Objective The present study was aimed at investigating the effects of cholera toxin(CTx) on promoting the regeneration of retinal ganglion cells(RGCs) in hamster retina. Methods After optic nerve (ON) transection, an autologus sciatic nerve (attached graft, AG) was removed and sutured to the proximal stump of the ON. CT X was injected or (and) a small segment of sciatic nerve (SN) inserted intravitrously. Animals were separated into 5 groups:regenerating control group(AG groups and solution groups); AG+CT X groups; AG+SN groups; AG+CT X+SN groups; Effect and Dosage groups; Animals in the former 4 groups were allowed to survive for 2-6 weeks respectively. The regenerated RGCs were labeled retrogradely by granular blue, and the changes in number of regenerating RGCs in each retina were observed under fluorescent microscope. Results The mean numbers of regenerating RGCs in AG+CT X groups were increased and significantly higher than those in AG groups and solution groups at each time point( P

【Objective】 To examine the regeneration of NPY-immuno reactivity (IR) retinal ganglion cells (RGCs) and the effects of cholera toxin ( CTx) injected or/and peripheral nerve implanted on regeneration of NPY-IR RGCs. 【Methods】 16 adult golden hamsters were ramdomly divided into 4 groups. Optic nerve (ON) was transacted and a segment of autologous sciatic nerve (attached g raft, AG) was removed and sutured to the proximal stump of ON in regenerating co ntrol group (AG group). The animals in experimental groups were further treated with CTx injection or/and implantation of a short of segment sciatic nerve (SN) intravitrously. By using the retrograde labeling with fluorogold (FG) combined w ith fluorescent immunocytochemistry, the regenerated NPY-IR RGCs were observed and counted under fluorescent microscope. 【Results】 At 4 weeks after surgery, the mean number of regenerated NPY-IR RGCs per retina in AG group was 58±22 wh ich constitutes (3.36±0.65)% of the total regenerated RGCs. In AG+CTx, AG+SN and AG+CTx+SN experimental groups, the mean numbers of regenerated NPY-IR RGCs per retina were 143±47, 125±37 and 437±77 ordinally which constitute (5.15± 0.89)%, (5.34±0.37)% and (8.11±0.85)% of the total regenerated RGCs respec tively, which were increased significantly compared with those in AG group. 【Co nclusion】 The results show that the axotomized NPY-IR RGCs have th e capability of regenerating their axons into the attached PN graft, CTx and/or SN could enhance the regeneration of the NPY-IR RGCs.

BACKGROUND: Recent studies show that the neurons in central nervous system have the regenerating ability under certain suitable environment such as elevating the c-AMP level of the neuron. But the mechanism is unclear.OBJECTIVE: To investigate the effect of protein kinase A inhibitor H-89and PI3-kinase inhibitor, wortmannin, on Cholera Toxin (CTx) in promoting the axon regeneration of retinal ganglion cells (RGCs) after distal axotomy of the optic nerve in adult hamsters.DESIGN: A randomized and controlled animal experiment SETTING: Department of Histology and Embryology of Guangzhou Medical College and Sun Yat-sen University of Medical Sciences MATERIALS: This experiment was conducted in the Guangzhou Medical College and Sun Yat-sen University of Medical Sciences between January 2000 and December 2001. Totally 25healthy adult male hamsters were chosen and randomly divided into 5 groups: model group; experimental control group; CTx group; CTx +protein kinase A inhibitor group; CTx +PI3-K inhibitor group, with 5 animals in each group.METHODS: A 2 cm segment of autologus sciatic nerve was removed and desheathed. The proximal end of the sciatic nerve was connected to the proximal stump of the optic nerve (ON). .The wound was enveloped with gelatin sponge. The remaining portion of sciatic nerve was placed on the top of the skull. Model group: a segment of autologus sciatic nerve was connected to the ON proximal stump. Experimental control group: Received an injection of Na2EDTA/NaCL solution introvitreally based on the treatment in control group. CTx group: CTx (1000 pg/eye)was injected introvitreally on the basis of the treatment in the control group. CTx+ protein kinase A inhibitor group: 3μL protein kinase A inhibitor H89 (60μmol/L)was injected introvitreally 30 minutes before operation, the other treatment was like mannin (1μmol/L) was injected introvitreally 30 minutes before operation,and the other treatment was like that of CTx group.⑤CTx+PI3-K inhibitor group: 3 μL PI3-K inhibitor wortmannin (1μmol/L) was injected introvitreally 30 minutes before operation,and the other treatment was like that of CTx group. Animals in each group survived for 4 weeks. Administration was given every other 5 days. H-89,wortmannin were given once five days and CTx was also given 30 minutes later every time. There were four times in total. 3 days before operation, a piece of gel foam soaked with 30g/L GB was applied to the proximal end of transected PN to label the RGCs conversely. Observation was performed under the fluorescence microscope and the number of GB-labeled cells was counted.MAIN OUTCOME MEASURES: The quantity of retrograde labeled axon regenerating RGCs in each groupRESULTS: There were fewer regenerating RGCs in the model group and experimental control group (2.6±0.87,2.4±0.95),and the number of them was significantly higher in the CTx group than in the control group and experimental group [(43.2±1.36),q=73.294 and 73.655,P ＜ 0.001]. There was no significant difference of the mean number of axon regenerating RGCs between CTx + protein kinase A inhibitor H-89 group and model group and experimental control group [(3.2±0.16), q=1.083 and 1.444, P＞ 0.05]; The mean number of axon regenerating RGCs in the three groups was significantly lower than that in the CTX group, with significant difference (q=72.211, P ＜ 0.001). The mean number of axon regenerating RGCs was higher in the CTx+PI3-K inhibitor group (9.6±1.85)than in the model group and experimental control group (q=12.637 and 12.998, P ＜ 0.05);but significantly lower than in the CTx group (q=60.657, P ＜ 0.001).CONCLUSION: CTx can promote the axon regeneration of RGCs after distal axotomy of the optic nerve; its promoting function can be blocked by H-89 and Wortmannin

