| Objective To explore the effects of neural stem cells (NSC) and olfactory ensheathing cells (OEC) co-transplantation on the neurons of primary motor cortex and the locomotor functions in the rats subjected to spinal cord transection (SCT). And elucidate the underlying molecular mechanisms.Method The cells culture, Immunohistochemistry (IHC), Terminal deoxynucleotidyl transferase mediated dUTP Nick end labeling (TUNEL), Western-blot, Reverse Transcription polymerase Chain Reaction (RT-PCR) and Behavioral test (BBB locomotor score and Motor evoked potential) were employed to test the hindlimb locomotor functions, the surviving or dying of the neurons of primary motor cortex , the expression of apoptosis associated factors and the molecular signal transduction molecular in the rats subjected to SCT and received NSC and OEC co-transplantation.Result1. Rats subjected to SCT at spinal level T9, which hindlimbs showed flaccid paralysis, indicating that the transection of the cord was complete. There was spontaneous, howbeit incomplete, recovery of the hindlimb locomotor functions after SCT. The BBB score of operation group was significant lower than sham operation group at all of the different time points (P<0.05). At 4 weeks after SCT, transcranial magnetic motor-evoked potentials (MEP) were not recorded in 80% experiment rats. Whereas all of the sham operation rats had the successful MEP records. At 2 weeks after transection, the TUNEL staining showed the operation group rats had more positive staining cells in the V layer of cortex than the sham operation group rats (P<0.05). Immunohistochemical staining revealed that the operation group rats expressed specific neuronal marker (Neun) in the V layer of cortex was significantly less than sham operation group rats at 4 weeks post operation (P<0.05). And the quantitative analysis demonstrated that the number of Bax and Caspase-3 immunoreactive products were more in the operation group rats than sham operation group rats in the V layer of cortex (P<0.05). Whereas the Bcl-2 immunoreactive products have no statistical difference between the operation group and sham operation group (P>0.05). The result of RT-PCR showed as follows: Compared with sham-operation group, the expression of Bcl-2 mRNA in the primary motor cortex decreased significantly in operation group at 14 and 21 days post operation (dpo), while a significant increase on the expression of Caspase-3 mRNA was found at 3dpo in the operation group rats (P<0.05). The expression of Bax, however, had no statistical difference between two groups (P>0.05).2. The rats received NSC and OEC co-transplantation had a better recovery of hindlimb locomotor functions than operation group rats from 3 weeks after transplantation (P<0.05). All of the rats received cell transplantation had a significant improvement of BBB locomotor score. The rats received NSC and OEC co-transplantation had the highest BBB score among three cell transplantation groups at 3 weeks after operation (P<0.05) . At 4 weeks post operation, NSC and OEC co-transplantation group rats had higher BBB score than NSC transplantation group rats (P<0.05). The BBB score of NSC transplantation group and OEC transplantation group was higher than operation group at 4 weeks post operation, respectively (P<0.05). At 4 weeks post transplantation, all of the rats received NSC and OEC co-transplantation had the successful MEP records. However, the amplitudes were lower than sham operation group (P<0.01). There was only one half rats had the MEP records in NSC transplantation group and OEC transplantation group. Both had lower amplitudes than sham operation group rats (P<0.01). The latencies of OEC transplantation group rats were the longest among all the experimental rats (P<0.05). Two weeks after cell transplantation, the TUNEL staining revealed the operation group rats had the most numbers of TUNEL positive staining cells in the V layer of motor cortex among all the experimental animals (P<0.05). The TUNEL staining cells of NSC and OEC co-transplantation rats were less than OEC transplantation group rats (P<0.05). Four weeks following cell transplantation, the statistical result showed the sham operation group rats had the most number of NeuN immunoreactivity in the V layer of motor cortex among all of the experiment rats except for NSC and OEC co-transplantation group (P<0.05). There was, however, no statistical difference of NeuN immunoreactive between sham operation group and co-transplantation group (P>0.05). Which suggested NSC and OEC co-transplantation have the potential to rescue the neurons of primary motor cortex. Significantly, the number of the Bcl-2 immunoreactive products in NSC and OEC co-transplantation group were the most among all of the experimental group rats (P<0.05). In the V layer of motor cortex, all of the cell transplantation groups rats had less Bax immunoreactive products than the operation group rats (P<0.05). NSC and OEC co-transplantation group and OEC transplantation group had less Caspase-3 immunoreactive products than operation group (P<0.05). Moreover, NSC and OEC co-transplantation group had the