Objective To investigate the pattern and the role of cytoskeletal genes expression during nerve regeneration. Methods We examined the changes of expression of ? tubulin(? Tub)and three neurofilament(NF)subunits mRNA in rat corticospinal neurons of sensorimotor cortex on the 1st,3rd,5th,7th,10th,14th,21st,28th and 56th days after spinal cord semitransection injury by using in situ hybridization. Results Levels of ? Tub,NF L,NF M and NF H mRNA were dramatically downregulated within large sized pyramidal neurons in layer V of the sensorimotor cortex of side lateral after spinal cord injury.The level of ? Tub mRNA was decreased as early as 1 day postinjury,whereas the levels of NF subunits mRNA were not decreased until later time(on the 3rd days postinjury),and both of them did not return to control levels on the 56th days following the lesion. Conclusion ? Tub gene expression if inhibited in CNS neurons postinjury. The axotomy signeal and the continuance downregulation of NF mRNA expressioon following spinal cord injury may contribute to the ineffective regeneration response of CNS neurons.

Objective In order to explore the mechanisms that regulate the expression of genes coding for cytoskeletal proteins during nerve regeneration. Methods In the present study, in situ hybridization was used to examine the changes of light(NF L),medium(NF M)and heavy (NF H)neurofilament protein subunits mRNA in L 4-6 dosal root ganglion (DRG) sensory neurons during nerve regeneration following a unilateral crush of the sciatic nerve. Results The hybridization signals of each neurofilament subunit mRNA were dramatic decrees in DRG sensory neurons post axotomy by light microscope.The signals of NF\|L and NF\|M mRNA were located in cytoplasm of neurons,whereas NF H mRNA was found in both nucleus and cytoplasm of neurons. Conclusion There are the different mechanisms of regulation of neurofilament subunit genes expression,the reduced neurofilament gene expression may represent a general response to axonal injury and plays an important role in effective nerve regeneration.

Objective On the basis that olfactory ensheathing cells (OECs) transplanted into injured spinal cord may facilitate axonal regeneration, the OECs were cultured from olfactory bulb and nasal olfactory mucosa in the present study, in order to explore if the olfactory mucosa could be a new donation for transplanting the olfactory ensheathing cells. Methods OECs were harvested from olfactory bulb and mucosa based on the differing rates of attachment of the various cell types, following GFAP and NGFRp75 immunocytochemistry. Results Three morphological and immunohistochmically distinct types of cell which appeared bipolar,tripolar and flat morphology were present in primary cultures of adult rat olfactory bulb and olfactory mucosa.Conclusion The method of purification for OECs based on the different rates of attachment among the various cell types is simple, inexpensive and practical. The OECs from nasal olfactory mucosa like ones from the olfactory bulb is an accessible source of tissue for autologous grafting in human spinal paralegia in the future.

Objective To study effects of GDNF and HSV GDNF on apoptosis of spinal cord motoneurons after scratch injury in vitro. Methods In the period of culture cell,motor neurons were periodically observed and counted.Scratch injury was executed on culturing 12th day,in the same time,cultured neurons were divided into 4 groups,and each group was given corresponding medium(medium serum free control group,serum group,HSV GDNF group,GDNF group).On the 4th and 7th day after scratch injury,TUNEL staining was respectively performed,and the number and the mean densities of apoptotic motoneurons were observed. Results The number of living motoneurons was in inverse proportion to time of scratch injury in each group.The number of apoptotic motoneurons from control group,HSV GDNF group to GDNF group was successively decreased as well as the mean densities of apoptotic motoneurons on the 4th and 7th day after scratch injury.Furthermore,the effects of groups with serum were no better than those of medium serum free groups,in the same time,difference was not obviously in HSV GDNF group and GDNF group. Conclusion GDNF and HSV GDNF can decrease apoptosis of injured motoneurons in vitro .It suggests that GDNF and HSV GDNF might play an important role in the growth and development of motor neurons.

Objective To investigate the expression of GDNF in the olfactory ensheathing glia cells of the adult rats and golden hamster for studying the mechanisms of the OECs in the CNS regeneration. Methods The immunohistochemical staining was performed on the slides of adult olfactory bulb from 3 rats and 3 golden hamsters.According to the primary antibody,tissue slides were divided into 3 groups:rabbit polyclonal GDNF,the rabbit polyclonal GFAP and rabbit polyclonal NGFRp75(Santa Cruz Biotechnology)respectively,and followed by the rabbit ABC immunoreactive staining system. Results It revealed that GDNF was expressed by cells in the first two lines of olfactory bulb from rat and golden hamster.The GFAP and NGFRp75 were also expressed in the same position from other two groups of slides respectively.Conclusion The olfactory ensheathing cell could secret GDNF in the progress of mediating neuronal survival and axonal elongation.

