and spinal curve in high fluoride group were significantly increased(P < 0.05),and body length was significantly shortened(P < 0.05).(2)Compared with the control group,the mineralized bone area,IOD,stained vertebrae number in low fluoride group were significantly increased(P < 0.05),while the chondrogenesis area was significantly decreased(P < 0.05),the mineralized bone area,IOD and stained vertebrae number in high fluoride group were significantly decreased(P < 0.05),the chondrogenesis area was significantly increased(P < 0.05).(3)There was no significant difference about the mRNA expression levels of Sox9 a and Sox9 b among all groups(P > 0.05).Compared with the control group,the mRNA expression levels of OC,ALP,Runx2 b,OPG and OPG/RANKL in low fluoride group were significantly increased(P < 0.05),the mRNA expression levels of OC,ALP,Runx2 b and OPG/RANKL in high fluoride group were significantly decreased(P < 0.05),while the mRNA expression level of RANKL in high fluoride group was significantly increased(P < 0.05).(4)The fluorescence intensity of ROS in low fluoride group was significantly increased compared with the control group,and that in high fluoride group was significantly increased compared with the low fluoride group(P < 0.05).Compared with the control group,DLAV loss number in low fluoride group was significantly decreased,while that in high fluoride group was significantly increased(P < 0.05).Compared with the control group,the number of endothelial cells on DLAV in low fluoride group was significantly increased,while that in high fluoride group was significantly decreased(P < 0.05).Compared with the control group,the mRNA expression level of EGFP in low fluoride group was significantly increased,while that in high fluoride group was significantly decreased(P < 0.05).(5)There was a significant negative correlation between DLAV loss number and the mineralized bone area(r =-0.796,P < 0.05),a significant positive correlation between DLAV loss number and the chondrogenesis area(r = 0.726,P < 0.05),a significant positive correlation between the number of DLAV endothelial cells and the mineralized bone area(r = 0.705,P < 0.05),a significant negative correlation between the number of DLAV endothelial cells and the chondrogenesis area(r =-0.528,P < 0.05).There was a significant negative correlation between EGFP mRNA and OC,ALP,Runx2 b mRNA(r = 0.966,0.993,0.990,P < 0.01).EGFP mRNA had no significant correlation with Sox9 a and Sox9 b mRNA(r =-0.602,0.374,P >0.05).Conclusions:Short time fluoride exposure during embryonic stage can’t only establish a bone damage model of zebrafish larvae,but also affects DLAV production in zebrafish larvae.The bone damage caused by abnormal bone mineralization and bone absorption through endochondral ossification may be related to DLAV formation loss.