OBJECTIVETo constitute the animal model of unilateral olfactory nerve transection and observe the expression level and distribution of odorant receptors.

METHODSThirty-two rats were divided into two groups: the olfactory nerve transection group (20) and the control group (12). The former group received the operation to transect the left olfactory nerve following the left olfactory bulb was exposed under microscope and the latter group did not give any disposal. At every stage of five days, two weeks, four weeks and six weeks after the operation, five rats from the nerve transection group and three from the control group were anaesthetized simultaneously, and olfactory epithelium were taken out after transcardial perfusion, then paraffin imbedding. Coronal sections were sliced for HE staining to observe the thickness changes of the olfactory epithelium, and for in situ hybridization (ISHs) to investigate the expression of olfactory receptor genes (Olr287, Olr226, Olr1493 and Olr1654) in the epithelium, also to evaluate the changes of the expression level and location of the selected receptors during the regeneration of olfactory epithelium.

RESULTSHE staining showed that 5 days after the operation cell quantity and thickness of the olfactory epithelium decreased obviously, which increased gradually 2 or 4 weeks after operation. After 6 weeks' recovery, the thickness of the epithelium could reach the control level. The pattern of cell staining by ISH showed a specific spatial distribution along the anteroposterior (AP) and dorsoventral (DV) axis. Evidence suggested that odorant receptors were distributed in continuous and multiple overlapping bands in the normal or nerve transected-recovered epithelium rather than in the conventionally accepted three or four zones. The data also demonstrated that the distribution of sensory neuron types, as identified and defined by odorant receptor expression, was restored to normal or nearly so by 6 weeks after operation. Likewise, the numbers of probe-labeled neurons in the nerve transected-recovered had an obvious decrease 5 days after olfactory nerve transection. Reactive cells (x(-) +/- s) of Olr1493 in the operated side was (53.9 +/- 19.9), compared with (419.0 +/- 21.2) in the unoperated side, there was statistic significance between them (t = 63.960, P < 0.01). Reactive cells increased gradually according to the regeneration of the epithelium, and were nearly equivalent to the normal side 6 weeks later without significant differentiation (t = 2.600, P > 0.05), according to the absolute positive cells in the operated and unoperated side of (417.8 +/- 32.4) and (445.3 +/- 10.0) respectively.

CONCLUSIONThe regeneration of the sensory neurons and receptors, both the number and the distribution, can recover to normal after olfactory nerve transection.

Animals ; Male ; Olfactory Mucosa ; metabolism ; Olfactory Nerve ; metabolism ; surgery ; Olfactory Nerve Injuries ; Olfactory Receptor Neurons ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Odorant ; genetics ; metabolism

The treatment of spinal cord injury is always a stubborn problem for neurosurgeons because nerve cell cannot regenerate and the glia scar can prevent the axonal regeneration. Olfactory ensheathing cells (OECs) is a kind of especial glia cell, which possesses the character of horizontal cell of central nervous system and schwann cell. Many foundational and clinical studies showed that the olfactory ensheathing cellscan promote axonal regeneration and prove axonal growth, some progress is made and this is bringing hope for treatment of spine injury.
Animals ; Cell Transplantation ; Cells, Cultured ; Humans ; Nerve Regeneration ; Neuroglia ; cytology ; transplantation ; Olfactory Mucosa ; cytology ; transplantation ; Spinal Cord Injuries ; therapy

OBJECTIVETo explore the impact and mechanism of electro-acupuncture (EA) on olfactory ensheathing cells (OECs) transplantation of spinal cord injury (SCI) axonal regeneration.

METHODSIn the experiment, 72 adult Sprague Dawley male rats weighted (220±20) g underwent contusion and transection method to cause the T9 model of spinal cord injury, were randomly divided into four groups involving model group, EA group,OECs group,and EA+OECs group. 5% fluorescein gold (FG) solution of 0.5 µl was injected into rats' spinal cord at 4 weeks and 8 weeks after SCI, a series of tests were performed including fluorescein gold(FG) retrograde tagging, BBB scores.

RESULTS(1)The BBB scores level among four groups had no differences from the 1st day to the 1st week after the SCI (P>0.05). From the 3rd week after the SCI, the BBB scores level in EA+ OECs group were obviously higher than that of other groups (P<0.05). (2)The result of the fluorescein gold (FG) retrograde tagging showed at 4 weeks and 8 weeks after treatment FG positive nerve fibers were observed in SCI region. In EA+OECs group the number of FG positive nerve fibers was more than other three groups, and the fibers were more regularly arranged than other three groups.

