The Role Of MAPK In Vitreous Hemorrhage Induced By Blunt Ocular Contusion In Rabbits And Its Relationship With Iron Overload

Objectives:To investigate the relationship between iron overload caused by vitreous hemorrhage caused by blunt trauma and MAPK pathway.The changes and expression levels of HO-1,p38MAPK,ERK1/2 and JNK1/2 in the retina were analyzed by dynamically observing the content of retinal iron.The relationship among iron overload,MAPK pathway and retinal injury and protective mechanism were discussed.It provides a new theoretical basis for the treatment of patients with closed ocular trauma.Methods:There were 40 adult clean New Zealand white rabbits with a weight of about 2-2.5kg,both male and female.The experimental group was divided into 21 rabbits,and the positive control group was divided into 19 rabbits.Three were removed from the experimental group and one from the positive control group.Exclusion criteria:if keratitis,corneal leukophora,cataract,endophthalmitis and other eye diseases are found in the experimental rabbit during the experiment,or death occurs due to model failure,the experiment will be excluded.All rabbits were anesthetized with 3%pentobarbital sodium(lml/kg)intravenously injected into the ear margin,and the right eye was injured.Finally the experimental rabbits were randomly divided into three groups:(1)the experimental group(n=18)blunt trauma vitreous hemorrhage group,after anesthesia,lie in the test bench,the eyelid opener fixed eyes,eye 1 meters from about 5 kg steel ball vertical drop,at the same quality,the same speed,the same height struck central eye corneal,batch production into severe blunt trauma model of vitreous hemorrhage.The corneal edema was observed under the microscope,the pupil was medium big,the vitreous body was completely hemorrhage,and the peep of the fundus was not clear.(2)positive control group(n=18),after anesthesia,lay flat on the operating table,he compound topicmide was used to dilate the pupil and prick the blood vessels on the retina with a lml syringe under the microscope to form retinal hemorrhage,which was made into non-traumatic vitreous hemorrhage.Under the microscope,retinal hemorrhage was observed to diffuse into the vitreous body,and the fundus was partially visible or the peep was not clear.(3)healthy control group(36 eyes),the contralateral eyes were treated as blank control group without any treatment.Immunohistochemistry was used to locate HO-1,p38MAPK,ERK1/2,JNK1/2 and semi-quantitatively western blot HO-1,p38MAPK,ERK1/2 and JNK1/2.Results:(1)ICP-OES was used to quantify and compare the iron content in the retinas of each group of rabbits at 1,2,4 and 8 weeks.The results showed that:compared with the healthy control group,the iron content in the experimental group increased significantly at 1,2,4 weeks,and slightly decreased in the experimental group at 8 weeks,but still increased significantly compared with the healthy control group,the difference was statistically significant(P<0.05).Compared with the healthy control group,the iron content in the positive control group increased slightly at week 1,2 and 4,decreased slightly at week 8,and was still significantly higher than that in the healthy control group(P<0.05).Compared with the positive control group,the iron content in the experimental group increased slightly at 1,2 and 4 weeks,and increased slightly at 8 weeks.The difference was statistically significant(P<0.05).The results were consistent with similar analyses of human retina and rat retina,with significant differences.Iron levels in the retina increase over time,regardless of whether the retina is damaged or not,as long as there is vitreous hemorrhage.But over time,it may be due to vitreous hemorrhage absorption or decreased retinal iron content.By means of the experimental group,positive control group and healthy control group at different times of the Fe content is found that the experimental group and positive control group 4 weeks of Fe content is the highest,as a result,HE staining,Prussian blue staining,immunohistochemistry and western blot adopt retinal samples of three groups of 4 weeks(2)then HE staining,experimental section 4 weeks observation found that thinning of the retina,and the structure of the disorder,cone rod cell layer fracture disappear,inner and outer nuclear layer cavity change obviously,the nucleus number decreased significantly,inner and outer plexiform layer thin obviously,some even disappear,ganglion cell nucleus pycnosis.RPE has not changed.The positive control group had less retinal changes than the experimental group.The healthy control group was normal with reference to intact retina.Prussian blue staining showed that retinal iron deposition in the experimental group was mainly concentrated in the neuro-retinal layer(inner layer,outer plexus layer and outer nuclear layer).In the positive control group,retinal iron deposition was mainly concentrated in the neuroretina(inner plexus layer).Healthy control group had no deposition.(3)immunohistochemistry showed that p38MAPK,JNK1/2 were expressed in the nerve fiber layer and ganglion cell layer in the experimental group,ERK1/2 was expressed in the inner boundary membrane,and HO-1 was expressed in ganglion cells and rods.P38MAPK,JNK1/2,ERK1/2,and HO-1were almost not expressed in the retina of the positive control group and healthy control group.(4)the gray level of SDS-PAGE electrophoresis bands was analyzed in the three groups of rabbits at week 4.Compared with the positive control group,the protein expression levels of JNK1/2,HO-1 and p38MAPK in the experimental group were increased,P<0.05,and the difference was statistically significant.The expression level of ERK1/2 protein in the experimental group was similar to that in the positive control group,but still increased,P<0.05,and the difference was statistically significant.Prolonged iron overload in vitreous hemorrhage caused by blunt trauma,especially the sustained high level of free iron,leads to long-term iron ion-mediated oxidative damage and persistent damage to the retinal neuroepithelium,especially the photoreceptor cell layer.Iron overload after vitreous hemorrhage was significantly correlated with the up-regulation of HO-1 expression level,indicating that the increase of HO-1 can protect retinal cells by significantly reducing the production of reactive oxygen species.The activation of p38MAPK,JNK1/2 and ERK1/2 after hemorrhage indicated that MAPKs signaling pathway played an important role in the injury of vitreous retinal ganglion cells and Muller glial cells caused by blunt injury and the protective mechanism.The positive control group,namely the non-traumatic hemorrhage group,also significantly activated the expression of p38MAPK and JNK1/2 in the rabbit retina,but could not up-regulate the expression of ERK1/2 in the retina.JNK1/2 and p38MAPK signals may be related to the damage and protection mechanism of retinal ganglion cells after hemorrhage,while ERK1/2 signal pathway may mediate the protection and repair effect of Muller glial cells after hemorrhage The mechanism of MAPKs signaling pathway in iron overload remains to be further studied.Conclusions:Iron overload may damage the retinal nerve epithelium by activating the MAPK signaling pathway.Therefore,this experiment confirmed the toxic effect of iron overload caused by vitreous injury on the retina.The enlightenment for clinical treatment is that vitrectomy should be carried out as soon as possible when conditions permit,to clean up excess vitreous hemorrhage and reduce the damage of iron ion to the retina.