Integral Absorbance Research Articles

Research on Polycaprolactone-Gelatin Composite Scaffolds Carrying Nerve Growth Factor for the Repair of Spinal Cord Injury.

Objective This study was to investigate the mechanism of action of polycaprolactone/gelatin (PCL/GE) composite fiber scaffold with nerve growth factor (NGF) in the recovery of spinal cord injury (SCI). Methods Sixty female Sprague-Dawley (SD) rats were randomly assigned to the negative control group, the positive control group, the PCL/GE scaffold group, and the collagen-binding structural domain nerve growth factor (CBD-NGF)/PCL/GE scaffold group, with 15 rats in each group. Spinal cord transection was used to establish SCI models in rats. The negative control group received sham surgery, while the other three groups were given spinal cord transection at the tenth thoracic vertebra (T10) segment. The rats in the PCL/GE scaffold group were implanted with a 4 mm PCL/GE composite fiber scaffold, and those in the CBD-NGF/PCL/GE scaffold group were implanted with a CBD-NGF/PCL/GE composite fiber scaffold. The Basso–Beattie–Bresnahan (BBB) locomotor rating scale was used to evaluate the locomotor ability of the hind limbs of the rats, and the amplitude and latency of motor evoked potentials (MEP) were recorded by neurophysiological testing at 12 w postoperatively. The levels of growth-associated protein 43 (GAP43) and neurofilament protein 200 (NF200) in the spinal cord tissue of the injury site were determined using Western Blot at 12 w after surgery. Spinal cord tissues of 2 cm within the injury site, the thoracic segment above the injury site, and the lumbar segment below the injury site were collected from the measurement of axonal transport using fluorescent retrograde tracer fluorogold, and the integrated absorbance (IA) values of FC-positive cells were calculated. Results After treatment, the negative control rats showed normal locomotion function of the hind limb with the highest BBB scores, while the positive control rats had the lowest BBB scores and showed paraplegia. The scaffold groups exhibited better locomotion function of the hind limb and higher BBB scores than the positive controls, with greater improvement observed in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Compared with the positive controls, the PCL/GE scaffold group and CBD-NGF/PCL/GE scaffold group exhibited significantly shorter latency and increased amplitude of MEP, with more significant changes observed in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Compared with the positive control group, the GAP43 and NF200 levels of spinal cord tissue were significantly elevated in both the PCL/GE scaffold group and the CBD-NGF/PCL/GE scaffold group, and the changes were more pronounced in the CBD-NGF/PCL/GE scaffold group (P < 0.05). The differences in the IA values of FC-positive cells in the spinal cord tissue of the lumbar segment below the injury site among the four groups did not come up to the statistical standard (P > 0.05). Compared with the positive control group, the FC-positive cell IA values of spinal cord tissue in the thoracic segment above the injury area were markedly increased in the PCL/GE scaffold group and the CBD-NGF/PCL/GE scaffold group, and the alterations were more significant in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Conclusion PCL/GE composite fiber scaffold with NGF significantly improves motor and neurological functions in the hind limbs of SCI rats and promotes the recovery of axonal transport, and the mechanism may be associated with the upregulation of GAP43 and NF200 levels in spinal cord injury site tissues.

Anti-tumor and analgesic activity evaluation and mechanism of Compound Kushen Injection

This study aims to evaluate the anti-tumor and analgesic activities of Compound Kushen Injection(CKI) based on zebrafish model in vivo and investigate the anti-tumor mechanism. To be specific, zebrafish tumor xenotransplantation model was established by microinjection of murine LPC H12 cells into yolk sac. Then the high-dose CKI(H-CKI), medium-dose CKI(M-CKI), low-dose CKI(L-CKI) groups, and the model group were set. The anti-tumor activity of CKI was evaluated with the tumor area growth fold and integral absorbance(IA) growth fold 72 h after administration. The peripheral pain and central pain in zebrafish were respectively induced with acetic acid(AA) and phorbol myristate acetate(PMA). Zebralab ViewPoint system was employed to monitor behavioral trajectory of zebrafish, and movement times, movement time, movement distance, and movement velocity were used to evaluate the analgesic activity of CKI. Finally, real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression levels of apoptosis-related B lymphocyte tumor-2(Bcl-2) and phosphatidylinositol-3-kinase(PI3 K)/protein kinase B(Akt or PKB) pathway-related genes, for the verification of the anti-tumor mechanism. Compared with the model group, M-CKI and H-CKI significantly reduced the growth folds of tumor area and IA, relief the peripheral pain and central pain. The mechanism was that CKI can up-regulate the expression of cysteine aspartic acid specific protease-3(caspase-3, Casp3) and caspase-9(Casp9), down-regulate the expression of phosphoinositide 3-kinase(PI3 K) and Akt, and significantly reduce the expression of Bcl-2, hypoxia-inducible factor-1α(HIF-1α), and vascular endothelial growth factor(VEGF). In conclusion, CKI has significant inhibitory effect on tumor growth and pain, which is related to the PI3 K/Akt signaling pathway. The pathway mediates cell apoptosis, suppresses tumor growth, and alleviates tumor pain.