CONCLUSIONThe combination of electro-acupuncture and OECs transplantation can recover the pathway of nerve conduction and promote nerve fibers regeneration and hind limb function recovery for SCI rat, and can guide the trend of the axonal regeneration.

Animals ; Axons ; physiology ; Cell Transplantation ; Combined Modality Therapy ; Electroacupuncture ; Humans ; Male ; Nerve Regeneration ; Olfactory Nerve ; physiopathology ; transplantation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; physiopathology ; therapy

OBJECTIVETo investigate whether or not allografted olfactory mucosa gliacytes could repair peripheral nerve injure.

METHODSOlfactory mucosa gliacytes had been cultured in vitro for 2 weeks, then purified and condensed for later transplantation.Sixty adult female Wistar rats were randomized into 2 groups of 30 rats each, A (control) and B (test). Rats' left sciatic nerves were excised 25 mm long axons and retained epineurium lumen anastomosed to proximal ends. Culture mediums, and olfactory mucosa gliacytes were transplanted into epineurium lumen of A and B groups respectively. At 3 months postoperatively, the regenerations of injured sciatic nerves were evaluated by methods of macroscopy, photomicroscopy, transmission electron microscopy, retro-marked fluorescence red, the condensation of glial fibre acid protein (GFAP) and nerve growth factors (NF) assayed by immunofluorescence, and the concentration of myelin basic protein (MBP) and neurofilament protein (NF) assayed by enzyme linked immunosorbent assay.

RESULTSThe regenerations of injured sciatic nerves were superior in B group to in A group; the transportation distance of retro-marked fluorescence red were longer in B group than in A group (P < 0.01). The condensations of GFAP and NGF were more dense in B group than in A group. The concentrations of MBP and NF were more high in B group than in A group (P < 0.01). The function scores of injured limbs were superior in B group to in A group (P < 0.01). The quantifications of nerve fibers and myelin fibers of injured sciatic nerve were larger in B group than in A group (P < 0.01).

CONCLUSIONAllografted olfactory mucosa gliacytes could repair injured nerve defect.

Animals ; Cell Transplantation ; Cells, Cultured ; Disease Models, Animal ; Female ; Nerve Regeneration ; Neuroglia ; cytology ; Olfactory Mucosa ; cytology ; Random Allocation ; Rats ; Rats, Wistar ; Sciatic Nerve ; injuries ; Transplantation, Homologous

OBJECTIVETo observe the effects of cryopreserved olfactory ensheathing cells (OECs) transplantation on axonal regeneration and functional recovery following spinal cord injury in adult rats.

METHODSTwenty-four rats were divided into experimental and control groups, each group having 12 rats. The spinal cord injury was established by transecting the spinal cord at T10 level with microsurgery scissors. OECs were purified from SD rat olfactory bulb and cultured in DMEM (Dulbecco's minimum essential medium) and cryopreserved (-120 degree) for two weeks. OECs suspension [(1-1.4)x10(5)/ul] was transplanted into transected spinal cord, while the DMEM solution was injected instead in the control group. At 6 and 12 weeks after transplantation, the rats were evaluated with climbing test and MEP (moter evoked potentials) monitoring. The samples of spinal cord were procured and studied with histological and immunohistochemical stainings.

RESULTSAt 6 weeks after transplantation, all of the rats in both transplanted and control groups were paraplegic, and MEPs could not be recorded. Morphology of transplanted OECs was normal, and OECs were interfused with host well. Axons could regrow into gap tissue between the spinal cords. Both OECs and regrown axons were immunoreactive for MBP. No regrown axons were found in the control group. At 12 weeks after transplantation, 2 rats (2/7) had lower extremities muscle contraction, 2 rats (2/7) had hip and/or knee active movement, and MEP of 5 rats (5/7) could be recorded in the calf in the transplantation group. None of the rats (7/7) in the control group had functional improvement, and none had MEPs recorded. In the transplanted group, histological and immunohistochemical methods showed the number of transplanted OECs reduced and some regrown axons had reached the end of transected spinal cord. However, no regrown axons could be seen except scar formation in the control group.

CONCLUSIONSCryopreserved OECs could integrated with the host and promote regrowing axons across the transected spinal cord ends.

