Expression Of HHV6、HPV16 And HSV In Human Focal Cortical Dysplasia

BACKGROUND AND PURPOSE:Malformations of cortical development(MCDs),which is also called cortical dysplasia, cortical malformations[1], is a set of abnormal lesions of focal or diffuse cortical structure[2]and can often causes a variety of clinical manifestations, like epilepsy, developmental delays, local neurological deficits and mental retardation. Abnormal cortical formation, ion channel disease(channelopathies) often causes abnormal neurons potential formation, imbalance of stimulation and inhibition in neurons and lead to toxic effects of excitement, which is an important pathophysiological mechanism of seizures. MCDs accounted for 24% of pathogenesis in epilepsy, and that is about 40% in drug-resistant epilepsy, it is the main cause of refractory epilepsy.Focal cortical dysplasia(FCD), which was first described in details by Ta ylor and his colleagues 1971[3], is an important subtype of MCDs. FCDs often result in medically intractable epilepsy, in fact, it’s the most common cortical malformation encountered in epilepsy surgery[4]. FCD mainly affects the brain cortex and characterized by abnormal cortical lamination and Cell abnormity, based on these characteristics it was divided into three types(FCDI,FCDII, FCDIII), FCD subtype II is the most important one, which characterized by dysmorphic neurons(FCDIIa) and balloon cells(FCDIIb). FCD has a close relationship with refractory epilepsy [4].As the most important cause of refractory epilepsy in children, the specific pathogenesis of FCD is still unclear. At present, there are reports demonstrate that disruption of the mammalian target of rapamycin(m TOR) signaling comprises a common pathway underlying the structural disturbances of FCD. Other pathogenesises, for example, gene mutation or external environment induced damage(such as viral infection, ischemia, or anoxia) in the embryonic period was considered to be the main pathogenic cause of FCD[5]. The role of gene mutation has been confirmed in clinical specimens and animal models[5,6].External environment–induced injury, such as teratogenic chemical agents and trauma, has also been tested in animals[6,7]. However, viral infection in FCD has not been well documented.Researches show that viral infection and the inflammation it induced are important causes of epilepsy and viral infection can lead to clinical seizures. epilepsy model was established based on viral infection. At the same time, viral infection in embryonic stage and the inflammatory response it induced can activate the pathways of NF?B and m TOR signaling, which can influence the proliferation, migration and differentiation of neural stem cells, resulting in abnormal brain development and FCD formation. Experiments show that several common virus-susceptible groups in embryo stage including Herpes simplex virus(HSV), Human papillomavirus16(HPV16) and Human herpesvirus6(HHV6), their detection rates in the brain tissue of epileptic patients are significantly higher than that of normal. Which further support that viral infection in embryonic stage may be an important cause of FCD and epilepsy. But viral infection in FCD was not confirmed by surgical removal specimen yet. Our project will explore the expression of the above virus in FCD and explore the relationship between viral infection in embryo stage and FCD related epilepsy, thus provide new thoughts for the treatment of FCD related epilepsy in clinic.METHODS:1. Collecting brain tissues samples from different patients(including FCDIIa, FCDIIb and control);2. Extracting DNA from the above patients’ brain tissues, using Nested-PCR todetect HSV1, HSV2, HHV6 A, HHV6 B, HPV16 in the specimens.3. Selecting the positive results of Nested-PCR as experimental group and normal brain tissue as control group, using immunohistochemical and immunofluorescence methods to verify the results of Nested-PCR and observe the morphological distribution of viral infection.4. Using Western Blotting method to further verify the results before.5. Extracting RNA in specimens with two or more viral infection as the experimental group, RNA extracting from normal cerebral cortex as control group. Using Real-time q PCR to detect the expression level of TLR3 and TRIF, which are the receptor and primary signaling molecules in the immunoreaction induced by viral infection.RESULTS:1. Collecting experimental samples 37 cases and control samples 22 cases successfully. According to the clinical and pathological classification standards, in the experimental group: FCDIIa20 cases, FCDIIb17 cases; In the control group: trauma 8 cases, cerebral hemorrhage 4 cases, and 10 cases of normal tissues surrounding other tumors.2. By Nested-PCR, the results of HHV6 A are listed as follows: the infection rate of FCDII group was 20/37(54.1%), FCDIIa was 9/20(45.0%), FCDIIb was 11/17(64.7%), and control group was 2/22(9.1%)(Significant differences compared with the FCD group, P<0.05); The results of HHV6 B are listed as follows: the infection rate of FCDII group was 25/37(67.6%), FCDIIa was 14/20(70.0%), FCDIIb was 11/17(64.7%), and control group was 5/22(22.7%)(Significant differences compared with the FCD group, P<0.05); Gene sequencing, immunohistochemistry and Western Blot tests confirmed the existence of HHV6 A and HHV6 B and their expression products.3. By Nested-PCR, the results of HPV16 are listed as follows: the infection rate of FCDII group was 18/37(48.6%), FCDIIa was 9/20(45.0%), FCDIIb was 9/17(52.9%), and control group was 4/22(18.2%)(Significant differences compared with the FCD group, P<0.05); Immunohistochemistry and Western Blot tests confirmed the existence of HPV16 and its expression products.4. By Nested-PCR, the results of HSV1 are listed as follows: the infection rate of FCDII group was 33/37(89.2%), FCDIIa was18/20(90.0%), FCDIIb was 15/17(88.2%), and control group was 5/22(22.7%)(Significant differences compared with the FCD group, P<0.05); The results of HSV2 are listed as follows: the infection rate of FCDII group was 30/37(81.1%), FCDIIa was 16/20(80.0%), FCDIIb was 14/17(82.4%), and control group was 2/22(9.1%)(Significant differences compared with the FCD group, P<0.05); Immunohistochemistry and Western Blot tests confirmed the existence of HSV1 and HSV2 and their expression products.5. In the positive specimens selected, the expression level of TLR3 and TRIF, which are the primary signaling molecules in the immunoreaction induced by viral infection, are significantly increased than that in normal control group(TLR3:t=39.220, P=0.000;TRIF:t=22.226,p=0.000).CONCLUSIONS:1. The infection rates of HHV6 A, HHV6 B, HPV16, HSV1 and HSV2 in FCDII group are all higher than that of control group, which indicate a close relationship between embryonic period viral infection and FCD, and viral infection may be an important cause of FCD and epilepsy.2. Obvious TLR3-TRIF inflammatory signaling pathway activation was observed in viral infection specimens of FCD, suggest viral activated inflammation response in FCD, which may be an important cause of FCD related epilepsy.