Objective To investigate the influence of different transplantating times on the survival and immigration of the bone marrow mesenchymal stem cells (BMSCs) in injured spinal cord by subarachnoid administration, and to evaluate the most optimal subarachnoid administration times for BMSCs. Methods Eight adult male rats (weighing 120 g) were used to isolate BMSCs that were cultured, purified and labeled with Hoechst 33342 in vitro. Another 75 adult Wistar rats (weighing 220 g) were made the spinal cord injury (SCI) models at T9,10 level according to the improved Allen’s method and were randomly divided into 5 groups (groups A, B, C, D, and E, n=15). The labeled BMSCs at 1 × 107/mL 0.1 mL were injected into subarachnoid space of the rats via a catheters under the subarachnoid space in groups A (one time at 1 week), B ( two times at 1 and 3 weeks), C (3 times at 1, 3, and 5 weeks) and D (5 times at 1, 3, 5, 7, and 9 weeks) and 0.2 mL phosphate-buffered sal ine (PBS) was injected in group E (5 times at 1, 3, 5, 7, and 9 weeks) as blank control. The neurological functions were evaluated using the Basso-Beattie-Bresnahan (BBB) scale 1, 3, 5, 7, 9, and 12 weeks after transplantation. The migration, survival, differentiation, and histomorphological changes of BMSCs were observed by HE, immunohistochemistry, and fluorescence microscopy. Results At 3 weeks after injury, there were significant differences in the BBB scores between group E and groups A, B, C, D (P lt; 0.01), and between groups A, B and groups C, D (P lt; 0.01). At 7, 9, and 12 weeks, the BBB scores were significantly higher in groups C and D than in groups A and B (P lt; 0.01), and in group B than in group A (P lt; 0.01). There were no significant differences in the BBB scores between groups C and D (P gt; 0.05). The fluorescence microscopy showed that the transplanted BMSCs survived and grew in the injured region at 3 weeks after injury and as time went on, the transplanted cells gradually decreased in group A; in groups B, C, and D, BMSCs count reached the peak values at 5 and 7 weeks and then gradually decreased. At 12 weeks, the survival BMSCs were significantly more in groups C and D than in groups A and B (P lt; 0.01). HE staining showed that the formation of cavity was observed in each group at 3 weeks after injury and the area of cavity gradually decreased in groups A, B, C, and D. At 12 weeks, the area of cavity was the miximal in groups C and D, moderate in groups A and B, and the maximal in group E. The immunohistochemistry staining indicated that the expression of NF-200 was more intense in groups C and D than in groups A and B. The expression of NF-200-positive fibers was more intense in group C. Conclusion Multiple administration of BMSCs promotes the restoration of injured spinal cord and improves neurological functions, and three times for BMSCs transplantation is best
Objective To investigate the mechanism of adenosine-tri phosphate (ATP) activated mammal ian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signal pathway in the physiology and pathology of spinal cord injury (SCI). Methods Ninety-six adult healthy female Sprague-Dawley rats were randomly divided into 4 groups (groups A, B, C and D, n=24). In groups A, B and C, the rats were made the SCI models at T8-10 levels by using a modified Allen’ s stall, and in group D, rats were given laminectomy without SCI. The rats were subjected to the administration of ATP (40 mg/kg) for 7 days in group A, to the administration of physiological sal ine (equal-volume) for 7 days in group B, to the administration of ATP (40 mg/kg) and rapamycin (3 mg/kg) for 7 days in group C, and to the administration of physiological sal ine (equal-volume) for 7 days in group D. Locomotor activity was evaluated using the Basso-Beattie-Bresnahan rating scale at the postoperative 1st, 2nd, 3rd, and 4th weeks. Then, the expressions of spinal cord cell marker [Nestin, neuron-specific enolase (NSE), gl ial fibrillary acidic protein (GFAP)] and the mTOR/STAT3 pathway factors (mTOR, STAT3) were detected at the postoperative 1st, 2nd, 3rd, and 4th weeks by immunohistochemistry analysis, Western blot assay, and real-time fluorescence