Animals ; Axons ; pathology ; physiology ; Cell Transplantation ; Cryopreservation ; Immunohistochemistry ; Male ; Nerve Regeneration ; Olfactory Nerve ; cytology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; physiopathology ; surgery

OBJECTIVESTo observe olfactory ensheathing glia (OEG) survival and repair in vivo for spinal cord injury after OEG transplantation.

METHODSThe OEG was cultured with the olfactory bulb of Wistar neonate rats. The spinal cords contusion was made in group A, B, and C with the New York University impactor, then complete transection was performed in the contusion area in group A. OEG labeled by Hoechst was transplanted in group A and B. In group C, DMEM were injected. In group D, laminectomies were done without cord contusion and transection. The functional recovery of the spinal cord injury [Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale scores] and changes of body weight were observed. The tissue sections were done 24 weeks postoperatively. HE staining, neurofibril (NF) immunohistochemical staining, and silver staining were performed respectively to observe the pathologic changes and axon regeneration. The survival of OEG labeled by Hoechst was observed under the fluorescence microscope.

RESULTSLocomotive behaviour improved 4 weeks postoperatively. The BBB locomotion scores of group A and B were significantly higher than that of group C in all periods (from 4 weeks to 24 weeks) (P < 0.01). Sixteen weeks after operation, the BBB locomotion scores became stable and showed no change. HE staining showed that the area of spinal cord injury was disorder and the number of nerve cell was more in group A and B. In group C, there was the obvious cavum and few wring nerve fiber in the area of spinal cord injury. The nerve fibers innervated to the injuried area in group A and B were more than that of group C, but less than that of group D. A great number of OEG labeled by Hoechst were observed around spinal injuried area under fluorescence microscope. After operation, the body weight reduced in every group. The body weight of group D had recovered after 2 weeks and gradully increased. After 4 weeks, the body weight in group A, B, and C decreased to the minimum and were significantly less than that of group D (P < 0.01). After this, body weight in group A and B increased and was significantly more than that of group C (P < 0.05).

CONCLUSIONSOEG transplantation can promote the axons regeneration and the recovery of locomotion function in experimental spinal cord injuries.

Animals ; Animals, Newborn ; Axons ; physiology ; Cells, Cultured ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Nerve Regeneration ; Neuroglia ; cytology ; transplantation ; Olfactory Bulb ; cytology ; transplantation ; Olfactory Mucosa ; cytology ; transplantation ; Rats ; Rats, Wistar ; Spinal Cord ; physiopathology ; surgery ; Spinal Cord Injuries ; surgery

OBJECTIVETo observe the survival and the number of olfactory ensheathing cells (OECs) transplanted in the contused spinal cord, so as to provide a basis for further studying the biological action of OECs.

METHODSThe rat spinal cords were contused with NYU-impactor II at T10 level by dropping a 10 g rod from a height of 25 mm. At the 1st week after injury, OECs isolated freshly from green fluorecense protein (GFP) of the rats were transplanted into the spinal cord at injured site and other two sites 1 mm apart from the caudal and rostral ends with the OECs number of 30000/μl x 3 = 90000. The survival and the number of OECs were qualitatively and semi-quantitatively observed under the fluorescense microscope from 1 week to 13 weeks after transplantation. The motor function of the cord was evaluated with BBB score.

RESULTSGFP-OECs could survive at least for 13 weeks within the contused spinal cord. Their arrangement was from tight to loose and their number was decreased from 1 week to 13 weeks after injury. The average number of GFP-OECs was 536 at the 1st week, which was less than 1% of the number as compared with original transplantation. After then, the number of GFP-OECs was continually decreased, but the most obvious decrease was found during 1 week to 2 weeks. The extent of decrease at other time points was relatively mild. In contrast to the cell number, motor function of the cord was gradually recovered after transplantation.

CONCLUSIONSThe survival and the number of GFP-OECs are different between the animals and are affected by the pathological reaction of the host cord. Also it is related to the motor function recovery of the contused cord.

Animals ; Cell Count ; Cell Survival ; Cell Transplantation ; Motor Activity ; Nerve Degeneration ; Olfactory Bulb ; cytology ; transplantation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; physiopathology ; Spinal Cord Injuries ; physiopathology ; surgery

OBJECTIVETo observe whether olfactory ensheathing cells could be used to promote axonal regeneration in a spontaneously nonregenerating system.

