The Neuroprotective Effects Of CNTF On Hippocampal Neurons Against L-NMDA Via Unknown Transduction Pathway

The neuro-protective effects of CNTF on hippocampal neurons via unknown transduction pathway were observed and its mechanism was investigated. Damaged hippocampal neurons, which induced by ionotrophic glutamate receptor agonist L-NMDA, were used as damaged model. JAK/STATs inhibitor PTPi-2 was used to block the classical transduction pathway of CNTF. By primary cultured hippocampal neurons,running photograph of living cells, count number of living cells, detection of intracellular free Ca~2+ and immunohistochemical detection of gpl30, the main lesults are as follows:1. L-NMDA (100μmol/L, 200μmol/L, 400μmol/L, 500μmol/L) could damaged neurons. This damaged effect was dose-dependent when the process went on l~2h and was time-dependent when the concentration of L-NMDA arranged from 0μmol/L to 500μmol/L.2. CNTF could decrease damaged effects on hippocampal neurons induced by L-NMDA.2.1 Running photograph of living cell. CNTF (1μg/μ1) was administrated to every group, 5min later, administrated L-NMDA (200μmol/ L), there was no significant changes in cell bodies.2.2 Count number of living cell. The number of living cells in L-NMDA group was significantly fewer than that in normal group (P<0.01). Compared with normal group, there was no significant difference between the number of livingcells in CNTF and CNTF+L-NMDA group(P>0.05). The results suggested that CNTF could decrease damaged effects on hippocampal neurons induced by L-NMDA.3. The protective effects of CNTF on hippocampal neurons still existed after JAK/STATs was blocked by PTPi-2. This result intensively supported the conjecture that there was an unknown pathway on the upstream of already known signal transduction pathway, where the neuro-protective effects could take place.3.1 Running photograph of living cells. PTPi-2 (lOQug/L) was incubated to primarily cultured neurons for 30min. After CNTF(l/jg//A) was administrated for 5min, L-NMDA(20QMmol/L) was administrated. There was no significant change in hippocampal was observed after the neurons were carried out under the same condition for 2h.3.2 Count number of living cells. The number of living cells in L-NMDA group was significantly fewer than in normal group (P<0. 01). Compared with normal group, there was no significant difference between the number of living cells in CNTF + L-NMDA, PTPi-2(100//g/L) + CNTF(l^ul) + L-NMDA (200/anol/L) , CNTF(Wy?l) + L-NMDA(20Qumol/L) and PTPi-2(100/￡/L) + CNTF(l/4g//A) + L-NMDA(20Qumol/L) group. The results suggested that the unknown transduction pathway has the effect on suppressing hippocampal neurons damaging induced by L-NMDA.4. Detection of intracellular free [Ca2+]i. [Ca2+]i was increased within 10s after L-NMDA (20Qamol/L) was administrated to hippocampal neurons. CNTF could significantly suppress the intracellular [Ca2+]i content induced by L-NMDA in the neurons. There was no significant effect on [Ca2+]i increasing induced by L-NMDA after the hippocampul neurons incubated with PTPi-2 (100,ug/L,for 30min) and CNTF(l/^/il for 5min),the high level of [Ca2+]i induced by L-NMDA was suppressed.This result suggested that when JAK/STATs pathway was blocked,CNTF played neuron-protective effects via other signal transduction pathway,which was on upstream of JAK/STATs.5. Immunohistochemical detection of gpl30. Hippocampal neurons of L-NMDA...