The Influence Of Intraperitoneal Injection HMGB1 On Cortical Dysplasia And Neurobehavior During Pregnancy

Backgroud:The development of brain is a complex and sequential process: including the proliferation,migration,differentiation of neural precursor cells(NPCs)and the formation of neural synapses.In the embryonic period,the abnormal proliferation,migration and differentiation of NPCs could induce the malformation of cortical dysplasia(MCD)by the various internal and external fators.Research had shown the dysmorphic neurons are pacemaker cells in the MCD,which decrease the inhibitory neurotransmitter,increase the excitatory neurotransmitter and activate the glial cells.And then,increase the excitability of neurons,spontaneous discharge and eventually lead to seizures.Studies had shown the incidence of MCD with intractable epilepsy is 25%-53% postoperative,and the incidence of MCD was 80% in children with intractable epilepsy before the age of 3.Therefore,MCD is the primary pathological change in intractable epilepsy,and research the formation of MCD has become an important segment of epilepsy.MCD models were used to be induced by freezing injury,teratogenic agent(such as BCNU,MAM)and irradiation,which disturbed the development of NPCs by external injury,chemical and radiation.These exogenous teratogenic factors were widely used in the current MCD research.In addition,there are some endogenous fators also cloud induce MCD,which including genetic alterations,ischemia,hypoxia,infection,inflammation and internal environment changes.It is remarkable the endogenous brain injury and the subordinate inflammatory response have an important role in the formation of MCD by endogenous fators intervene on the embryonic.Some studies had shown high mobility group B1(HMGB1)is a common result of various endogenous damage and an primary inducer in the endogenous inflammatory response.As a primary ligand in the innate immunity,which could launch the Toll like receptor(TLRs)or advanced glycation end products(RAGE)to mediate the immune responses.HMGB1 is a highly conserved nonhistone chromosomal binding protein,which mainly distribute in the nucleus and cloud specifically bind to DNA.Under physiological conditions,HMGB1 has involved in gene transcription and gene repair.In addition to natural secretion,it also cloud be released to the outside when cell injury and death.Furthermore,it has a intense inflammatory activity,which could trigger and amplify the inflammatory response.Previous studies had shown that embryonic injury and endogenous inflammation cloud interfere the proliferation,migration and differentiation of NPCs,and it is an important initial signal when the TLRs and RAGE receptors were activated by HMGB1.Therefore,the study intends to investigate the effects of the development of the brain and behaviors during the critical period of brain development by intraperitoneal injection HMGB1,which simulates endogenous injury and activates inflammatory response.It also could provide a new endogenous mechanism to establish MCD animal model.Objective:Through activating the endogenous injury by intraperitoneal injection HMGB1 during pregnancy in vivo,and then to knowledge the optimal time and dose of HMGB1 on encephalodysplasia and abnormal behaviors.Study on the effect of HMGB1 on pathogeny structure.Study on the effect of HMGB1 on the epileptic susceptibility and electrophysiologic characteristics.Study on the effect of HMGB1 on emotional and cognitive behaviorals.Methods:1.The SD rats were respectively injected intraperitoneally with HMGB1 on the 7 days and 14 day of gestation.The dose was 1μg/2ml PBS,5μg/2ml PBS,10μg/2ml PBS,and the control group were intraperitoneally injected with 2ml PBS.2.At 90 days of postpartum,after treated with Brd U by 100mg/kg intraperitoneal injection for 3 days,used the Liu Jin-Giemsa dye and immunohistochemistry(NeuN)to observe the structure of brain tissue.The cell proliferation,migration and differentiation were detected by DCX-BrdU and Neu N-Brd U double labeled technique.With pilocarpine induced seizures(270mg/kg)to observe the epileptic susceptibility and electrophysiologic characteristics.3.At 90 days of postpartum,used the open field test,novel object recognition test and Morris water maze test(including the navigation experiment and the space exploration experiment)to study the effects of HMGB1 on the depression and anxiety,learning and memory behaviors of the offsprings.Results:1.The rats with high doses HMGB1 which were injected on the 14 days of pregnancy had showed irritability,agitation,"face like" movements and less activity.It had shown the malformation of the brain tissue,small size and weight loss;Nissl staining showed the cortical layer structure disappeared,the cells arranged disordered,the dysmorphic neurons and the hyperplastic nodules appeared.Immunohistochemistry and immunofluorescence showed the neurogenesis of NPCs decreased in dentate gyrus.Pilocarpine induced seizures in a shorter time,More severe symptoms and appeared status epilepticus in high dose group.As well as the spontaneous seizures and epileptic discharges appeared in these rats on the chronic seizures phase.2.The open field test,novel object recognition test and Morris water maze(navigation test and spatial probe test)detection the depression and anxiety,learning and memory on 90 days old.The rats with high doses HMGB1 which on the 14 days of pregnancy had shown anxiety and depression tendency.Accompany with the dose increased,anxiety and depression tendency was more obvious.In addition,these rats significantly reduced the novel object recognition,the learning and memory.With the doses increased,the result was worse.Conclusion:1.Intraperitoneal injection high dose HMGB1 during pregnancy could induce cortical structure disordered,neurogenesis decreased in the dentate gyrus,dysmorphic neurons and hyperplastic nodules appeared,and accompany with abnormal general behavior performance and increased seizure susceptibility.The results shown HMGB1 could induce the basic pathological and electrophysiological characteristics of MCD.Therefore,E14 is the suitable time and 10μg is suitable dose for this research.2.High dose of HMGB1 could induce the learning and memory deficit after intraperitoneal injection during pregnancy,and offsprings showed anxiety and depression tendency.3.The offsprings which were injected on the 14 days of pregnancy with intraperitoneal injection High dose HMGB1 could be induced the typical pathological characteristics of MCD and the cognitive deficit,increased the anxiety and depression.This is preferably MCD model by HMGB1 imitate the endogenous injury mechanism.