ISOLATION AND CHARACTERIZATION OF CULTURED ENSHEATHING CELLS FROM ADULT RAT OLFACTORY BULBS AND NASAL OLFACTORY MUCOSA / 解剖学报

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.

AN EXPERIMENTAL STUDY ON THE TRANSPLANTATION OFAUTOLOGOUS OLFACTORY MUCOSA TO THE INJURED SPINAL CORD IN ADULT RATS / 解剖学报

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

CELL DEATH IN HIPPOCAMPUS FORMATION INDUCED BY AUDIOGENIC KINDLING / 解剖学报

Objective To investigate if the cell death in hippocampus can occur after single and kindled audiogenic seizure,and the pertinent mechanisms involved. Methods HE staining was used to show the distribution of the dead cells and the immunocytochemistry was used to show the expression of the apoptosis\|related proteins Bcl\|2 and Bax. Results After single audiogenic seizure,there were a few dead cells in hippocampus.After kindled seizure a lots of pyknotic,acidophilic cells were found in hippocampal CA\-1,CA\-2,CA\-4 subregions and the granular layer of dentate gyrus.The CA (corrected optic density) values of Bax immunoreactivities in CA\-2 and CA\-4 subregions were increased significantly in kindled rats, in contrast with that in control rats.Conclusions\ Audiogenic kindling can induce marked cell death in hippocampus.Apoptosis may be one of the mechanisms underling this kind of cell death.

EXPRESSION OF N-METHYL-D-ASPARTATE RECEPTOR SUBUNIT-1 IN EPENDYMAL CELLS OF THE THIRD VENTRICLE OF RAT / 解剖学报

Objective To explore the expression of N-methyl-D-aspartate receptor subunit-1 (NMDAR1) in the ependymal cells of the third ventricle of rat. Methods The immunohistochemistry technique was used. Results (1)NMDAR1-immunoreaction (NMDAR1-IR) was strongly expressed in the ependymal cells of the third ventricle; (2)There was no significant sex difference in morphology and distribution of the NMDAR1-IR ependymal cells between male and female. Conclusion The present investigation provided the morphological evidence supporting that glutamate of CSF might regulate ependymal cells via NMDAR.

CHANGES OF STAT3 EXPRESSION IN RETINA AFTER TRANSECTION OF OPTIC NERVE / 解剖学报

Objective To study the expression and distribution of protein STAT3 in the retina after optic nerve transection. Methods Immunocytochemistry, Western blot and computer image analysis techniques were used. Results After optic nerve transection, STAT3 levels in the retina was highly up-regulated, the peak of which appeared at day 1 postaxotomy, then decreased gradually to the normal level 5 days later. More translocations of STAT3 to the nucleus were seen.Conclusion JAK-STAT signal transduction pathway was involved in the pathological processes in the retinal after transection of the optic nerve.

CELLULAR LOCALIZATION OF SUPPRESSOR OF CYTOKINE SIGNALING-3 IN ADULT RAT RETINA / 解剖学报

Objective To study the basic expression and cellular localization of SOCS-3 in normal rat retina. Methods Neuro-immunocytochemistry techniques were used. Results SOCS-3 positive cells were widely distributed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in the retina. In the GCL, SOCS-3 immunoreactivity was mainly in the neuclei of the ganglion cells.Some of the SOCS-3 positive cells in INL were M?ller cells.Conclusion Basal expression of SOCS-3 is widely present in the neurons and glia in normal adult rat retina and mainly in the form of nuclear protein.

EXPRESSION OF THE Nogo PROTEIN IN THE PRIMARY CULTURED ENSHEATHING CELLS FROM OLFACTORY BULB OF ADULT RAT BY CONFOCAL LASER SCANNING MICROSCOPE / 解剖学报

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.\;[

EXPRESSION OF GDNF,GFAP AND NGFRp75 BY OLFACTORY ENSHEATHING CELLS IN ADULT RAT AND GOLDEN HAMSTER / 解剖学报

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.