Objective Investigate the morphological features and distribution of olfactory ensheathing cells(Ecs) to study the relationship with CNS regeneration. Methods Luxol fast blue,Mallory special staining methods and TEM were emploed. Results ECs are distributed within the first two layers of olfactory bulb(OB) and olfactory epithelium along the olfactory nerve.The majority of cells are flattened with extended cytoplasm,although some are bipolar or tripolar with long and thin processes.NGFRp75 immunocytochemical reactive are sequentially expressed by ECs in the first two layers of OB and olfactory epithelium.TEM showed that the ECs possessed an irregularly shaped nucleus with a prominent nucleolus,and the mesaxon of each ECs encloses densely packed bundles of unmyelinated axons.Conclusion\ The ECs are a type of macroglia exclusively located in the first two layers of OB and olfactory epithelium,the morphology of EC is flattened with extended cytoplasm and the mesaxon of each ECs encloses densely packed bundles of unmyelinated axons.

Objective Studying the expression of the Nogo(N\|18) in olfactory ensheathing cells(OECs) to investigate the functional relationship between Nogo and the OECs in promoting the regeneration of neurite axon. Methods The expression of the Nogo(N\|18) in the primary cultured ensheathing cells from olfactory bulb of the adult rats was studied with the method of immunocytochemical staining and double\|immunofluorescence staining examed under the confocal laser scanning microscope. Results The Nogo\|A protein was located in primary cultured ensheathing cells from the olfactory bulb.The protein was mainly distributed in the cytoplasm.The member and processes were less stained.Conclusion\ The ensheathing cells in vitro contain Nogo protein.It suggests that Nogo protein is not a depressive factor in the axon regeneration of olfactory system.\;[

Objective In order to explore the possibility of autologous transplantation of olfactory mucosa for spinal cord repair,the changes of autologous olfactory mucosa being transplanted into spinal cord and the effect of promoting axon regeneration in adult rats were investigated. Methods Forty male adult rats were divided into two groups randomly:olfactory mucosa transplantation and control groups.Olfactory mucosa (1*!mm+2) was taken from the posterior region of nasal septum,and this graft was transplanted into the posterior funiculus of cervical 1 segment of spinal cord and destructed the corticospinal tract(2*!mm?1^5*!mm).The control graft was derived from the respiratory lamina and then was grafted into the corresponding lesion.After being survived for 4-6 weeks,the animals were killed,the transplanted site was sectioned horizontally,immunofluorescence double labeled with the neurofilament(NF) and anti-NGFR-p75,investigated under the confocal microscope.Another set of slice was used immunohistochemistry to investigate the olfactory mucosa how to integrate with the surrounding tissue. Results The control lesions have the obvious hollow,no p75-positive reaction in the respiratory lamina and no NF-positive fibers grew through it;olfactory mucosa group showed that olfactory mucosa which is labeled p75-positive healed up with surrounded tissue,the hollow declined markedly,the labeled olfactory ensheathing cells(OECs) soakage into the nerve tissue.OECs induced a fine,unbranched,elongative growth of the cut CST axons.The axons were regenerated through the graft and continued to reenter into the caudal host part.Conclusion Autologous olfactory mucosa may be the donor which could repair the spinal cord injury for clinical situations.

Objective To investigate the effect of bFGF on the proliferation of olfactory epithelium ensheathing cells. Methods With BrdU incorporation method,the effect of bFGF on the proliferating rate of olfactory epithelium ensheathing cells was observed in the research. Results The olfactory epithelium ensheathing cells can proliferate without any proliferating factor.The bFGF(10?g/L)can enhance the proliferating rate in a moderate way.In this experiment BPE(bovine pituitary extract) can not enhance the stimulating effect of bFGF.Conclusion The bFGF(10?g/L)can stimulate the proliferating rate of the olfactory epithelium ensheathing cells in vitro.

Objective To investigate the technical methods for culturing and purifying the olfactory ensheathing cells(OECs) from the adult canine and human olfactory epithclium.To establish a basis for future studying the transplantation of peripheral(OECs) to repair the spinal cord injury in human. Methods Purifying the OECs from the olfactory epithelium of adult canine and man according to their different attachment time with other types of cells.Culturing for 25 days,observed at 6d,10d and 25d,and immunostained with NGFRp75 antibody to identify the OECs. Results The number of cultured olfactory epithelium OECs from both adult canine and man were increased much more after 10 days of culture,and its sharp showed to be bi-polar or tripe-polar and are immunopositive to NGFRp75 antibody.The in vitro OECs of canine grew better than that in man's in the present conditions.Conclusion\ The method of different attachment time seems available in purifying olfactory ensheathing cells from both the adult canine and man olfactory epithelium.