Objective To study the progressive development of the retinas through an observation on the histological changes of the retinas from neonatal mice of different day-ages. Methods The retinas from the mice of 1 to 20 days of age were examined by light microscopy,and from 1 to 3 days,by autoradiography. Results The retinas of the mice below 3 days of age only had the RPE cells layer,the neuroblast layer and the ganglion cell layer.With the increase in dayage,the retinas developed gradually and would be mature in the 20th day. Conclusions The retinas of mice is a kind of immature tissue before the 20th days,so it can be considered as transplantation donors. (Chin J Ocul Fundus Dis, 1999, 15: 174-176)
ObjectiveTo establisht a gut microbiota mice model for chronic obstructive pulmonary disease (COPD) with fecal microbiota transplantation (FMT) and its evaluation.MethodsThe mice received FMT from healthy individuals, COPD Ⅰ-Ⅱ subjects, or COPD Ⅲ–Ⅳ subjects. After microbiota depletion, the FMT was performed by a single oral administration of 100 μL per mouse every other day, for a total of 14 times in 28 days. On the 29th day, the peripheral blood mononuclear cells were analyzed, the gut microbiota of mice before and after FMT was analyzed by 16S rRNA sequencing, and the mice model were evaluated.ResultsThe operational taxonomic units, Chao 1 and Shannon indexes of mice all decreased significantly after antibiotic treatment (P<0.001), but increased significantly after FMT from healthy individuals, COPD Ⅰ-Ⅱ subjects, or COPD Ⅲ–Ⅳ subjects (P<0.05 or P<0.01). The abundance of Firmicutes, Proteobacteria and Actinobacteria in the guts of the mice in the healthy human FMT group, COPD Ⅰ-Ⅱ FMT group and COPD Ⅲ-Ⅳ FMT group were significantly different from those of the control group who only received phosphate buffer saline instead of FMT (P<0.05 or P<0.01). The auxiliary T lymphocytes and cytotoxic T lymphocytes were higher, but B lymphocytes decreased in the peripheral blood of the mice in the COPD Ⅰ-Ⅱ FMT group and COPD Ⅲ-Ⅳ FMT group (P<0.05 or P<0.01).ConclusionFMT can successfully establish a COPD gut microbiota research model.
Objective Isoflurane has an acute preconditioning effectiveness against ischemia in kidney, but this beneficial effectiveness can only last for 2-3 hours. To investigate whether isoflurane produces delayed preconditioningagainst renal ischemia/reperfusion (I/R) injury, and whether this process is mediated by hypoxia inducible factor 1α(HIF- 1α). Methods A total of 52 male C57BL/6 mice were randomly assigned to 4 groups (n=13 in each group): the controlgroup (group A), PBS/isoflurane treated group (group B), scrambled small interference RNA (siRNA)/isoflurane treated group (group C), and HIF-1α siRNA/isoflurane treated group (group D). In groups C and D, 1 mL RNase-free PBS containing 50 μg scrambled siRNA or HIF-1α siRNA was administered via tail vein 24 hours before gas exposure, respectively. Equivalent RNasefree PBS was given in groups A and B. Then the mice in groups B, C, and D were exposed to 1.5% isoflurne and 25%O2 for 2 hours; while the mice in group A received 25%O2 for 2 hours. After 24 hours, 5 mice in each group were sacrificed to assesse the expressions of HIF-1α and erythropoietin (EPO) in renal cortex by Western blot. Renal I/R injury was induced with bilateral renal pedicle occlusion for 25 minutes followed by 24 hours reperfusion on the other 8 mice. At the end of reperfusion, the serum creatinine (SCr), the blood urea nitrogen (BUN), and the histological grading were measured. Results The expressions of HIF-1α and EPO in groups B and C were significantly higher than those in group A (P lt; 0.01). The concentrations of SCr and BUN in groups B and C were significantly lower than those in group A, as well as the scores of tubules (P lt; 0.01), and the injury of kidney was amel iorated noticeably in groups B and C. The expressions of HIF-1α and the concentrations of SCr and BUN in group D were significantly lower than those in group A (P lt; 0.01). Compared with groups B and C, the expression of HIF- 1α and EPO in group D decreased markedly (P lt; 0.01), the concentrations of SCr and BUN were increased obviously, as well asthe scores of tubules (P lt; 0.01), and the renal injury was aggratived significantly. Conclusion Isoflurane produces delayed preconditioning against renal I/R injury, and this beneficial effectiveness may be mediated by HIF-1α.