METHODSAfter laminectomy at the lower thoracic level, the spinal cords of adult rats were exposed and completely transected at T10. A suspension of ensheathing cells was injected into the lesion site in 12 adult rats, and control D/F-12 (1:1 mixture of DMEM and Ham's F-12) was injected in 12 adult rats. Six weeks and ten weeks after cell transplantation, the rats were evaluated by climbing test and motor evoked potentials (MEPs) monitoring. The samples were procured and studied with histologicl and immunohistochemical methods.

RESULTSAt the 6th week after cell transplantation, all the rats in both the transplanted and control groups were paraplegic and the MEPs could not be recorded. At the 10th week after cell transplantation, of 7 rats in the control group, 2 rats had muscles' contraction of the lower extremities, 2 rats had hips and/or knees' active movement; and 5 rats' MEPs could be recorded in the hind limbs in the transplanted group (n=7). None of the rats in the control group had functional improvement and no MEPs recorded (n=7). Numerous regenerating axons were observed through the transplantation and continued to regenerate into the denervated host tract. Cell labelling using anti-Myelin Basic Protein (MBP) and anti-Nerve Growth Factor Receptor (anti-NGFR) indicated that the regenerated axons were derived from the appropriate neuronal source and that donor cells migrated into the denervated host tract. But axonal degeneration existed and regenerating axons were not observed within the spinal cords of the adult rats with only D/F-12 injection.

CONCLUSIONSThe axonal regeneration in the transected adult rat spinal cord is possible after ensheathing cells transplantation.

Animals ; Axons ; physiology ; Brain Tissue Transplantation ; Cell Transplantation ; Male ; Nerve Regeneration ; Olfactory Bulb ; cytology ; transplantation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; physiology ; Spinal Cord Injuries ; physiopathology ; surgery

OBJECTIVE: To investigate a new way to yield plenty of high purity olfactory ensheathing cells (OECs) and its biocompatibility with appropriate scaffolds. METHODS: OECs were prepared from neonatal Wister rats and co-cultured with poly [LA-co-(Glc-alt-Lys)] (PLGL). Its contact angle, adherent rate, and activity rate were tested. RESULTS: The contact angle of poly (D, L-lactic acid) (PDLLA) (84.5 degree+/-1.5 degree) was significantly higher than that of PLGL (52.6 degree+/-0.8 degree), the adherent rate of PLGL (80%) was significantly higher than that of the PDLLA (57%), and the activity rate of PLGL (88%) was much higher than that of the PDLLA (76%). CONCLUSION PLGL possesses better hydrophilicity and biocompatibility than PDLLA, and it can provide a better cell growth circumstance which is helpful for the effective treatment of nerve injury.
Animals ; Animals, Newborn ; Biocompatible Materials ; Cells, Cultured ; Lactic Acid ; chemical synthesis ; pharmacology ; Nerve Regeneration ; Olfactory Bulb ; cytology ; Polyglycolic Acid ; chemical synthesis ; pharmacology ; Polylactic Acid-Polyglycolic Acid Copolymer ; Rats ; Rats, Wistar ; Spinal Cord Injuries ; physiopathology ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVECombine olfactory ensheathing glia (OEG) implantation with ex vivo non-viral vector-based neurotrophin-3 (NT-3) gene therapy in attempting to enhance regeneration after thoracic spinal cord injury (SCI).

METHODSPrimary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1(+)-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord (T9) contusion by the New York University impactor. The animals in 3 different groups received 4x10(5) OEG transfected with pcDNA3.1(+)-NT3 or pcDNA3.1(+) plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted.

RESULTSNT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1(+)-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting.

CONCLUSIONNon-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT-3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.

Animals ; Animals, Newborn ; Brain Tissue Transplantation ; methods ; Cells, Cultured ; DNA, Recombinant ; therapeutic use ; Disease Models, Animal ; Female ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Graft Survival ; genetics ; Growth Cones ; metabolism ; ultrastructure ; Nerve Regeneration ; genetics ; Neuroglia ; metabolism ; transplantation ; Neurotrophin 3 ; biosynthesis ; genetics ; Olfactory Bulb ; cytology ; transplantation ; Paralysis ; metabolism ; physiopathology ; therapy ; Plasmids ; genetics ; Rats ; Rats, Wistar ; Recovery of Function ; genetics ; Spinal Cord Injuries ; metabolism ; physiopathology ; therapy ; Treatment Outcome ; Up-Regulation ; genetics