Immunomodulator On The Effects And Related Mechanisms In Seizure

Epilepsy is a kind of chronic diseases which is due to neurons of a sudden,intermittent epileptiform discharges lead to the short-term recurrent brain dysfunction. There are extensive contacts between the nervous system and the immune system. Since Besedovsky proposed the immune - nerve - endocrine regulation network theory, many research suggested that epilepsy associated with the disbalance of the immune - nerve - endocrine regulation network, particularly the relationship between immune and epilepsy had a new light in recent years. Cytokines are not only inherent immune response and the adaptive immune response major participant, but also shared information molecules between the immune system and the nervous system. Some animals research showed that IL-1, IL-2 and TNF-αare connected with seizures. Microglia and astrocytes have been identified as the source of CNS-specific inflammatory cytokines. Astrocytes can secreted a variety of cytokines (IL-1, TNF-α)by itself.Those cytokines are involved in the occurrence, development and prognosis of epilepsy. And microglial cells as the major source of the key pro-inflammatory cytokines (IL-1, TNF-α) and the immune regulatory cytokines (IL-12, IL-18) which are closely related to the occurrence and development of epilepsy. This preliminary study found that immunosuppressant glucocorticoids or cyclosporine have a certain effect on cytokine-induced epilepsy, but whether the role of regulation on the traditional-induced epileptic seizure are still rarely reported. Therefore, in order to fully explore the role of immune regulation on effects of epilepsy activities induced by pentylenetetrazol (PTZ), we use immunosuppressant dexamethasone (DEX) and levamisole immunostimulants levamisole (LMS) intervene PTZ-induced epilepsy, and study the effects on the related mechanisms of immuneregulator intervention on the epilepsy induced by PTZ from the neuron, astrocyte, microglia three aspects. Experiment is divided into two parts.PART ONE:EFFECTS OF IMMUNOLOREGULATION ON IMMUNOREACTION OF GLU AND PKC IN NEURON AND MICROGLIA OF RATS WITH EPILEPSYGlu was one of the major excitatory amino acid in the central nervous system. Excessive Glu can lead to excitability of nerve cells remarkably increase and cytotoxic damage. Thus Glu is closely related to the incidence of epilepsy. PKC was a kind of Ca2+, phospholipid-dependent protein kinase and played an important role in the central nervous system transmembrane signal transduction. It also participated in the signal transduction pathway in epilepsy. So in the first part of this study, we used immunosuppressant dexamethasone and immunoenhancer levamisole intervening epilepsy induced by PTZ. Methods of immunocytochemisty, cell culture and image analysis technology were applied to study the expression of Glu and PKC in neurons of rats brain and cultured microglias. Experiment was divided into two parts include in vivo and in vitro. Experimental rats in vivo were randomly divided into four groups, each group had 10 animals(:1)control group, treated with normal saline by intraperitoneal injection. (2)PTZ group, treated with pentylenetetrazol(60mg/kg/bw) by intraperitoneal injection. (3)DEX+PTZ group, pretreatment with the dexamethason(e5mg/kg/bw)injected intraperitoneally 30 min prior to the pentylenetetrazol(60mg/kg/bw)by intraperitoneal injection.(4) LMS+PTZ group, levamisole(25mg/kg/bw)by intragastric administration for 5 days before pentylenetetrazol(60mg/kg/bw)by intraperitoneal injection. The seizures were classified according to the Diehl scoreing system stage 0～V. In vitro: First screening the time point of most obvious response in microglia treatment by PTZ 2h, 6h, 12h. And then the purification cultured microglias in the most obvious time point were divided into four groups, namely the control group (no factors added), pentylenetetrazole group(final concentration of PTZ is 0.5mmol/L), levamisole + pentylenetetrazol group (final concentration of LMS is 0.5μg/ml and final concentration of PTZ is 0.5mmol/L), dexamethasone + pentylenetetrazol group (final concentration of DEX is 10-4 mol/L and final concentration of PTZ is 0.5 mmol/L) . Results: 1. The animal behavior: NS group displayed no seizures, whereas PTZ group showed stageⅢ～Ⅳ, DEX+PTZ group stageⅠ～Ⅱand LMS+PTZ group stageⅤ. 2. Results of immunocytochemistry staining and image analysis in vivo: the neurons in all layers of the cerebral cortex and the hippocampal pyramidal cells and granular cells all had the Glu and PKC positive staining in four groups. Glu immunoreactive positive cells are cytoplasm stained with a brownish yellow color. PKC immunoreactive positive cells showed that the membrane received the dark staining as buffy color, the plasma light staining. The average optical density of Glu and PKC positive cells had the same trend: the expressions of Glu and PKC in PTZ group were higher comparing with NS group, and the expressions were further enhanced in LMS+PTZ group, while the DEX+PTZ group is lower than both PTZ group and LMS+PTZ group. There are significant differences in the dense of Glu and PKC immunoreactivities among four groups (P<0.05). 