THE SEGMENTAL DISTRIBUTION OF THE LABELLED SYMPATHETIC PREGANGLIONIC NEURONS AFTER HORSERADISH PEROXIDASE(HRP)INJECTION INTO THE COELIAC GANGLION OF THE CAT / 解剖学报

Twelve adult cats were used in our experiment.Under sodium pentobarbitalanesthesia one to two mg of HRP(Sigma Type Ⅵ),dissolved in 7 to 15 microlitresof distilled water,was slowly injected into the ganglion at two or three sites.Theleft coeliac ganglion was injected in seven cats,the right in other four and in oneboth sides were injected.After postoperative survival of 2～5 days,the animalswere perfused through the left ventricle with a mixture of 2% paraformaldehydeand 1.25% glutaraldehyde in 0.1 M phosphate buffer(pH 7.4).Serial frozen crosssections of the spinal cord were cut at 48 micra after postfixation and all sectionsof even numbers were selected and processed with the blue reaction according toMesulam's eighth procedure(1976)and counterstained with neutral red.The labelled cells were found in ipsilateral cord of the injected ganglion,andwere distributed in a transverse zone which may be divided into four nuclei:1.thenucleus intermediolateralis pars principalis(IL_p)2.the nucleus intermediolateralispars funicularis(IL_f)3.the nucleus intercalatus(IC)and 4.the nucleus interca-latus pars paraependymalis(IC_(pe)).The total number of the labelled cells in eachcase varies from 1,179 to 92.The segmental range of the labelled cells extendedfrom the T_1 to the L_1 segments in the longest and from the T_4 to the T_(10)or fromthe T_3 to T_9 segments in the shortest.The data were treated with the statisticalweighted method,and the result shows:about 69.93%?0.57 of the total labelledcells are located in the spinal segments from T_5 to T_8,and about 21.61%?0.52 inthe T_6 segment.The percentage of labelled cells in the four nuclei given above isas follows:IL_p:77%,IL_f:12%,IC:9% and IC_(pe):2%.Furthermore the majorityof labelled cells in IC and IC_(pe)were found in T_(5～8)segments,the greatest densityof them was in the T_6 segment of spinal cord.We found no obvious difference between the left and right in the pattern of thedistribution and the segmental arrangment of the labelled sympathetic preganglionicneurons.

TRANSECTION OF THE OPTIC NERVE INDUCES EXPRESSION OF THE SMALL HEAT SHOCK PROTEIN HSP27 IN THE OPTIC PATHWAY OF GOLDEN HAMSTER / 解剖学报

Objective To observe the expression of the small heat shock protein (HSP27) in the optic chiasma (OC), optic tract (OT), dorsal lateral geniculate body(LG) and superior colliculus (SC) of the adult golden hamster after intraorbital transection of the left optic nerve (ON). Methods The experimental animals were left to survive for l, 2, 3, 4, 5, 6, 8 weeks following ON transection. The animals were perfused with formol-saline and brains were excised, sectioned and stained with the immunohistochemistry. The sections were observed under the light microscope, the optical density (A) was measured and the data were analysed statistically. Results Immunohistochemical results indicated that the HSP27-expressions were not different between the right and left side of the OC, OT, LG and SC in normal or sham-operation controls. However, following transection of the left ON, HSP27 immunohistochemical stainings in the right site of OC, OT,LG and SC were increased, comparing with the left side. The maximum difference of HSP27 immunostaining between the right and left side appeared in the lst week following left ON axotomy. The sharply decrease of the A difference occurred at the 2nd week after axotomy with insignificant changes in the subsequent several weeks. And the significant A difference was observed in most time except 6th week. Most of HSP27-positive cells had morphological appearances similar to astrocytes with smaller cell body and numerous processes. Conclusion After the transection of monolateral ON, HSP27 expressions in the contralateral optic pathway of brain increased and persisted up to 8 weeks. This result suggested that the increase of HSP27 expression had something to do with the injury of the optic pathway, but the mechanism and biological significance of the increase in HSP27 expression level required to be studied further.;

THE SEGMENTAL DISTRIBUTION OF THE LABELLED SPINAL GANGLIONIC CELLS AFTER HRP INJECTION INTO THE COELIAC GANGLION OF THE CAT / 解剖学报