Recovery integral absorbance method in the full concentration range to eliminate the interference of background gas

At present, gas sensors are extremely susceptible to interference from background gases in the field environment, which leads to greatly reduced accuracy. For this reason, we propose an improved method of recovering integral absorbance (IA) using Y component of first harmonic to achieve accurate prediction of the full range of concentration (not reaching absorption saturation). This approach can eliminate the interference of background gas at a low modulation depth (m < 0.25). When the background gas is pure nitrogen and a mixture of nitrogen and carbon dioxide, the recovery effect of this method on methane is both close to the theoretical value when the background gas is air. The linear fitting coefficients for the methane concentration range of 2000–7000 ppm are all greater than 0.999. The prediction effect is satisfactory regardless of the background gas, with a relative error of less than 1%. In summary, this method has considerable application prospects.

Expression of HuD protein and P2Y2 receptor in the rectum of children with middle-high anorectal malformation

Objective To observe the expression of HuD protein and P2Y2 receptor in the normal colon and the terminal rectum of children who suffered from anorectic malformation (ARM) in the middle and upper rank, and to explore the effects of HuD protein and P2Y2 receptor on the development of the enteric nervous system. Methods The terminal rectum specimens were collected from 25 children with ARM (malformation group) treated by surgerical procedures in affiliated hospital of Zunyi Medical college. In the control group, there were 15 cases. By hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), immunofluorescence and Western blotting, the expression of the two proteins was detected. Results Immunohistochemistry and immunofluorescence showed that the HuD protein and P2Y2 receptor mainly existed in intestinal myenteric plexus and submucosal plexus. The integral absorbance (IA) values of HuD protein and P2Y2 receptor in control group and malformation groups were: 43.48±7.11, 3.70±1.21 and 18.83±3.65, 8.49±2.68; IA values of HuD protein and P2Y2 receptor in the two groups were: 96.56±17.38, 22.32±4.36 and 78.13±7.86, 26.56±5.32; relative quantification of HuD protein and P2Y2 receptor in two groups were 1.13±0.32, 0.49±0.12 and 0.92±0.18, 0.36±0.09 (P=0.030, 0.030). Conclusion The expression of HuD protein and P2Y2 receptor in children with ARM in the middle and upper rank was significantly lower than that in normal colon. HuD protein and P2Y2 receptor have some influence on the development of enteric nervous system in children with ARM. Key words: Anorectic malformation; Enteric nervous system; HuD protein; P2Y2 receptor

Promotion of transplanted collagen scaffolds combined with brain-derived neurotrophic factor for axonal regeneration and motor function recovery in rats after transected spinal cord injury