PCR analysis. Results The BBB scores in group A showed a steady increase in the postoperative 1st-4th weeks and were significantly higher than those in groups B and C (P lt; 0.01), but were lower than that in group D (P lt; 0.01). Real-time fluorescence PCR results showed that the mRNA expressions of mTOR, STAT3, NSE of group A steadily increased, however, the Nestin mRNA expression gradually decreased in the postoperative 1st-4th weeks, which were all significantly higher than those of groups B, C, and D (P lt; 0.01). The mRNA expression of GFAP showed a steady increase in group A and was significantly less than those of groups B and C, but was higher than that of group D (P lt; 0.01). There were significant differences (Plt; 0.01) in all markers between groups B, C, and group D; there were significant differences in mTOR, P-mTOR, STAT3, and P-STAT3 mRNA between groups B and C at 1st-4th weeks (P lt; 0.05). The similar changes were found by Western blot assay. Conclusion ATP can activate the mTOR/STAT3 pathway to induce endogenic NSCs to prol iferate and differentiate into neurons in rats, it enhances the heal ing of SCI.
ObjectiveTo measure anatomical parameters related to cervical uncovertebral joint and provide data support for the design of uncovertebral joint fusion cage.MethodsAccording to the inclusion and exclusion criteria, raw DICOM data of cervical CT scan in 60 patients (30 males and 30 females, aged 39-60 years) were obtained, then the three-dimensional cervical spine model was reconstructed for anatomical measurement by using the Mimics19.0 software. The height of the uncinate process, the length of the uncinate process, the width of the uncinate process, and the length of the uncovertebral joint in the intervertebral foramen region were measured bilaterally from C3 to C7. The anterior and posterior distances between the uncinate processes were measured from C3 to C7. The height of the uncovertebral joint space, the central height of the intervertebral disc space, and the depth of the intervertebral disc space were also measured from C2, 3 to C6, 7. The mean, standard deviation, maximum, and minimum were calculated by using the SPSS22.0 statistical software for the design of uncovertebral joint fusion cage.ResultsThe height of the uncinate process, the length of the uncinate process, the width of the uncinate process, and the length of the uncovertebral joint in the intervertebral foramen region of C3-C7 and the height of the uncovertebral joint space of C2, 3-C6, 7 showed no significant difference between two sides (P>0.05). The height of the uncovertebral joint space also had no significant difference between females and males (P>0.05). The anterior distances between the uncinate processes of C3-C7 were significantly larger than the posterior distances between the uncinate processes (P<0.05), the uncovertebral joint presented a posterior cohesive shape. The central height of the intervertebral disc space in male group was slightly higher than that in female group, and the differences were significant (P<0.05) at C2, 3 and C5, 6; the depth of the intervertebral disc space in male group was significantly higher than that in female group (P<0.05). The central height of the intervertebral disc space was (4.94±0.49) mm (range, 3.81-5.90 mm), the depth of the intervertebral disc space was (15.78±1.23) mm (range, 12.94-18.85 mm), the anterior and posterior distances between the uncinate processes were (17.19±2.39) mm (range, 13.39-24.63 mm) and (10.84±2.12) mm (range, 7.19-16.64 mm), respectively. According to the results of the anatomical research, the height of the uncovertebral joint fusion cage was designed as 5, 6, 7, and 8 mm; the depth of the uncovertebral joint fusion cage was designed as 12, 13, 14, 15, and 16 mm; the width of the uncovertebral joint fusion cage was designed as 14-18 mm; and the two wings are designed as arc-shape with 2 and 3 mm in width.ConclusionThere are certain differences in the anatomical parameters of the uncovertebral joint between different segments. The uncovertebral joint fusion cage that designed based on the results of anatomical research is suitable for most patients.