Nine healthy adult cats were used in the investigation.Lumbar incision wasmade in one side and the coeliac ganglion was exposed under Nembutal anesthesia.One or two mg of HRP(Sigma Type Ⅵ)dissolved in 7 to 18 microlitres of distilled water was slowly injected into the ganglion via two or three insertions.Theleft coeliac ganglion was injected in four cats,the right in the other four,and theganglia of both sides were injected in the rest one.After a postoperative survivalof 4～5 days,the animals were perfused through the heart with 2% paraformalde-hyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer(pH 7.4).Frozen serialsections(48 micra thick)of the postfixed spinal ganglia(C_3～L_7)were made andprocessed for the blue reaction using benzidine according to the Mesulam's eighthprocedure.The sections were counterstained with neutral red.HRP granules in the labelled cells in the spinal ganglia were discrete and easilyidentified under bright-field microscope,although the number of the granules variedfrom cell to cell.After the injection into the coeliac ganglia in ten sides,a totalnumber of 12,381 labelled cells were observed in the spinal ganglia.The greatermajority of them(more than 99%)were found ipsilateral to the side of injection.The labelled cells were found in the spinal ganglia from T_2 to L_2,but most of thesecells(78.1%)were distributed in the T_5 to T_9 segments.Furthermore,56.26% ofthe total number of them was situated at the level of T_(6～8),with the highest densityof the labelled cells in the seventh thoracic ganglion,constituting 22.19% of thetotal number.Although the number of labelled cells observed varied from case tocase,their percentage in the constitution of each ganglion in every case was similarto the general plan given above.There is no conspicuous difference between theleft and right sides in respect to the segmental distribution of labelled cells.Thelabelled cells were mainly of medium and small sizes.The larger cells,whichaccount for only 2.78% of all labelled cells,were very rare,and most of themwere found in the T_(5～9)spinal ganglia.The results of our investigation show that the visceral sensory fibers passingthrough the coeliac ganglion in one side come mostly from the ipsilateral spinalganglia T_5 to T_9.And about half of these fibers originate from T_6 to T_8 ganglia.Considering the fact that the greater majority of labelled cell bodies are of mediumor small size,it has been inferred that the fibers that arise from them are finemyelinated or unmyelinated and a part of them,at least,mediate the visceral pain.The labelled larger cells might be related to Pacinian corpuscles in the mesentery.It is difficult for the superior mesenteric ganglion to be separated from the coeliacganglion in cat;therefore,the labelled sensory neurons described above include,at least,a certain number of cell bodies of afferent fibers which traverse thesuperior mesenteric ganglion.

STUDIES ON PURIFIACATION AND IDENTIFICATION OF THE OLFACTORY ENSHEATHING CELLS FROM BOTH ADULT MAN AND CANINE / 解剖学报

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.

EFFECT OF bFGF ON THE PROLIFERATION OF CULTURED OLFACTORY EPITHELIUM ENSHEATHING CELLS IN ADULT RAT / 解剖学报

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.

EXPRESSION OF STAT3 PROTEIN IN THE RETINA OF HAMSTER DURING EARLY POSTNATAL DEVELOPMENT / 解剖学报

Objective To investigate the expression and distribution of STAT3 in the retina during the early development of postnatal hamster. Methods Immunocytochemical method and Western blot analysis were used. Results The expression of STAT3 was found in all layers of the retina in all newborn hamsters,most pronounced in the ganglion cell layer(GCL),the inner plexiform layer(IPL)and the inner area of neuroblastic layer(NBL).With the development,the STAT3 immunoreactivity was gradually restricted in the cytoplasm and the nucleus of the retinal ganglion cells.The result of the Western blot showed that the STAT3 was highly expressed in the first week of postnatal development and then reduced gradually till the lowest point in the adulthood.Conclusion The expression of STAT3 protein may be closely related to the early development of the retina in the postnatal hamster.

MORPHOLOGICAL STUDY ON OLFACTORY ENSHEATHING CELLS DERIVERED FROM OLFACTORY BULBS AND NASAL OLFACTORY EPITHELIUM IN ADULT RAT / 解剖学报

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.

THE DISTRIBUTION OF NOS POSITIVE NEURONS AND FIBERS IN SPINAL INTERMEDIATE GRAY OF SPONTANEOUSLY HYPERTENSIVE RATS / 解剖学报

Objective To investigate the distribution of nitric oxide synthase (NOS) in the spinal intermediate gray and it's relationship with hypertension. Methods Using NADPH\|d histochemistry method, the distribution of NOS in the spinal intermediate gray was investigated under microscope and analyzed quantitatively with image analysis system. Results 1 NOS positive neurons were observed mainly in the nucleus intermediolateralis thoracolumbalis pars principalis(ILp) and paracentral gray, as well as in the area of nucleus intercalatus spinalis. NOS positive cells existed in groups in ILp. The size and density of NOS positive cell groups differed with different spinal segments, no significant difference could be found in the distribution pattern of NOS positive neurons in the spinal intermediate gray between SHR and WKY. 2 The A value of NOS positive substance in the ILp of SHR was significantly lower than that in WKY. Conclusion The results imply that lower expression of NO in the area of ILp in SHR might be related to the development and maintenance of hypertension in SHR.\;