To evaluate the effect of the combination of collagen scaffold and brain-derived neurotrophic factor (BDNF) on the repair of transected spinal cord injury in rats. Thirty-two Sprague-Dawley rats were randomly divided into 4 groups: group A (sham operation group), T 9, T 10 segments of the spinal cord was only exposed; group B, 4-mm T 9, T 10 segments of the spinal cord were resected; group C, 4-mm T 9, T 10 segments of the spinal cord were resected and linear ordered collagen scaffolds (LOCS) with corresponding length was transplanted into lesion site; group D, 4-mm T 9, T 10 segments of the spinal cord were resected and LOCS with collagen binding domain (CBD)-BDNF was transplanted into lesion site. During 3 months after operation, Basso-Beattie-Bresnahan (BBB) locomotor score assessment was performed for each rat once a week. At 3 months after operation, electrophysiological test of motor evoked potential (MEP) was performed for rats in each group. Subsequently, retrograde tracing was performed for each rat by injection of fluorogold (FG) at the L 2 spinal cord below the injury level. One week later, brains and spinal cord tissues of rats were collected. Morphological observation was performed to spinal cord tissues after dehydration. The thoracic spinal cords including lesion area were collected and sliced horizontally. Thoracic spinal cords 1 cm above lesion area and lumbar spinal cords 1 cm below lesion area were collected and sliced coronally. Coronal spinal cord tissue sections were observed by the laser confocal scanning microscope and calculated the integral absorbance ( IA) value of FG-positive cells. Horizontal tissue sections of thoracic spinal cord underwent immunofluorescence staining to observe the building of transected spinal cord injury model, axonal regeneration in damaged area, and synapse formation of regenerated axons. During 3 months after operation, the BBB scores of groups B, C, and D were significantly lower than those of group A ( P<0.05). The BBB scores of group D at 2-12 weeks after operation were significantly higher than those of groups B and C ( P<0.05). Electrophysiological tests revealed that there was no MEP in group B; the latencies of MEP in groups C and D were significantly longer than that in group A ( P<0.05), and in group C than in group D ( P<0.05). Morphological observation of spinal cord tissues showed that the injured area of the spinal cord in group B extended to both two ends, and the lesion site was severely damaged. The morphologies of spinal cord tissues in groups C and D recovered well, and the morphology in group D was closer to normal tissue. Results of retrograde tracing showed that the gray matters of lumbar spinal cords below the lesion area in each group were filled with FG-positive cells; in thoracic spinal cords above lesion sites, the IA value of FG-positive cells in coronal section of spinal cord in group A was significantly larger than those in groups B, C, and D ( P<0.05), and in groups C and D than in group B ( P<0.05), but no significant difference was found between groups C and D ( P>0.05). Immunofluorescence staining results of spinal cord tissue sections selected from dorsal to ventral spinal cord showed transected injured areas of spinal cords which were significantly different from normal tissues. The numbers of NF-positive axons in lesion center of group A were significantly larger than those of groups B, C, and D ( P<0.05), and in groups C and D than in group B ( P<0.05), and in group D than in group C ( P<0.05). The combined therapeutic approach containing LOCS and CBD-BDNF can promote axonal regeneration and recovery of hind limb motor function after transected spinal cord injury in rats.

Effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells

To investigate the effect of FTY720-P on the differentiation and maturation of MC3T3-E1 cells. The MC3T3-E1 cells were divided into the experimental group and the control group. In the experimental group, the cells were induced by the medium containing 400 ng/mL FTY720-P (chloroform as solubilizer) in vitro. In the control group, the cells were cultured with the medium only containing chloroform. The cell morphology of 2 groups were observed by inverted phase contrast microscope; the expression of osteoblast related protein (collagen type Ⅰ and collagen type Ⅲ) was detected by immunofluorescence staining; the alkaline phosphatase (ALP) staining and alizarin red staining were used to observe the formation of osteoblasts and the formation of mineralized nodules in 2 groups; and the TUNEL fluorescence assay was used to detect the cell apoptosis. After 48 hours of culture, the cells of 2 groups had grown into slender fusiform at the bottom of the bottle, and there was no significant difference in cell morphology between 2 groups. Immunofluorescence staining showed that the expression of collagen type Ⅰ was positive in the experimental group and weakly positive in the control group; the integrated absorbance ( IA) value of the experimental group was 187 600±7 944, which was significantly higher than that of the control group (14 230±1 070) ( t=43.680, P=0.001). The expression of collagen type Ⅲ was weakly positive in the experimental group and the control group, and there was no significant difference in IA value between 2 groups ( t=1.976, P=0.119). ALP staining and alizarin red staining were positive in the experimental group and negative in the control group. TUNEL staining was positive in the experimental group and negative in the control group; the rate of TUNEL staining positive cells in the experimental group was 35.82%±2.99%, which was significantly higher than that in the control group (2.28%±0.51%) ( t=23.420, P=0.002). FTY720-P can promote the osteogenic differentiation of MC3T3-E1 cells with speeding up maturation and mineralization of extracellular matrix and affect the apoptosis of the cells.

