To study the effects of stereotaxic transplantation of induced neural stem cells (iNSCs) receiving traumatic brain injury (TBI) mouse serum pre-treatmenton complement activation following TBI.

Healthy adult male C57BL/6 mouse TBI models were established using a standardized weight-drop device. Mice having an neurological severity scores (NSS) of 4-8 were enrolled in TBI group (n=48). Twenty-four TBI mice were randomly selected for preparation of TBI mouse serum and heat-inactivated TBI mouse serum, andthe other 24 mice were randomly divided into 4 groups: the TBI-iNSC group (n=6), the HITBI-iNSC group (n=6), the phosphate buffered solution (PBS)-iNSC group (n=6) and the Control group (n=6). Sham-operated mice underwent the same procedures, but not head trauma (n=6). At 12 h after TBI, TBI-iNSCs, HITBI-iNSCs, PBS-iNSCsor PBS were separately injected into the brains of TBI mice via stereotactic method. On day 14 after TBI, animals were sacrificed for molecular-biological andmorphological analysis. We performed western blot to analyze the expression of the C3d, C9, Crry and active Caspase-3 in brain tissues. Next, immunofluorescent stainingand terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining were utilized to determine the effects of TBI-iNSC, HITBI-iNSC and PBS-iNSC grafts on NeuN⁺ and TUNEL⁺ cells in the brains of TBI mice.

Western blot analysis revealed that the expression of C3d, C9 and active Caspase-3 in the brains of the Sham group was significantly lower than that in PBS-iNSC, HITBI-iNSC, TBI-iNSC and Control groups (P<0.05). In contrast, the expression of C3d, C9 and active Caspase-3 in the brains of the Control group was significantly higher than that in PBS-iNSC, HITBI-iNSC and TBI-iNSC groups (P<0.05). Moreover, the levels of C3d, C9 and active Caspase-3 in the brain were substantially higher in the PBS-iNSC and HITBI-iNSC groups than in the TBI-iNSC group (P<0.05). Additionally, the expression of Crry in the brain of the Control group was significantly lower than that in the other four groups (P<0.05). Furthermore, the levels of Crry in the brain tissues of mice in the PBS-iNSC and HITBI-iNSC groups were significantly higher than in the Sham group but substantially lower than those in the TBI-iNSC group (P<0.05). Immunofluorescent stainingand TUNEL staining showed that there were no significant differences in the levels of NeuN⁺/TUNEL⁺ cells between the PBS-iNSC and HITBI-iNSC groups. However, the levels of NeuN⁺/TUNEL⁺ cells in the TBI-iNSC group were markedly lower than that in PBS-iNSC and HITBI-iNSC groups (P<0.05).

Stereotaxic transplantation of iNSC sreceiving TBI mouse serum pre-treatmentcould increase Crry expression, inhibit complement activation and reduce brain damage following TBI.