Yali Xu

Altered Expression of CX3CL1 in Patients with Epilepsy and in a Rat Model

Chemokine C-X3-C motif ligand 1 (CX3CL1, alias fractalkine), is highly expressed in the central nervous system and participates in inflammatory responses. Recent studies indicated that inflammatory processes within the brain constitute a common and crucial mechanism in the pathophysiological characteristics of epilepsy. This study investigated the expression pattern of CX3CL1 in epilepsy and its relationship with neuronal loss. Double immunolabeling, IHC, and immunoblotting results showed that CX3CL1 expression was up-regulated in the temporal neocortex of patients with temporal lobe epilepsy. In a rat model of epilepsy, CX3CL1 up-regulation began 6 hours after epilepsy, with relatively high expression for 60 days. In addition, ELISA revealed that the concentrations of CX3CL1 in cerebrospinal fluid and serum were higher in epileptic patients than in patients with neurosis but lower than in patients with inflammatory neurological diseases. Moreover, H&E staining demonstrated significant neuronal loss in the brains of epileptic patients and in the rat model. Finally, the expression of tumor necrosis factor-related apoptosis-inducing ligand was significantly increased in both patients and the animal model, suggesting that tumor necrosis factor-related apoptosis-inducing ligand may play a role in CX3CL1-induced cell death. Thus, our results indicate that CX3CL1 may serve as a possible biomarker of brain inflammation in epileptic patients.

Increased expression of calponin-3 in epileptic patients and experimental rats.

Calponin-3 is an actin-interacting protein and is expressed in the brain. Our previous microarray scan has found an up-regulation of calponin-3 gene CNN3 in the temporal lobe of patients with drug-resistant epilepsy. Here we investigated in epileptic patients the changes of brain and cerebrospinal fluid (CSF) calponin-3 expressions, and assessed calponin-3 expression pattern in a rat model of pilocarpine-induced epilepsy. We showed that in the temporal neocortices of 30 patients with drug-resistant epilepsy, both mRNA and protein level of calponin-3 were significantly increased. In addition, the augmentation of CSF calponin-3 from 126 epileptic patients was closely correlated with disease duration. Moreover, in the cortices of temporal lobes of pilocarpine-treated rats, calponin-3 increased along with the time and maintained at significant high levels for up to 2 months, while the up-regulation of hippocampal calponin-3 only occurred at 24h and 1 week. The elevated calponin-3 suggests that deregulation of actin filament dynamics in axonal and dendritic outgrowth and synaptic rearrangement may contribute to pathophysiology of epilepsy.

Expression pattern of Mical-1 in the temporal neocortex of patients with intractable temporal epilepsy and pilocarpine-induced rat model.

Mical‐1 is a novel F‐actin‐disassembly factor that is critical in actin reorganization. It provides a molecular conduit through which actin reorganizes‐a hallmark of cell morphological changes, including axon navigation. However, whether Mical‐1 is involved in the epileptogenesis remains unknown. Here, we investigate Mical‐1 expression pattern in patients with intractable temporal lobe epilepsy (TLE) and pilocarpine‐induced rat model. We used double‐labeled immunoflurescence, immunohistochemistry, and Western blotting to assess the location and expression of Mical‐1 in temporal neocortex of patients with intractable TLE, and the expression pattern of Mical‐1 at different time point in the hippocampus and temporal lobe cortex of the pilocarpine‐induced rat model. Double‐labeled immunofluorescence showed that Mical‐1 was coexpressed with neuron‐specific enolase (NSE) in the cytoplasm of neurons in temporal neocortex of patients with TLE and hippocampus of rat model. Faint and scattered immunoreactivity for Mical‐1 in the neuron of temporal neocortex in TLE group, but strong immunoreactivity for Mical‐1 was shown in control subjects. To quantitatively evaluate the Mical‐1 immunoreactivity, we measured the mean optical density (OD) of Mical‐1. In the hippocampus of pilocarpine‐induced rat model, the OD values transient increased at 6 h after seizure then decreased from 1 day to 14 days, and returned to a subnormal level at 60 days. The lowest level of Mical‐1 expression occurred at 14 days after seizure in the hippocampus. In the temporal lobe cortex of rat model, the OD values decreased at all time point after kindling compared to the normal group. Furthermore, our Western blot analysis confirmed these expression patterns of Mical‐1 from latent stage to chronic stage. Our results indicate that in patients with TLE and pilocarpine‐induced rat model, the expression of Mical‐1 were followed a downtrend from the latent stage to chronic stage after seizure evoke. Thus, as an effect factor participated in F‐actin disassemble, Mical‐1 may associate with inner pathophysiological modulation in epilepsy. Synapse, 2011.