Correlation analysis between sperm-associated antigen 9 and epithelial-mesenchymal transition, extracelluar matrix-associated protein in prostate cancer

Objective To study correlation between sperm-associated antigen 9 (SPAG9) and epithelial-mesenchymal transition (EMT), extracelluar matrix (ECM)-associated protein in prostate cancer. Methods The immunohistochermical analysis was used to detect the expression of SPAG9, E-cadherin, Vimentin and matrix metalloproteinase-2 (MMP-2) in 60 specimens of prostatic carcinoma (PCa) and 30 specimens of benign prostate hyperplasia (BPH). Statistical analyses were used to evaluate the relationship between SPAG9 and E-cadherin, Vimentin, MMP-2. Results The immunohistochermical analysis showed the average integral absorbance (IA) of each protein in metastatic PCa, nonmetastatic PCa and BPH as follows: for SPAG9 (466.34±13.52, 284.98±25.69 and 106.01±13.58, the differences were all statistically significant, P=0.000); for Vimentin (285.79±15.85, 184.20±21.31 and 79.56±7.96, the differences were all statistically significant, P=0.000); for MMP-2 (269.97±15.74, 175.82±21.81 and 87.86±17.61, the differences were all statistically significant, P=0.000). The IA values of these proteins were reduced successively in the three groups; however, the IA values of E-cadherin (148.62±28.31, 286.73±18.73 and 417.73±12.90, the differences were all statistically significant, P=0.000) were increased successively in the three groups. Pairwise comparisons of each protein’s IA within the three groups revealed the statistically significant differences. There was a positive correlation between SPAG9 and Vimentin (rs=0.443, P=0.014), MMP-2 (rs=0.443, P=0.001) while negative correlation was existed between SPAG9 and E-cadherin (rs=-0.431, P=0.017). Conclusion SPAG9 may promote the occurrence and development of PCa by regulating EMT, ECM-associated proteins. Key words: Sperm-associated antigen 9; Epithelial-mesenchymal transition; Extracelluar matrix; Prostatic carcinoma

Expressions of interleukin-21 and interleukin-22 in Kimura's disease

To investigate the expressions of interleukin (IL)-21 (IL-21) and IL-22 in patients with Kimura's disease (KD). Expressions of IL-21 and IL-22 were examined immunohistochemically in 36 patients with KD and 7 normal controls. The integral absorbance (IA) of the two groups was compared. Meanwhile, clinical data were reviewed. The IA of IL-21 [M(Q): 1 373 418 (1 800 926)] and IL-22 [M(Q): 462 086(484 672)] in KD was significantly higher than those in normal controls [M(Q): 70 445(44 658), 51 599(71 241), P < 0.05]. The overexpression of IL-21 was significantly associated with pruritus (Z = -1.993, P < 0.05). Moreover, IL-21 was identified for disease recurrence (Z = -2.303, P < 0.05). There was a significant association between the expression of IL-22 and the number of affected sites (Z = -1.979, P < 0.05). In addition, IL-22 was significantly higher in the high-eosinophils group than in the low-eosinophils group (Z = -2.025, P < 0.05). There was no association between IL-21, IL-22 and age, gender, laterality, maximum size. IL-21 and IL-22 may be involved in the pathogenesis of KD.

Effect of mycophenolate mofetilglial on glial scar formation into the hippocampus and learning and memory functions of the rats with diffuse axonal injury

Objective To determine the intervention of mycophenolate mofetil(MMF) in glial scar formation and learning and memory function in a rat model of diffuse axonal injury(DAI). Methods Ninety-six SD rats were randomly divided into sham group, normal saline (NS) group and mycophenolate mofetil (MMF) group according to the random number table, with 32 rats per group. Immunohistochemistry was used to detect activated microglia cells, activated astrocytes and chondroitin sulphate proteoglycanns (CSPGs) in the hippocampus. Image-Pro Plus software was used to quantitatively assess the changes of activated microglia cells, activated astrocytes and CSPGs. Morris water maze was applied for testing rat learning and memory function. Integrated absorbance (IA) of major constituents (microglia, astrosyte, chondroitin sulphate proteoglycan) of the glial scar was determined and analyzed for the correlation with the parameters of MWM. Results At 7, 14 and 28 days after injury, MMF group showed decreased IA of activated microglia in the hippocampus compared to sham and NS groups (P<0. 05). At 7-11 days after injury, percent distance and percent time in the target quadrant of Morris water maze did not differ significantly among the three groups and were not related to the IA of glial scar. At 28-32 days after injury, percent distance and percent time in the target quadrant of Morris water maze lowered significantly in MMF group. At 28 days after injury, IA of the glial scar had a positive correlation with mean speed and mean escape latency, but negative correlation with percent distance and time in the target quadrant that measured in Morris water maze at 28-32 days after injury. Conclusion MMF significantly attenuates glial scar formation into the hippocampus and improves learning and memory function in rats during the recovery stage when administered in the early stage after DAI. Key words: Diffuse axonal injury; Hippocampus; Mycophenolate mofetil

Effects of hypoxia preconditioning on inflammatory responses and apoptosis in the rat model after spinal cord injury