Objective To evaluate the effects and the molecular mechanism of Liuwei dihuang pills in preventing steroid-induced osteonecrosis of the femoral head (ONFH) so as to provide an expremental basis for preventing ONFH cl inically. Methods Thirty-six adult Kunming mice (weighing 40-50 g, 46 g on average) were randomly divided into three groups (n=12): group A (control group), group B (model group) and group C (prevention group). In groups B and C, ONFHmice models were produced by intraperitoneal injection of horse serum at first (10 mL/kg) and a second injection of horse serum intraperitoneally (5 mL/kg) and prednisolone intramuscularly [45 mL/(kg?day), for 5 days] 2 weeks later. At the same time, the mice in group C were given Liuwei dihuang pills intragastrically [2 g/(kg?day)] and were given normal sal ine [10 mL/(kg?day)] in group B. In group A, mice were given normal sal ine intramuscularly and intragastrically as controls. The animals were sacrificed 2, 4, and 8 weeks after first treatment with prednisone, and femoral heads and l ivers were harvested to do histopathology analysis and apoptosis assay. Results Other mice survived throughout the experiment period except two death at 7 and 11 days after second injection of horse serum intraperitoneally in group B and one death at 24 hours after second injection of horse serum intraperitoneally in group C. The appearance and shape of the femoral head and the surface of cartilages were all normal. The histological observation showed: normal structures of l iver and femoral head were seen in group A at each time point; swell ing l iver cells with small fat vacuole, unclear structure of hepatic cords and narrower sinus hepaticus were seen, the bone trabeculae of femoral head was thin, sparse and collapsed in some regions and the changes became more obvious with time in group B; group C had similar results to group A. The percentage of empty osteocyte lacunae was significantly higher in group B than in groups A and C (P lt; 0.01). The osteoprotegrin expression significantly decreased and the osteoprotegrin l igand expressionsignificantly increased in group B when compared with groups A and C (P lt; 0.01). Apoptosis analysis showed that the apoptosis index in group B was significantly higher than that in groups A and C (P lt; 0.01). Conclusion Liuwei dihuang pills can prevent steroid-induced ONFH by improving l ipid metabol ism, releiving bone lesion, and protecting against cell death.
Objective To investigate the role and mechanism of heat shock protein 60 (HSP60) in induction of murine skin allograft tolerance. Methods At the age of 8-12 weeks, inbred female BALB/C (H-2d) mice (n=45) and CBA/N (H-2k)mice (n=15) were used as transplantation donors and C57BL/6 (H-2b) mice (n=60) as recipients. Recipients C57BL/6 (H-2b) mice were randomized into 4 groups (n=15). In group A, 1 cm × 1 cm Wolfe-Krause skin graft was excised from the back of BALB/C (H-2d) mice and hypoderma was scraped off aseptically, and then transplanted to the back of C57BL/6 (H-2b)mice. The method of skin transplantation in the other 3 groups was the same as to group A. In group B, C57BL/6 (H-2b) mice were treated with imcompleted Freund’s adjuvant (IFA) administration into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group C, C57BL/6 (H-2b) mice were administered HSP60 emulsified in IFA into the back 2 weeks before transplantation of BALB/C (H-2d) mice skin. In group D, C57BL/6 (H-2b) mice were treated with HSP60 emulsified in IFA into the back and followed by skin transplantation of CBA/N (H-2k) mice 2 weeks later. The delayed type hypersensitivity was determined 7 days after transplantation. One-way mixed lymphocyte reaction, the concentration of cytokines in the mixed lymphocyte reaction culture supernatant was determined 7 days and 25 days after transplantation. The survival time of skin allograft was observed. Results The survival time of skin allograft in groups A, B, C and D was 12.4 ± 0.5, 11.6 ± 0.8, 29.3 ± 2.6 and 27.6 ± 2.1 days, respectively. There was significant difference between groups A, B and groups C, D (P﹤0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). The counts of per minute impulse (cpm) of mixed lymphocyte reaction 7 days after transplantation in groups A, B, C and D was 12 836 ± 1 357, 11 876 ±1 265, 6 581 ± 573 and 6 843 ± 612, respectively. There was significant difference between groups A, B and group C and group D (P lt; 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). The cpm of mixed lymphocyte reaction at 25 days after transplantation in group A, B, C and D was 13 286 ±1 498, 12 960 ± 1 376, 11 936 ± 1 265 and 12 374 ± 1269, respectively. There was no significant difference among 4 groups (P gt;0.05).The concentration of IL-10 in the mixed lymphocyte reaction culture supernatant in groups C, D were higher than that in groups A, B, and IL-2 and IFN-γ were lower than that in groups A, B 7 days after transplantation (P lt; 0.05), while there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). There was no significant difference in cytokines among the 4 groups 25 days after transplantation (P gt; 0.05). The delayed type hypersensitivity in groups A, B, C and D 7 days after transplantation was 0.84 ± 0.09, 0.81 ± 0.07, 0.43 ± 0.05 and 0.46 ± 0.03 mm, respectively. There was significant differences between groups A, B and groups C, D (P lt; 0.05). While there was no significant difference between group A and group B as well as between group C and group D (P gt; 0.05). Conclusion HSP60 may play a role in induction and maintenance of murine skin allograft tolerance.
