| Malformations of cortical development(MCD) have been considered major causes of intractable epilepsy clinically among young patients. Nowadays, more and more new diagnostic techniques were developed clinically, especially the emergence of high-resolution MRI, more and more MCD patients are diagnosed. MCD characterized by cortical dyslamination and the presence of cytomegalic neurons, such as dysmorphic neurons(DN) and balloon cells(BC), and is a well-recognized cause of intractable epilepsy. Focal cortical dysplasia(FCD) and tuberous sclerosis complexes(TSC) are two major subtypes of MCD. Previous studies have indicated that cortical lesions in FCD are possible epileptogenic foci; however, the exact molecular mechanisms underlying the pathogenesis of these cortical lesions remain largely unknown. Purinergic ligand-gated ion channel 7 receptor(P2X7R) has been considered as a potential target for intractable epilepsy.However, the expression of P2X7 R has not been well characterized in human epilepsy, and little is known about the expression of this receptor in non-temporal lobe epilepsies. In the present study, we analyzed the expression and cellular distribution of P2X7 R and its downstream target IL-1β in surgically resected cortical lesions of patients with FCD and TSC. We then compared these expression levels with samples from normal control cortex(CTX) that was obtained from autopsies to elucidate the contribution of P2X7 R to the pathogenesis and/or progression of human FCD and TSC. And methylazoxymethanol(MAM) was administered intraperitoneally by injecting to rats who were pregnant at the 15 th gestational day to produce animal MCD model and P2X7 R, IL-1β and Caspase-1 expressions were detected. Main results are listed as follows:1. The cellular distribution and expression of P2X7 R in FCD1.1 Both Quantitative real-time PCR analysis and Western blot analyses were performed to quantify the expression of P2X7 R in the total homogenates of the neocortex from the CTX, FCD I, and FCD II specimens. P2X7 R mRNA expression was significantly increased in FCD specimens compared with the CTX specimens. Additionally, P2X7 R mRNA levels were significantly higher in the FCD II specimens than in the FCD I specimens. The P2X7 R protein expression levels within the cortical lesions of the FCD specimens were increased compared with the expression levels in the CTX samples. Representative Immunoblots of P2X7 R in total homogenates from the FCD I and FCD II cortical lesions displayed a denser band than did the homogenates from CTX tissues. Statistical analysis indicated that the levels of P2X7 R protein in the FCD I specimens were significantly higher than those in the CTX and that the protein expression levels from the FCDII specimens were significantly higher than those in the FCD I specimens.1.2 Immunohistochemistry and immunofluorescence showed that weak P2X7 R expression was observed in gray matter(GM) neurons and in glia-like cells in the white matter(WM) in the control CTX. In the FCD Ia specimens, moderate to strong P2X7 R expression was encountered throughout the microcolumns in the GM and junction area and in the HNs, as well as the glia-like cells in the junction area. In addition, strong P2X7 R immunoreactivity was observed in the glia-like cells and the malformed cells(MCs), which included DN in the FCD II specimens. Confocal images in FCD IIa and IIb specimens displayed a co-expression of P2X7 R with both the glial fibrillary acidic protein(GFAP) in astrocytes and NeuN. This confirmed that P2X7 R was expressed in both neurons and astrocytes. Double-labeling experiments also confirmed that the majority of P2X7R-positive DN and BC in the FCD II specimens had a neuronal lineage(NeuN positive; GFAP negative). Additionally, P2X7 R was also co-expressed with human leukocyte antigen DR(HLA-DR) in microglia.1.3 Quantitative real-time PCR analysis and Western blot analyses were also performed to quantify the expression of P2X7 R in the total homogenates of the neocortex from the TSC specimens. Results were showed that P2X7 R mRNA expression was significantly increased in TSC specimens compared with the CTX specimens. Additionally, P2X7 R protein levels were significantly higher in the TSC specimens than in the CTX samples. Immunohistochemistry and immunofluorescence showed that strong P2X7 R expression was observed in DN in the sample of TSC. And strong P2X7 R immunoreactivity was observed in the MCs. Confocal images in TSC specimens displayed a co-expression of P2X7 R with GFAP in astrocytes and NF200 in neuron.2. The cellular distribution and expression of IL-1β and IL-1R1 in FCD2.1 We further investigated the expression of IL-1β, which is considered to be the master regulator of neuroinflammation and has been identified as a key mediator of apoptosis in neuronal cells. We observed a diffuse distribution of IL-1β throughout the dysplastic regions in most of the FCD specimens. A moderate to strong level of IL-1β expression was shown in the microcolumns in the GM, and a strong level of IL-1β expression was detected in the BC and DN in the cortical lesions of FCDII specimens. Double-labeled immunofluorescence image of FCD specimens indicated that IL-1β co-expression with NF200 in most DN, GFAP in some astrocytes, and HLA in some microglia, confirming that IL-1β is expressed in microglia.2.2 Both Quantitative real-time PCR analysis and Western blot analyses were performed to quantify the expression of IL-1β, IL-1R1 and Caspase-1 in the total homogenates of the neocortex from the CTX, FCD I, and FCD II specimens. All of IL-1β, IL-1R1 and Caspase-1 mRNA expression was significantly increased in FCD specimens compared with the CTX specimens. Additionally, their mRNA levels were significantly higher in the FCD II specimens than in the FCD I specimens. The protein expressions were in accordance with the expressions of mRNA. Statistical analysis indicated that the protein levels of IL-1β, IL-1R1 and Caspase-1 in the FCD I specimens were significantly higher than those in the CTX and that the protein expression levels from the FCDII specimens were significantly higher than those in the FCD I specimens.3. The cellular distribution and expression of P2X7 R in methylazoxymethanol acetate(MAM) animal model3.1 MAM intraperitoneal injection was prenatally administered to pregnant rats at gestational day 15 to induce developmental brain dysgeneses. As a result, the offsprings showed microcephaly, reduced autonomic activities and, showed a slow response to the outside stimulation. And the flexibility of crawling, avoidance and feeding ability were all decreased. The MAM animal model showed a lighter weight compared the normal rats. The main pathological changes included abnormalities of extensive cortical layers, there were more heterotopic neurons appeared in layer I, and subcortical nodules including heterotopic neurons could be seen from the hippocampus to periventricular region and neocortex. And normal polarity was disappeared as well as neuronal morphology was abnormal, the neuronal cell body was increased. All the above abnormal morphology could be seen. And in the molecular layer, numerous ectopic neurons could be seen, and a mass like aggregation and visible pathological nodules appeared.3.2 Both Quantitative real-time PCR analysis and Western blot analyses were performed to quantify the expression of P2X7 R in the total homogenates of the neocortex from the MAM animal model and control rats. P2X7 R mRNA expression was significantly increased in MAM specimens compared with the CTX specimens. Additionally, Caspase-1、IL-1β mRNA and protein levels were significantly higher in the MAM specimens than in the control specimens. Immunohistochemistry and immunofluorescence showed that weak to moderate P2X7 R expression was observed in normal rat brain and only less P2X7 R were observed in neuron-like cells and weak P2X7 R expression was observed in glial cells. Strong IL-1β immunoreactivity was observed MAM compared to control.In summary, we have provided evidence that supports increased P2X7 R expression in human patients with FCD, and together with previously animal studies, we assume that the up-regulation of P2X7R/Caspase-1/IL-1β pathway involves in the pathogenesis of FCD. |
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