Objective To observe the effects of hypoxia preconditioning on inflammatory cytokines and apoptosis after spinal cord injury in the rat model.Methods 154 adult female wistar rats were randomly divided into three groups,including the spinal cord injury group,the spinal cord injury ± hypoxia precondition (HP) group,the sham operation group.Immunohistochemistry was performed to determine the expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)in spinal cord injury area at five time points (2,6,12 h,1,3 d).BBB score was used to evaluate the function recovery of hind limb.The method of immunohistochemistry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) were adopted to detect the level of B cell lymphoma/leukemia-2 (bcl-2) protein and the apoptotic cells at five time points (6 h,1,3,7,14 d)after spinal cord injury.Results The integral absorbance (IA) of TNF-α in HP group was decreased at 6 h and 12 h,compared with that in spinal cord injury group (5.187±0.628 vs.7.962±2.185; 3.219±0.724 vs.5.361 ±1.246,P＜0.05).The IA ofIL-1β in HP group was lower than spinal cord injury group at three time points (6,12 h,1 d) (3.845 ± 1.669 vs.6.674 ± 1.537 ; 6.241 ± 1.142 vs.9.961 ± 2.814 ; 2.264 ± 1.476 vs.4.418 ± 0.973,P ＜ 0.05).In HP group,the IA of bcl-2 protein was significantly higher than spinal cord injury group at any time point (4.335 ±0.563 vs.3.256 ± 1.024,P ＜0.05; 6.873 ±2.032 vs.5.029 ± 1.537,P ＜0.01 ; 11.869 ± 2.560 vs.6.893 ± 2.164,P ＜ 0.01 ; 10.340 ± 1.469 vs.8.074 ± 2.175,P ＜ 0.05 ; 8.143 ± 2.376 vs.5.238 ± 1.467,P ＜0.05).The apoptosis index (AI) of HP group was decreased on 1 d,3 d,7 d,14 d (20.63 ±5.86 vs.26.37 ±4.76,P＜0.05; 29.85 ±8.12 vs.37.96 ±3.81,P＜0.05; 31.56 ±4.01 vs.41.74 ±6.69,P ＜0.01 ; 22.66 ±5.24 vs.32.87 ±9.90,P ＜0.05),compared with the spinal cord injury group.The BBB scores of HP group was higher than spinal cord injury group on 7 d and 14 d (5.68 ±1.83 vs.3.06 ±1.21; 10.21 ±2.64 vs.6.45 ±1.97,P＜0.05).Conclusion Hypoxiapreconditioning can inhibit the inflammatory response of local tissue damage and reduce apoptosis through the high expression of bcl-2 protein,thus promote the neural functional recovery after spinal cord injury. Key words: Hypoxia precondition ; Spinal cord injury; Inflammation ; Apoptosis

Repair of transversal spinal cord injury in rats by porous silk fibroin scaffolds seeded with bone mesenchymal stem cells

Objective To investigate the therapeutic effects of porous silk fibroin scaffolds (PSFSs) seeded with bone mesenchymal stem cells (BMSCs) on the repair of transversal spinal cord injury (SCI) in rats.Methods Ninety-six SD rats were randomly divided into control group,PSFSs group,BMSCs group and PSFSs plus BMSCs group (24 each).The function repair was evaluated by Basso-Beattie-Bresnahan (BBB) score system at lst,2nd and 4th week.Immunohistochemistry and Western blotting were applied to detect the expression of growth associated protein 43 (GAP-43) and myelin basic protein (MBP) in the injured spinal cord.The intensity of GAP-43 expression was measured by immuno-fluorescence staining.Results At 4th week after injury,the implanted groups exhibited a more significant improvement in hindlimb locomotor function than in control group in BBB scores (P ＜0.05).The BBB sores in control group,PSFSs group,BMSCs group and PSFSs plus BMSCs group were 5.23 ± 1.13,7.17 ±1.11,7.35 ± 1.59 and 9.43 ± 2.16 respectively.Immunohistochemistry showed that the integral absorbance (IA) values of GAP-43 and MBP in implanted groups were significantly higher than those in control group (P ＜ 0.05).In control group,PSFSs group,BMSCs group and PSFSs plus BMSCs group,the IA values of GAP-43 were 13.94 ±3.63,26.07 ±5.31,22.14 ±4.56 and 33.03 ±7.58,and those of MBP were 13.80 ± 3.41,21.16 ± 3.36,32.96 ± 7.74 and 44.06 ± 7.99,respectively.The immuno-fluorescence staining indicated that there were more GAP-43 positive growth cones at the lesion site in PSFSs plus BMSCs group.Western blotting results showed that there was significant difference in the expression of GAP-43 and MBP proteins between control group and implanted groups (P ＜ 0.05).In control group,PSFSs group,BMSCs group and PSFSs plus BMSCs group,the gray value ratio of GAP-432 was 0.16 ± 0.04,0.41 ±0.05,0.33 ±0.06 and 0.72 ±0.16,and that of MBP was 0.96 ±0.11,1.85 ±0.08,2.14 ±0.16 and 2.43 ± 0.31,respectively.Conclusion Transplantation of PSFSs seeded with BMSCs can promote the axonal regeneration,myelination and functional recovery after SCI in rats. Key words: Spinal cord injury; Bone mesenchymal stem cells; Silk fibroin; Transplantation