ObjectiveTo study the possibility of the C17.2 neural stem cells (NSCs) differentiating into neural cells induced by serum-free condition medium of olfactory ensheathing cells (OECs) and to detect the cell viability of the differentiated cells. MethodsOECs were isloated and cultured from the olfactory bulbs of 3-day-old postnatal mouse to prepare serum-free condition medium of OECs. After C17.2 NSCs were cultured with H-DMEM/F12 medium containing 15% FBS and the cell fusion reached 80%, the 3rd passage cells were induced by serum-free condition medium of OECs in the experimental group, by H-DMEM/F12 in the control group, and non-induced C17.2 NSCs served as the blank control group. The growth condition of cells was observed with inverted microscope. After 5 days, the immunofluorescence staining[microtubule-associated protein 2 (MAP-2) and β-tubulin-Ⅲ] and Western blot (Nestin, β-tubulin-Ⅲ, and MAP-2) were carried out to identify the neural cells derived from NSCs. The cell viabilities were measured by MTT assay and the quantity of lactate dehydrogenase (LDH) release in the medium. ResultsIn the experimental group, the C17.2 NSCs bodies began to contract at 24 hours after induction, and the differentiated cells increased obviously with long synapse at 3 days after induction; in the control group, the cell morphology showed no obvious change at 24 hours, cell body shrinkage, condensation of nuclear chromatin, and lysis were observed at 3 days. The immunofluorescence staining showed that β-tubulin-Ⅲ and MAP-2 of C17.2 NSCs were positive at 5 days after induction, and Western blot suggested that the expression of Nestin protein declined significantly and the expressions of β-tubulin-Ⅲ and MAP-2 protein were increased in the experimental group, showing significant differences when compared with those in the control group and blank control group (P<0.05). The LDH release and the cell viability were 130.60%±6.86% and 62.20%±3.82% in the experimental group, and were 178.20%±5.44% and 18.00%±3.83% in the control group respectively, showing significant differences between 2 groups (P<0.05). The LDH release and the cell viability of experimental group and control group were significantly lower than those of blank control group (100%) (P<0.05). ConclusionNeurotrophic factors from OECs play an important role in inducing C17.2 NSCs differentiation into neural cells and keeping the viability of differentiated cells after induction.