Up-regulation of apelin in brain tissue of patients with epilepsy and an epileptic rat model

Prolonged epileptic seizures or SE can cause neuronal cell death. However, the exact role of neuroprotectant against brain injury during epileptic seizure needs to be further elucidated. The aim of this study was to investigate the expression of the apelin, a novel neuroprotective peptide, in brain tissues of the patients with temporal lobe epilepsy (TLE) and experimental rats using immunohistochemistry, immunofluorescence and Western blotting analysis and to discuss the possible role of apelin in TLE. Thirty temporal neocortical tissue samples from the patients with drug-refractory TLE underwent surgical therapy and nine histologically normal temporal lobes tissues as controls were used in our study. Fifty-six Sprague-Dawley rats were randomly divided into seven groups, including one control group and six groups with epilepsy induced by lithium-pilocarpine. Hippocampus and adjacent cortex were taken from the controls and epileptic rats at 1, 3, 7, 14, 30, and 60 days after onset of seizures. Apelin was mainly expressed in the neurons of TLE patients and controls, and was significantly increased in TLE patients compared with the controls. Apelin was also expressed in the neurons of experimental and control rats, it was gradually increased in the experimental rat post-seizure and reached a stable high level in chronic epileptic phase. Our results demonstrated that the increased expression of apelin in the brain may be involved in human TLE.

Up-regulation of serum- and glucocorticoid-induced protein kinase 1 in the brain tissue of human and experimental epilepsy

Several studies have shown that serum- and glucocorticoid-induced protein kinase 1(SGK1) can regulate both glutamate receptors and glutamate transporters and may participate in the regulation of neuroexcitability in neuronal diseases. In our previous study, we analyzed differential gene expression in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients using a complementary DNA microarray and found that the SGK1 gene was up-regulated more than twofold in the brain tissues of epileptic patients. In the current study, we measured SGK1 expression in the brain tissues of humans and in an experimental model of rat epilepsy in order to explore the relationship between SGK1 expression and epilepsy. The SGK1 expression was detected in thirty human brain tissues derived from patients undergoing operation for drug-refractory epilepsy and was also detected in eight samples from autopsies. Meanwhile, we investigated SGK1 expression during the epileptic process in rats using immunofluorescence, RT-PCR and western blot analysis. SGK1 expression was enhanced in the temporal neocortex of patients with drug-refractory epilepsy and was also highly expressed in the rat brain during different phases of the epileptic process. SGK1 expression was also related with the elevation of EAAT3, which expression reduced after knockdown SGK1. These results provide new insight into the potential role of SGK1 in the pathophysiology of epilepsy.

Abnormal expression of stathmin 1 in brain tissue of patients with intractable temporal lobe epilepsy and a rat model.

Microtubule dynamics have been shown to contribute to neurite outgrowth, branching, and guidance. Stathmin 1 is a potent microtubule‐destabilizing factor that is involved in the regulation of microtubule dynamics and plays an essential role in neurite elongation and synaptic plasticity. Here, we investigate the expression of stathmin 1 in the brain tissues of patients with intractable temporal lobe epilepsy (TLE) and experimental animals using immunohistochemistry, immunofluorescence and western blotting. We obtained 32 temporal neocortex tissue samples from patients with intractable TLE and 12 histologically normal temporal lobe tissues as controls. In addition, 48 Sprague Dawley rats were randomly divided into six groups, including one control group and five groups with epilepsy induced by lithium chloride‐pilocarpine. Hippocampal and temporal lobe tissues were obtained from control and epileptic rats on Days 1, 7, 14, 30, and 60 after kindling. Stathmin 1 was mainly expressed in the neuronal membrane and cytoplasm in the human controls, and its expression levels were significantly higher in patients with intractable TLE. Moreover, stathmin 1 was also expressed in the neurons of both the control and the experimental rats. Stathmin 1 expression was decreased in the experimental animals from 1 to 14 days postseizure and then significantly increased at Days 30 and 60 compared with the control group. Many protruding neuronal processes were observed in the TLE patients and in the chronic stage epileptic rats. These data suggest that stathmin 1 may participate in the abnormal network reorganization of synapses and contribute to the pathogenesis of TLE. Synapse 66:781–791, 2012.