Protective effect on rat retinal ganglion cells and the safety of intravitreal injected acteoside

Objective To observe the protective effect on retinal ganglion cells (RGC) and the safety of intravitreal injected acteoside in rats.Methods A total of 50 male Sprague Dawley rats with the weight of 190-210 g were used in this study.Fifteen rats were used for safety experiment of intravitreal injection of acteoside.The rats were divided into group A,B,C,control group and blank group,three rats in each group.The rats in group A,B and C were received intravitreal injection of 5 μl acteoside at the concentration of 1,2,and 5 mg/ml,respectively.Phosphate buffer solution (PBS) was injected in rats of control group.No treatment was performed for blank group.The retinal structure was examined by hematoxylin-eosin (HE) staining of retinal frozen sections at one,two and three weeks after injection.The retinal ultrastructure was examined by ultrathin section under transmission electron microscope at one and three weeks after injection.Others 35 rats were used for experiment of protective effect of acteoside on RGC.The rats were divided into operation group A and B (n=8),sham operation group C and D (n=8),and blank group (n=3).The optic nerve of rats in operation group was clamped for 10 seconds after optic nerve exposure,while the optic nerve of rats in sham operation group was exposed only.The rats in operation group A and B were received intravitreal injection with 5 μl acteoside (1 mg/ml) and 5 μl PBS respectively.The rats in sham operation group C and D were received intravitreal injection with 1 μl acteoside (1 mg/ml) and 1 μl PBS respectively.No treatment was performed for blank group.The retinal structure was examined by HE staining of retinal frozen sections at one,two and four weeks after injection.Immunohistochemistry was used to measure the expression of growth associated protein 43 (GAP-43).RGC apoptosis was assessed by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labelling (TUNEL) method.Software of SPSS 13.0 was used for the data statistical analysis in this study.Results In the safety experiment of intravitreal injected acteoside,there was no abnormity of cornea,anterior chamber,lens,vitreous cavity and retina after injection.At one,two and three weeks after injection,the retina structure was normal without significant apoptosis,necrosis and inflammatory cell infiltration.The ganglion cell layer showed slightly edema; there was no obvious change of retinal ultrastructure after injection of acteoside with 5 mg/ml and 2 mg/ml,but slight change with the format of 1 mg/ml.Transmission electron microscopy showed that intravitreal injection of 5μl acteoside at the concentration of 2 or 5 mg/ml can induce significant changes of micro-structures of retina,while injections at 1mg/ml can only induce minor changes.In the experiment of protective effect of acteoside on RGC,light microscope revealed that the cell showed typical changes of apoptosis in operation group,but not in sham operation group and blank group.At the first and second week after injection,compared with the sham operation group and blank group,the RGC number was decreased in operation group.The difference of RGC numbers between operation group A and B was statistically different (F=26.206,P＜0.05).The RGC numbers in operation group continues to decrease at the fourth week after injection,there was obvious difference compared with the first and second week after injection (F=17.364,P＜0.05),but there was no difference of RGC numbers among sham operation intra-group and between sham operation group and blank group at all the time points.Immunohistochemistry showed that at the first week after injection,the integrated absorbance (IA) value in operation group was higher than that in other groups (F=33.466,P＜0.05) ; there was no difference of IA value between operation group A and B.At the second week after injection,IA value in operation group A had slightly declined,but higher than that in operation group B (F=14.391,P＜0.05).At the fourth week after injection,IA value in operation group A declined further,but also higher than that in other groups (F=4.178,P＜0.05).TUNEL showed that on the first week after injection,RGC apoptosis rate in operation group was increased than that in other groups (F=15.365,P＜0.05).At the second week after injection,RGC apoptosis rate in operation group was decreased,and it in operation group A was lower than that in operation group B (F=15.365,P＜0.05).At the fourth week after injection,RGC apoptosis rate in operation group was decreased obviously,there was no difference compared with other groups (F =2.057,P ＞ 0.05).There was no difference of RGC apoptosis rate between sham operation group and blank group at all the time points.Conclusion Intravitreal injection of 5 μl acteoside (1mg/ml) is safe for rat retina,and can up-regulate GAP-43 expression and inhibit RGC apoptosis in optic nerve crush rats. Key words: Polyphenols/therapeutic use; Retinal ganglion cells/drug effects; Retinal ganglion cells/pathology; GAP-43 protein