Objective To observe the preventing effect of intraocular injection of Bevacizumab (Avastin) to retinal microvascular proliferation in non-obese diabetes mice. Methods In the study, thirty non-obese diabetes mice (NOD mice) were selected. The left eyes of mice were selected as treatment group with 1mu;l A vastin (25mg/1ml) injected, and right eyes were selected as control group with 1 mu;l saline injected. One week, one month, two months after injection, ten mice were selected randomly, and then enucleated two eyes, in which the retinal microvascular endothelial cells ultrastructure and immunohistochemistry of retinal CD34 and VEGF, were observed and measured. The differences of dense of positive sta ining between two groups were compared by digital image analysis. Results The positive expression of VEGF and CD34 were brown staining, and the positive staining of CD34 located in vascular endothelial cells. There was statistically significant difference in VEGF expression between two groups in 1 week and 1 month after injection（t=21.6, t=13.5; P＜0.01), and no statistically significant difference in 2 months after injection （t=0.9, P＞0.05）. There was statistically significant difference in CD34 expression between two groups in 1 month and 2 months af ter injection（t=3.2, P＜0.01; t=2.7, P＜0.05） and no statistically significant difference in 1 week after injection（t=1.3, P＞0.05）. In every time point after injection, there was no obvious change in the microstructure of retinal vascular endothelial cells. Conclusion Intraocular injection of Avastin could prevent the abnormal proliferation of retinal microvascular in NOD mice. （Chin J Ocul Fundus Dis，2008，24：180-183）
ObjectiveTo observe the anticancer efficacy of ginsenoside Rg3 on colorectal cancer in vitro and in vivo. MethodsMice colorectal cell line (CT26) was incubated in 96-well plates (3×103-4×103 per well) with various concentrations of ginsenoside Rg3 (0, 5, 10, 15, 20 μg/mL) for 24 hours and 48 hours. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dipheny 1-2-H-tetrazolium bromide assay was used to detect the inhibitory rate of cells. Xenograft models were established by subcutaneous implantation of CT26 cells into BABL/c mice. Each mouse was injected with 1×107 cells suspended in serum-free medium. Xenograft mice were randomized into four groups: physiological saline group, ginsenoside Rg3 5 mg/kg group, ginsenoside Rg3 10 mg/kg group, and ginsenoside Rg3 20 mg/kg group. Ginsenoside Rg3 was administrated to mice by intragastric gavage. All animals were observed for activity, body weight, tumor size, survival time, mental state and adverse effect of ginsenoside Rg3. Hematoxylin-eosin stain was used for comparing necrosis rate among groups. ResultsThe inhibitory rates of cells were increasing following the elevating concentrations of ginsenoside Rg3. The anti-proliferation effect of ginsenoside Rg3 for 48 hours was weaker than the anti-proliferation effect for 24 hours. The decrease of mice body weight was slower than physiological saline group after administration of ginsenoside Rg3, and the number of mice with worse physiological state, lack of activity and loss of appetite in physiological saline group were more than that in ginsenoside Rg3 groups. However, these results among four groups were not significantly different (P>0.05). There were no obvious adverse effects of ginsenoside Rg3 found during the whole study. The necrosis rate of physiological saline group, Rg3 10 mg/kg group and Rg3 20 mg/kg group was 20%, 60% and 80% respectively. ConclusionGinsenoside Rg3, as a single agent, still has anticancer activity. The anticancer efficacy is increasing following the elevating concentrations of ginsenoside Rg3. Ginsenoside Rg3 is a dose dependent agent.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
ObjectiveTo investigate the effects of resveratrol on airway remodeling in mice with chronic asthma. MethodsTwenty-four female BALB/c mice were randomly divided into three groups (8 mice in each group), namely a control group, an asthma group and a resveratrol (RV) group. All mice were sensitized with ovalbumin (OVA). The sensitized mice were then challenged with OVA while the control group were challenged with phosphate-buffered saline. The mice in the RV group were intraperitoneally injected with RV 30 min before OVA challenge, while the mice in the control and the asthma group were intraperitoneally injected with equal volume of dimethylsulfoxide. Periodic acid-Schiff (PAS) staining was performed to evaluate goblet cell hyperplasia, and Masson-trichrome staining was used to evaluate the deposition of collagen matrix. In addition, immunohistochemical analysis of the α-smooth muscle actin (α-SMA) was applied to examine airway smooth muscle cell hyperplasia and hypertrophy. The positive staining with PAS, Masson, α-SMA areas (μm 2/μm) of per bronchial basement membrane perimeter was used to indicate the degree of airway remodeling. ResultsIn the asthma group and the RV group, the degree of the goblet cell hyperplasia was significantly higher than that in the control group (5.44±1.13, 4.18±0.85vs. 0.00±0.00,P<0.01), and the level of goblet cell hyperplasia in the RV group was lower than that in the asthma group (P<0.05). The Masson staining showed that the deposition of collagen in the asthma group and the RV group was significantly higher than that in the control group (9.80±2.78, 5.71±0.68vs. 1.67±0.65,P<0.01), and the collagen deposition in the RV group was further lower than that in the asthma group (P<0.01). The α-SMA immunohistochemical analysis demonstrated that the expression of α-SMA in the asthma group and the RV group was significantly higher than that in the control group (10.39±1.65, 7.57±1.98vs. 2.41±1.06,P<0.01), and the level of α-SMA in the RV group was also lower than that in the asthma group (P<0.05). ConclusionThese findings suggest that resveratrol has an inhibitory effect on the process of airway remodeling in mice with chronic asthma.