Objective The present study was aimed at investigating the regeneration promoting mechanism of forskolin and IBMX on injured retinal ganglion cells(RGCs). Methods Fluorescent retrograde labeling and autologus sciatic nerve transplanting techniques were used to observe the effects of H 89 or/and wortmannin on the regeneration promoting effects of forskolin and IBMX on injured retinal ganglion cells by intravitreal injection. Results Both H 89 and wortmannin could partly block the promoting effects of forskolin and IBMX on regeneration of injured RGCs, the effects of wortmannin might be more powerful, if united, they could inhibit the effects of forskolin and IBMX completely.Conclusion The regeneration promoting effects of forskolin and IBMX on injured RGCs might be realized by PKA CREB and PI3 K Akt accesses.

The localization and development of substance P (SP) like immunoreactive retinal neurons in the adult, newborn and postnatal New Zealand albino rabbits were studied with immuaocytochemistry ABC method. We found most of the SPlike immunoreactive somas in adult rabbit retina located in inner margin of inner nuclear layer (INL) and ganglion cell layer (GCL). Their processes ramified in the lamina 1,3 and 5 of inner plexiform layer (IPL). Some immunoreaetive fibers in the layer of optic nerve fiber (LON) were occasionally observed. The highest cell density of SP-like somas occured in the visual streak (VS). The cell density gradually decreased from the VS to the ventral and dorsal periphery. In the new born rabbit retina, the labelled cell bodies and their processes appeared. Most of them located in the GCL, and a few of them located in the' INL. Their processes formed discontinuous layer in the lamina 5 and never seen in the lamina 3. The cell density of SP positive somas gradually increased from newborn to postnatal 4 th day. From postnatal 6 th day to 12 th day, the cell density gradually decreased. From postnatal 12 th day the somas mainly located in the INL. On the 20th day after birth the distribution and morphology of SP-like immunoreactive somas and processes had approached that of mature age. These results suggest that rabbit retinal SP-like immunoreactive neurons had appeared at prenatal period and continuously developed after birth.

Objective To investigate the effects of Colera Toxin(CTx) on the regeneration of retinal ganglion cells (RGCs) after distal axotomy in adult hamsters. Method After transecting the optic nerve(ON) intracranically,an autologus sciatic nerve (attached graft,AG) was removed and connected to the proximal stump of the ON.CTx was injected and/or a 2mm segment of sciatic nerve(SN) was inserted intravitreally.Animals were divideded into six groups:control group 1(AG group) and control group 2(solution group);AG+SN group;AG+CTx group;AG+SN+CTx group;effect and dosage group.Animals in the former five groups were allowed to survive for 4-6 weeks respectively.Granular blue fluorescent retrograde labeling method was used to measure the quantity of regenerating RGCs of control and experiment animals. Results The mean number of regenerating RGCs in AG+CTx groups were increased and significantly higher than those in control group 1 and control group 2 at each time point(P