First harmonic with wavelength modulation spectroscopy to measure integrated absorbance under low absorption

Integrated absorbance (IA) can be used to infer gas temperature and concentration directly, in this paper, we proposed a new method that uses the 1st harmonic to measure the IA under low absorption conditions (<10%). Subsequently, a large number of numerical simulations are used to validate the reliability and accuracy of this method, and several absorption lines of CO₂ and H₂O molecules near 6981 cm⁻¹ are selected to determine the IA and species concentration in experiments. Calculation and experiment results show that the proposed method can accurately measure IA in actual measurements.

Curative effect of the combined application of 5-fluorouracil and diprospan in the treatment of keloid

Objective To explore the curative effect of the combined application of 5-fluorouracil (5-Fu) and diprospan in the treatment of keloid.Methods Five square-shaped skin defects were made on each ear of 12 New zealand rabbits and Kelodi model was established on the ventral side of ears through 6 mm of each side.The120 models were equally divided into three groups:5-Fu and diprospan,diprospan and untreated control.The expression levels of proliferating cell nuclear antigen (PCNA) in scars were detected by using immunohistochemistry.Results The integrated absorbance (IA) values of PCNA expression were 116.00 ± 13.84,161.00 ± 14.78 and 211.00± 18.88 in 5-Fu and diprospan group,diprospan group and untreated control group,respectively (all P ＜ 0.05).Conclusion The combined application of 5-Fu and diprospan had a good curative effect in curing keloid. Key words: Keloid; 5-fluorouracil; Proliferating cell nuclear antigen

Establishment of experimental angiogenic models with applications of quantitative digital image analysis

To establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study. An endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry. Digital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01). The endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

Intrauterine hyperglycemia and leptin resistance of offsprings.

To study the effect of intrauterine hyperglycemia on leptin level and offspring development in rats. Female and male adult Wistar rats were mated, streptozotocin (STZ, 50 mg/kg) was administered intraperitoneally on 5th day of gestation to induce diabetic model, diabetic pups (DP) were exposed to intrauterine hyperglycemia; control pups (CP) were exposed to controls, which was injected with citrate buffer, 8 pups were choosed from each group. Weight gain between 3 - 10 weeks were recorded. Plasma leptin was detected by enzyme-linked immunosorbent assay (ELISA) when the rats were 11 weeks old, and the expression of leptin receptor in hypothalamus was measured at protein level by histomorphology and mRNA level measured by realtime PCR [expressed with integral absorbance (IA)] in 11 weeks in order to discuss the relation of leptin and offspring development. The fasting blood glucose level was significantly higher in diabetic mother compared with the controls [(28.3 +/- 5.1) mmol/L vs. (6.3 +/- 1.4) mmol/L, P < 0.05]. However, there was no difference between the fasting blood glucose level in DP group and CP group [(5.1 +/- 0.8) mmol/L vs. (5.3 +/- 0.6) mmol/L, P > 0.05]. The growth rate between 3-10 weeks was significantly higher in DP group 649.7% than CP group 479.2%, P < 0.05. The base insulin level was lower in DP group [(0.76 +/- 0.37) microg/L vs. (1.06 +/- 0.14) microg/L, P < 0.05]; while there was no difference in plasma leptin and the expression of leptin receptor in hypothalamus [(113 +/- 37) microg/L vs. (128 +/- 40) microg/L, P > 0.05]. The growth rate was not associated with plasma leptin in DP group (r = -0.501, P = 0.311) but associated in CP group (r = -0.553, P = 0.001). The protein level of DP group (4125 +/- 414) did not significantly differ from that of CP group (4244 +/- 511). The median of mRNA of leptin receptor in hypothalamus in DP group did not altered significantly compared with that of CP group (1.25 vs 1.80, P > 0.05). Intrauterine hyperglycemia accelerated growth rate of offsprings between 3 and 10 weeks, however, plasma leptin was not discreased, which indicated leptin resistance. Intrauterine hyperglycemia did not influence the expression of leptin receptor in hypopthalamus in offsprings, this suggested the leptin resistance may be not caused by the quantity of leptin receptors.