Time-Dependent Decrease of Clusterin as a Potential Cerebrospinal Fluid Biomarker for Drug-Resistant Epilepsy

Our previous study on proteomic analysis has shown that clusterin (CLU) is significantly decreased in the cerebrospinal fluid (CSF) of patients with epilepsy. Therefore, the present study aimed to confirm CLU concentration reduction in the CSF of patients with drug-resistant epilepsy and drug-responsive epilepsy. Fifty-two patients with epilepsy (23 drug resistance and 29 drug effectivity) and 20 control individuals were recruited. The concentrations of CSF and serum CLU were detected. Moreover, alteration of CLU was detected in the rat hippocampus over time after pilocarpine-induced status epilepticus (SE). Our results showed that human CSF-CLU levels were decreased in patients with both drug-resistant epilepsy and drug-responsive epilepsy compared to controls, and concentration of CSF-CLU was obviously lower in drug-resistant epilepsy than in drug-responsive epilepsy. In the pilocarpine-induced seizure rats, expression of neuronal CLU was gradually decreased in a time-dependent manner from acute phase to chronic phase after the onset of SE. In conclusion, CLU level is decreased in the CSF of human epilepsy and the similar alteration is confirmed in a rat model with epilepsy. Therefore, CLU might contribute to the development of epilepsy and be a potential CSF biomarker for resistant epilepsy.

Increased expression of placental growth factor in patients with temporal lobe epilepsy and a rat model

Placental growth factor (PIGF) plays a role in angiogenesis and neuroprotection. It has been suggested that angiogenesis and blood-brain barrier damage are involved in the pathophysiology of epilepsy. In this study, we investigated the PIGF expression in the temporal neocortices of 11 patients with pharmaco-resistant temporal lobe epilepsy (TLE) and 6 non-epileptic controls, using double immunofluorescence labeling, immunohistochemistry and Western blotting. We also assessed PIGF expression pattern in a rat model of TLE induced by lithium chloride-pilocarpine. We found that PIGF expression was significantly elevated in patients with TLE than in control. TLE patients with initial injuries had significantly higher PIGF level than those without initial injuries. In the TLE rat model, PIGF upregulation started at 6h after status epilepticus and maintained at significant high level for up to 60 days. These results suggest that the augmentation of brain PIGF is associated with development of epilepsy.

Upregulation of dysbindin in temporal lobe epileptic foci of human and experimental animals.

The gene encoding dystrobrevin‐binding‐protein‐1 (dysbindin) is expressed in many areas of the central nervous system and plays a role in intracellular vesicle trafficking, synaptic vesicle trafficking, and neurotransmitter release. At a cellular level, dysbindin is thought to mediate presynaptic glutamatergic transmission. Using Western blotting and immunofluorescence, we investigated dysbindin expression in brain tissues of the patients with temporal lobe epilepsy (TLE) and rats with TLE (lithium chloride pilocarpine model) to explore its possible role in epileptogenesis. Twenty‐five samples of temporal neocortex from patients undergoing surgery for drug‐refractory TLE epilepsy and 10 histologically normal temporal lobes tissues from control subjects were used in our study. We also examined dysbindin expression in the hippocampus and adjacent cortex from experimental Sprague–Dawley rats. Dysbindin was expressed in the cytoplasm of neurons from epileptic specimens, and levels of dysbindin proteins were significantly increased in patients with TLE. Dysbindin was also expressed in the neurons of the hippocampus and adjacent cortex from experimental and control rats. Western blotting of rat brain tissue showed that dysbindin was upregulated gradually from 6 h after kindling. Maximal expression was seen around 2 months in chronic epileptic phase. These results demonstrated that the increased expression of dysbindin might play a role in the pathogenesis of drug‐refractory TLE. Synapse, 2012.

Altered Norbin Expression in Patients with Epilepsy and a Rat Model

Norbin is widely distributed in neuronal tissues, is a regulator of Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation. Norbin is also an important endogenous modulator of metabotropic glutamate receptor 5 (mGluR5) signaling, and nervous system-specific homozygous gene disruptions, result in epileptic seizures. In this study, we aimed to investigate norbin expression patterns in epilepsy and to elucidate the relationships between norbin and mGluR5 and p-CaMKII in epilepsy. Double-immunolabeling, immunohistochemistry and immunoblotting studies showed that norbin was downregulated in the temporal neocortex of patients with temporal lobe epilepsy (TLE) compared with control subjects. Moreover, in a rat model of lithium chloride-pilocarpine-induced epilepsy, norbin expression began to decrease at 6 h after the onset of status epilepticus and remained at a low level until 60 days. In addition, p-CaMKII expression was significantly increased in both patients with TLE and in animal model. Norbin and mGluR5 were found to be co-expressed in neurons of epileptic tissues. Finally, norbin over-expression facilitated by injections of adeno-associated viral vector into the rat hippocampus increased latency and survival in the lithium chloride-pilocarpine model. Thus, our results indicate norbin participates in the pathogenesis of epilepsy, perhaps by modulating mGluR5 signaling, regulating CaMKII phosphorylation, and may exert antiepileptic effects.