Objective To investigate the differentiation of mesenchymal stem cells(rMSCs) of young rat into neuron-like cells with Ligustrazin hydrochloride. Methods rMSCs were separated from femurs marrow flushed out with DMEM(low glucose) by using a needle and syringe, then planted in plastic culture flask. Through expanded to 5 passages, rMSCs were induced to differentiate into neuron-like cells with Ligustrazin hydrochloride. Anti-neurofilament(NF-M), nestin, neuron-specific enolase(NSE), MAP-2,GAP-43 and glial fibrillary acidic protein(GFAP) antibodies were detected by immunohistochemistry. Results rMSCs were comprised a single phenotypic population and displayed a fibroblast-like morphology after 5 passage in culture. With 10*!?g/L bFGF pre-induce for 24*!h, then the medium was replaced with induction medium containing Ligustrazin hydrochloride. The induced-rMSCs exhibited neuronal morphological characteristics from the first half an hour to 5*!h. The neuron-like cells expressed NF-M, NSE, MAP-2,GAP-43 and nestin positive, but didn't express glial astrocyte marker GFAP.Conclusion rMSCs can be induced to differentiate into neuron like cells with Ligustrazin hydrochloride in vitro.

Objective To investigate the effects of transplantation of sciatic nerve(SN) and injection of 8-(4-Chlorophenylthio)-cAMP(CPT-cAMP) into the viteous on axotomized retinal ganglion cells(RGCs) in adult hamsters. Methods Twenty adult golden hamsters were ramdomly divided into 4 groups.Optic nerve(ON) was transected and a segment of autologous sciatic nerve(attached graft,AG) was removed and sutured to the proximal stump of ON in regenerating control group(AG group).The animals in experimental groups were further treated with CPT-cAMP injection and/or implantation of a short of segment sciatic nerve(SN) intravitrously.Fluorescent retrograde labeling method and quantitative anatomical techniques were used to measure the number of RGCs. Results 1.In control (AG) retinas,the mean number of regenerating RGCs was 1*!428?284/retina.2.In AG+SN group,the mean number of regenerating RGCs was 2*!220?415/retina.3.In AG+cAMP group,the mean number of regenerating RGCs was 2*!175?364/retina.4.The mean number of regenerating RGCs in AG+cAMP+SN group were 4*!255?820/retina.AG+SN,AG+cAMP groups were significantly different from AG group(P

Objective Use three inhibitors to block three different signaling pathway to explore the mechanisms of 3-isobutyl-1-methylxanthine(IBMX) and 8 (4 Chlorophenylthio) cAMP(CPT-cAMP) on the regeneration of retinal ganglion cells. Method Fluorescent retrogratde tracing method and quantitative anatomical techniques were used to measure the numbers of RGCs in control one,control two group and experiment groups. Results 1^ In control group 1(AG),the mean number of regenerating RGC was 1428??284/retina in 4 weeks after surgery; 2^ In control group 2(AG+IBMX+CPT-cAMP) the mean number of regenerating RGC was 4?917?1 489/retina in 4 weeks after surger; 3^ The mean number of regenerating RGC of experiment groups in 4 weeks after surgery were (1) H89 group(AG+IBMX+cAMP+H89):1?426?320/retina; (2)Wortmannin group(AG+IBMX+cAMP+Wortmannin):4143?1?057/retina; (3)PD98059 group(AG+IBMX+cAMP+PD98059):4?154?965/retina. Conclusion The promoting effects of IBMX and CPT-cAMP on regeneration of RGCs can be interrupted by H89 and can't be blocked by wortmannin and PD98059.

Objective To investigate the differentiation potenitial of ES cell derived epidermal like stem cells,to lay a base for the study of their differentiation mechanism,as well as seek new source to provide seed cells for skin engineering. Methods Mouse ES cells labeled or unlabeled by Hoechst 33342 were cocultrued with human amnion for 4 days.The epidermal like stem cell clones formed on the surface of amnion were digested with trypsin and transplanted into hypodermis of nude mice for 10,20 and 45 days,then the differentiation pattern of the donor cells were observed and estimated with morphological and immunohistochemical method. Results The grafts may differentiate into tubular or follicular like structures lined with simple or stratified epithelial like cells which expressed ? 1 integin,CK19,CK15,PK involucrin and CEA respectively after 10 to 30 days of transplantation.Keratinized stratified squamous epithelium,sebaceous gland like,sweat gland like and hair follicle like structures were observed after 45 days ofter transplantation.Conclusion ES cell derived epidermal like cells might have differentation potential to diffreentiate into keratinized stratified squamous epithelium,sebaceous like,sweat gland like and hair follicle like structures.