周围神经损伤
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Figure 1｜Differentiation and MCSA of postnatal and fetal ENCCs on days 2 and 5.
Both postnatal and fetal ENCCs proliferated rapidly and formed neurosphere-like bodies on day 2 (Figure 1A and B) and free-floating neurospheres on day 5 (Figure 1C and D). However, the MCSA of neurospheres derived from fetal ENCCs was larger than those from postnatal ENCCs (P = 0.0045; Figure 1E).
Figure 2｜Differentiation, apoptosis and migration analysis of the postnatal and fetal ENCCs in vitro.
Differentiation potency of fetal ENCCs is similar to that of postnatal ENCCs
During culture in differentiation medium, neurospheres derived from postnatal ENCCs adhered gradually and showed neurite outgrowth and radial migration at day 7 (Figure 2A). Most postnatal ENCCs extended axon-like projections (Figure 2B) and differentiated into neuron- or glial-like cells (Figure 2C and D). Given the similar differentiation potential of fetal and postnatal ENCCs, we did not evaluate fetal ENCC differentiation in this study. Further analysis revealed no significant difference in the number of peripherin/DAPI-positive cells per field between fetal and postnatal ENCCs (P = 0.32; Figure 2E). These results suggest that the neuronal differentiation potential of ENCCs is similar in the embryonic to postnatal stages.
Fetal ENCCs have better migration ability than postnatal ENCCs
Cell migration was evaluated using Hanging Cell Culture Inserts. The fetal (Figure 2F) and postnatal ENCCs (Figure 2G) had migrated through the membrane to the lower surface. The number of migrating fetal ENCCs was significantly greater than the number of migrating postnatal ENCCs (P < 0.0001; Figure 2H). These results suggest that fetal and postnatal ENCCs have different migratory abilities.
The number of apoptotic fetal ENCCs is less than the number of postnatal ENCCs
To analyze the apoptosis of fetal and postnatal ENCCs, we conducted flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide staining. The number of apoptotic fetal ENCCs was significantly less than the number of apoptotic postnatal ENCCs (P = 0.0002; Figure 2I–K).Figure 3｜The morphological characteristics of the Hirschsprung’s disease rat model.
Figure 4｜Effects of fetal and postnatal ENCC transplantation on ganglionic cell numbers and distal intestinal pressure change in the Hirschsprung’s disease rat model.
Rats in all groups survived during the experimental period. We compared hematoxylin and eosin staining between the control (Figure 3A) and the other intervention groups (Figure 3B), and found constricted segments as well as dilatation and stool retention proximal to the treated bowel (Figure 3C). On day 84, autopsy of rats in the postnatal ENCCs group revealed dilatation of the colonic gut proximal to the treated bowel and adhesion between the BAC-treated intestinal segment and surrounding tissues (Figure 3C). Quantitative analysis revealed more enhanced green fluorescent protein/peripherin double-positive cells per ganglia in the fetal ENCCs group compared with the postnatal ENCCs group on days 56 and 84 (Figure 4A–E).
Distal intestinal pressure change is greater in HSCR rats treated with fetal ENCCs compared with those treated with postnatal ENCCs
In the control group, rats displayed more reflexive contractions or pressure changes when the proximal colon was stimulated. In comparison, rats in the other groups showed less reflexive contractions and decreased intraluminal pressure changes. The pressure in the fetal ENCCs group was significantly higher than that in the postnatal ENCCs group (P = 0.0056; Figure 4F).