Effects on human leukocyte antigen-G on proliferation and invasion of JEG-3 cell line

To investigate the effect of human leukocyte antigen-G (HLA-G) on the growth and invasion of JEG-3 cell line and the role of HLA-G in the onset and development of pre-eclampsia. The experiment was composed of three groups: groups of transfection, negative control and blank control, which corresponded to groups of HLA-G siRNA transfection, negative siRNA transfection and no transfection. HLA-G overexpressed choriocarcinoma cell line JEG-3 was used. The role of HLA-G in JEG-3 cell monolayer was examined by RNA interference technology using HLA-G specific small interfering RNA (siRNA). Expression of HLA-G was detected by reverse transcriptase-polymerase chain reaction and western blot analysis. Changes of cell cycle, apoptosis, proliferation and invasion were respectively detected by methyl thiazolyl tetrazolium (MTT), flow cytometry assay and transwell test. (1) The mRNA and protein levels of HLA-G control group and blank control group were 0.0013 +/- 0.0014, 0.0163 +/- 0.0007 and 0.1923 +/- 0.0384, 0.2184 +/- 0.0153, respectively, which were both significantly different (P<0.05); the number of negative transfection group was 0.1606 +/- 0.0133 and 0.2020 +/- 0.0132, which had no significant difference compared with blank control group (P>0.05). (2) The integral absorbance (IA) values of the HLA-G transfection group and blank control group were 0.44 +/- 0.04 and 0.75 +/- 0.13 respectively, which was significantly different (P<0.01); the IA value of negative control group was 0.69 +/- 0.10, which was not significantly different compared with blank group (P>0.05). (3) The ratios of G2/M and S phase cells in transfection group were (10.9 +/- 2.2)% and (58.6 +/- 0.8)% respectively, significantly different compared with the blank control group [(15.4 +/- 1.9)% and (52.9 +/- 2.3)% respectively; P<0.01]. (4) The ratio of early apoptosis cells in transfection group [(14.5 +/- 2.7)%] was significantly increased compared with negative [(5.3 +/- 1.1)%] and blank control group [(4.7 +/- 0.6)% ; P<0.01]. (5) The invasion number of transfection group and blank control group were 121+/- 12 and 452 +/- 17 respectively, with a significant difference between them (P<0.01). HLA-G is probably involved in the onset of preeclampsia by regulating proliferation and invasion of trophoblast.

Image analysis for degradation of DNA in retinal nuclei of rat after death

The changes of retinal nuclear DNA content in rats after death was detected and the relationship between degradation of retinal nuclear DNA and postmortem interval (PMI) was analyzed. Ninety healthy adult SD rats, female, weighing 250+/-10 g, were randomly divided into 15 groups. At 20 degrees C, the retinal cells were withdrawn every 2 h within 0 to 28 h after death and stained with Feulgen-Vans. Index of density (ID), integral absorbance (IA) and average absorbance (AA) in retinal nucleus were analyzed by image analysis system. And the obtained data were subjected to linear regression analysis by using SPSS12.0 software. The results showed that in retinal nucleus, AA and IA were gradually declined with the prolongation of PMI, while ID had an increased tendency. Within 28 h after PMI, the regression equations were as follows: Y(AA )=-0.009X(AA )+0.590 (R(2)=0.949), Y(IA )=-0.097X(IA )+18.903 (R(2)=0.968), Y(ID)=0.122X(ID)+2.246 (R(2)=0.951). It was concluded that retinal nuclear DNA after death in rats was degraded gradually and had a good correlation with PMI.

Scan-to-scan nonreproducibility in GC/FTIR: An evaluation of several chromatogram calculation algorithms

A new “second difference” (SD) algorithm for calculating wavelength-selective GC/FTIR chromatograms is presented and compared with the Gram-Schmidt (GS), integrated absorbance (IA) and maximum absorbance (MA) algorithms. For an instrument with good scan-to-scan reproducibility, the sensitivity of the four algorithms generally decreases in the order GS>SD>MA>IA. The new algorithm is less affected by instrumental instability, so that in severe cases the sensitivity order becomes SD>GS>MA>IA.