3. Immunocytochemical staining and image analysis in microglias showed that microglias in normal control group received mycobacterium appearance and the small cell body. Glu and PKC immunoreactive in microglia increased at 2h following pentylenetetrazol treatment, while the cell morphology changes of microglia are processes retraction and cell body enlarged. The most obvious change of microglia at 6h, cell processes retracted even a round. The expression of Glu and PKC in PTZ group were higher comparing with NS group,the immunoenhancer enhanced the expression, while the immunodepressant attenuated the expression of Glu and PKC. These findings suggested that the onset statement of epilepsy might be affected by enhancing or suppressing an immune condition of organism through the expression of Glu and PKC in neurons and microglias.PART TWO :EFFECTS OF IMMUNOLOREGULATION ON IMMUNOREACTION OF NFκBp65 AND NMDAR1 IN NEURON AND ASTROCYTE OF RATS WITH EPILEPSYNFκB is a multi-polar gene regulation protein,which most common dimer is heterologous dimer of p50/RelA(p65), and its expression and nuclear translocation can be used as activated signs of neurons and astrocytes. NMDAR is the ion receptor of glutamate, which transfer excitatory synaptic activities and play an important role in the pathogenesis of epilepsy. So in the second part of this study, we use NFκBp65 and NMDAR1 as observation index. Methods of behavioral observation, immunocytochemisty, cell culture and image analysis technology were applied to study the expression of NFκBp65 and NMDAR1 in neurons of rats brain and cultured astrocytes which interfered by immunoregulant before treating with PTZ. 1.The experimental rats in vivo were randomly divided into four groups, each group had 8 animals:(1)control group, treated with normal saline by intraperitoneal injection. ( 2 ) PTZ group, treated with pentylenetetrazol(60mg/kg/bw) by intraperitoneal injection. (3)DEX+PTZ group, pretreatment with the dexamethasone(5mg/kg/bw)injected intraperitoneally 30 min prior to the pentylenetetrazol( 60mg/kg/bw ) by intraperitoneal injection. ( 4 ) LMS+PTZ group, levamisole( 25mg/kg/bw ) by intragastric administration for 5 days before pentylenetetrazol(60mg/kg/bw)by intraperitoneal injection. The seizures were classified according to the Diehl scoreing system stage 0～V. 2. Astrocytes were cultured and purified according to the methods of McCarthy. And then the purification cultured astrocytes in the most obvious time point were divided into four groups, namely the control group (no factors added), pentylenetetrazole group(final concentration of PTZ is 0.5mmol/L), levamisole + pentylenetetrazol group (final concentration of LMS is 0.5μg/ml and final concentration of PTZ is 0.5 mmol/L), dexamethasone + Pentylenetetrazol group (final concentration of DEX is 10-4 mol/L and final concentration of PTZ is 0.5 mmol/L) . Results: 1. The animal behavior is the same with first part. 2. Results of immunocytochemistry staining and image analysis in vivo: the neurons in all layers of the cerebral cortex and the hippocampal pyramidal cells and granular cells all had the NFκBp65 and NMDAR1 positive staining in four groups. NFκBp65 positive cells are cytoplasm or nucle stained with a brownish yellow color. NMDAR1 immunoreactive positive cells are membrane stained. The average optical density and image analysis of NFκBp65 and NMDAR1 immunoreactive cells in hippocampus and cerebral cortex of rats all groups revealed that the OD values of NFκBp65 and NMDAR1 positive cells had the same trend: the expressions of NFκBp65 and NMDAR1 in PTZ group were higher comparing with NS group, and the expressions were further enhanced in LMS+PTZ group, while the DEX+PTZ group is lower than both PTZ group and LMS+PTZ group. There are significant differences in the dense of NFκBp65 and NMDAR1 immunoreactivities among four groups (P<0.05). 3. Immunocytochemical staining and image analysis in astrocytes showed that the positive product of NMDAR1 was brown and the expression of NMDAR1 increased at 2h following pentylenetetrazol treatment, peaked at 6h, and then gradually attenuated at 12h. The positive product of NFκBp65 in control group was a brown granular in the cytoplasm. The expression of NFκBp65 began at 2h following pentylenetetrazol treatment, the average optical density of NFκBp65 immunoreactive peaked at 6h and had a significant difference compared with the control group (P<0.05). The immunoreactive of NFκBp65 and NMDAR1 in DEX + PTZ group was weaker than in PTZ group, but little active astrocyte still occurred in DEX + PTZ group. The immunoreactive of NFκBp65 and NMDAR1 in LMS+PTZ group is stronger than PTZ group, while astrocytes are further actived and the volume enlarged obviously. These results indicated that immunoenhancer or immunodepressant can respectively up-regulate or down-regulate the activation of NFκBp65 and the expression of NMDAR1 in neuros and astrocytes to interfere in the occurrence and development of epilepsy.Concolusion: Our experments studied the the immune regulator intervention on the epilepsy induced by PTZ from the neuron, microglia and astrocyte three aspects. We found that immunoenhancer or immunodepressant can respectively up-regulate or down-regulate the expression of NMDAR1, Glu and PKC and the activation of NFκBp65 in neurons of cerebral cortex and hippocampus, while influence the expression of Glu and PKC in microglias and the activation of NFκBp65 and the expression of NMDAR1 in astrocytes. The abvoe results showed that the two-way regulation existed in the nervous system and immune system, the changes of immune condition can affected the epilepsy state through the nuclear factor, excitatory neurotransmitter, messenger molecules in neurons, microglias and astrocytes. This study provided a new experimental data for epilepsy and immune - nerve - endocrine regulation network and a new thought for the prevention and treatment of epilepsy in clinical.