least number of Caspase-3 immunoreactive products among three cell transplantation groups (P<0.05). The result of RT-PCR showed that the expression of Bax mRNA in co-transplantation group was increased compared with NSC transplantation group, whereas decreased compared with OEC transplantation group at 3dpo (P<0.05). The expression of Bcl-2 mRNA in the NSC and OEC co-transplantation group was increased at 14dpo and 21dpo compared with OEC transplantation group and increased at 21 dpo compared with operation group (P<0.05). Compared with operation group, the expression of Caspase-3 in the motor cortex of NSC and OEC co-transplantation group rats were decreased at 3 and 7dpo (P<0.05). And lower than NSC transplantation group at 14dpo (P<0.05).3.The result of RT-PCR showed that the expression of PKB mRNA in the motor cortex of NSC transplantation group rats was lower than co-transplantation group, OEC transplantation group and operation group at 3 dpo (P<0.05). At the same time point, operation group was lower than sham operation group (P<0.05). At 14dpo, the expression of PKB mRNA in NSC transplantation group was reversed and was higher than OEC transplantation group (P<0.05). NSC and OEC co-transplantation group rats had the highest expression of PKB mRNA among three cell transplantation groups (P<0.05). And OEC transplantation group was lower than operation group and sham operation group (P<0.05) .There was no statistical difference in all of the experimental rats at 7 dpo and 28 dpo (P>0.05). Conversely, the expression of ERK1 mRNA was higher in the motor cortex of sham operation group and operation group than three cell transplantation groups (i.e. NSC transplantation group, OEC transplantation group and co-transplantation group) at all of the time points (P<0.01). Moreover, the expression of ERK mRNA was the highest in the motor cortex of NSC transplantation group among three cell transplantation groups at 3dpo (P<0.01). At 3dpo, the expression of Stat-3 mRNA in the motor cortex of co-transplantation group and OEC transplantation group was higher than operation group and sham operation group (P<0.05) . At 14 dpo, operation group was less than sham operation group (P <0.01). And each cell transplantation groups was higher than operation group, respectively (P<0.05) . At 21 dpo, the expression of Stat-3 mRNA of every cell transplantation group was less than sham operation group (P<0.05). NSC transplantation group showed a significant upregulation at 28 dpo compared with sham operation group and NSC and OEC co-transplantation group (P <0.05) . At 14 dpo, Western-blot analysis showed that the expression of Akt non-phosphorylated protein and ERK 1/2 phosphorylated protein in the motor cortex of NSC and OEC co-transplantation group was increased compared with operation group and sham-operation group (P<0.05). And the expression of Akt non-phosphorylated protein in NSC transplantation group was higher than operation group (P<0.05). Compared with sham operation group, operation group and NSC transplantation group, there was a significant upregulation of Stat-3 non-phosphorylated protein in the motor cortex of co-transplantation group (P<0.05). The result also showed there was no difference on the expressions of Akt phosphorylated protein, ERK1/2 non-phosphorylated protein and Stat-3 phosphorylated protein in all of the experiment animals (P>0.05).4. Immunocytochemical analysis showed that there was more NeuN immunoreactivity neurospheres and less Nestin-positive cells in the co-culture medium than NSC single culture medium at 7 days (P<0.05). There was, however, no statistical difference in the numbers of GFAP-positive cells and APC-positive cells between co-culture and single culture medium (P>0.05). Which suggested OEC are capable of promoting NSC differentiate into neurons.Conclusion1. There was spontaneous, howbeit very limited, recovery of the hindlimb locomotor functions after SCT. It is urgent to find an optional and effective measure to improve the motor functions after spinal cord injury. The neurons of motor cortex in the rats suffered to apoptosis and death after SCT. The abnormal expression of apoptosis genes in the motor cortex would be responsible for the result, which was triggered by SCT. Therefore, it is prerequisite to play light on the changes of cerebral cortex after SCI.2. NSC and OEC as two types of 'seed' cells. Both of them single or combinated engrafted into the rats subjected to SCT could effectively promote the recovery of the hindlimb locomotor functions. Whereas, co-transplantation had the better effects than single transplantation.3. The underlying mechanism of NSC and OEC co-transplantation maybe as follows: The first, OEC have the potential to promote NSC profileration and differentiate into neurons. The second, co-transplantation prevented the neurons of motor cortex from apoptosis in the rats subjected to SCT. The third, NSC and OEC co-transplantation promoted the expression of Bcl-2 mRNA and protein level. And the effect was associated with the activated of ERK signal transduction molecular. |
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