PURPOSE: Plasma neurotrophin-3 (NT-3) levels are associated with several neural disorders. We previously reported that neurotrophins were released from salivary glands following acute immobilization stress. While the salivary glands were the source of plasma neurotrophins in that situation, the association between the expression of neurotrophins and the salivary gland under chronic stress conditions is not well understood. In the present study, we investigated whether NT-3 levels in the salivary gland and plasma were influenced by chronic stress. MATERIALS AND METHODS: Expressions of NT-3 mRNA and protein were characterized, using real-time polymerase chain reactions, enzyme-linked immunosorbent assay, and immunohistochemistry, in the submandibular glands of male rats exposed to chronic stress (12 h daily for 22 days). RESULTS: Plasma NT-3 levels were significantly increased by chronic stress (p<0.05), and remained elevated in bilaterally sialoadenectomized rats under the same condition. Since chronic stress increases plasma NT-3 levels in the sialoadenectomized rat model, plasma NT-3 levels were not exclusively dependent on salivary glands. CONCLUSION: While the salivary gland was identified in our previous study as the source of plasma neurotrophins during acute stress, the exposure to long-term stress likely affects a variety of organs capable of releasing NT-3 into the bloodstream. In addition, the elevation of plasma NT-3 levels may play important roles in homeostasis under stress conditions.
Animals ; Male ; Neurotrophin 3/*blood/genetics ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological/*physiology ; Submandibular Gland/*metabolism

OBJECTIVETo explore a new method to treat brachial plexus root avulsion experimentally by reimplantation combined with transplantation of neural stem cells (NSCs) modified by neurotrophin-3 gene (NT-3).

METHODSThe total RNA was extracted from neonatal rat striatum and the NT-3 cDNA was obtained by reverse transcription and amplified by polymerase chain reaction. The NT-3 gene was transferred into NSCs via the pLEGFP-C1, an expression plasmid vectors. The untransfected NSCs, the pLEGFP-C1 treated NSCs, and the pLEGFP-C1-NT-3 treated NSCs were transplanted into corresponding spinal cord segment with brachial plexus root avulsion. The survival, differentiation, and migration of the transplanted cells were determined under confocal laser scanning microscope or by immunohistochemistry method. The nerve regeneration was evaluated by gross observation, electrophysiological examination and reverse horseradish peroxidase tracing.

RESULTSThe NT-3 gene was successfully amplified and transferred into neural stem cells via the plasmid vectors. The transplanted cells survived, differentiated, and migrated and NT-3 was expressed within the spinal cord. The animals regained some muscle strength which was less than 3-degree muscular strength according to the British Medical Research Council (BMRC) evaluating system. The results of electrophysiological examination and reverse horseradish peroxidase tracing were superior in the pLEGFP-C1-NT-3 group to the NSCs untransfected group or the pLEGFP-C1 group.

CONCLUSIONTransplantation of NSCs modified by NT-3 gene combined with reimplantation is a relatively effective way to treat brachial plexus root avulsion experimentally. It still need further study to improve the results.

Animals ; Brachial Plexus ; injuries ; Neurotrophin 3 ; genetics ; Radiculopathy ; surgery ; Rats ; Replantation ; Stem Cell Transplantation ; Transfection

Animals ; Cerebral Cortex ; metabolism ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred Strains ; Morphine Dependence ; metabolism ; Neurotrophin 3 ; metabolism ; Prosencephalon ; metabolism

OBJECTIVE: To investigate the effect of pretreatment with neurotrophin-3 (NT-3) on intrathecal ropivacaine in rats. METHODS: A total of 144 male Sprague Dawley rats weighing 280-320 g were successfully implanted with microspinal cather following the improved methods of Yaksh. The rats were randomly divided into 4 groups and given saline (Group NS, n=36), 0.5% ropivacaine (Group M, n=36), 1% ropivacaine (Group R, n=36), and ropivacaine+NT-3 (Group T, n=36). The rats received 0.12 mL/ kg body weight of ropivacaine at 0.5% or 1%, or normal saline only, via an implanted intrathecal catheter at 90-min interval for 12 h in Group NS, M, R and T. In the meantime the rats also received NT-3 0.1 mg/kg in group T. On days 1, 3, 5, 7, 14 and 28, we assessed the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), behavioural change and histopathological damage score changed for possible neuronal injury within the spinal cord. RESULTS: Compared with Group NS and Group M, the PWMT and PWTL were significantly higher on 1, 3, 5 d and the histopathological damage score was significantly higher on 1, 3, 5, 7, 14 d in Group R (P<0.05). Compared with Group T, the PWMT and PWTL in Group R were significantly higher on 1, 3, 5 d and histopathological damage score was significantly higher on 5, 7, 14 d (P<0.05). CONCLUSION NT-3 pretreatment in mice has obvious protective effect against repeated intrathecal injection of 1% ropivacaine in the spinal nerve.
Amides ; adverse effects ; Animals ; Injections, Spinal ; Male ; Neurotrophin 3 ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ropivacaine ; Spinal Cord ; drug effects

OBJECTIVE: To study the changes in facial nerve function, morphology and neurotrophic factor III (NT-3) expression following three types of facial nerve injury. METHOD: Changes in facial nerve function (in terms of blink reflex (BF), vibrissae movement (VM) and position of nasal tip) were assessed in 45 rats in response to three types of facial nerve injury: partial section of the extratemporal segment (group one), partial section of the facial canal segment (group two) and complete transection of the facial canal segment lesion (group three). All facial nerves specimen were then cut into two parts at the site of the lesion after being taken from the lesion site on 1st, 7th, 21st post-surgery-days (PSD). Changes of morphology and NT-3 expression were evaluated using the improved trichrome stain and immunohistochemistry techniques ,respectively. RESULT: Changes in facial nerve function: In group 1, all animals had no blink reflex (BF) and weak vibrissae movement (VM) at the 1st PSD; The blink reflex in 80% of the rats recovered partly and the vibrissae movement in 40% of the rats returned to normal at the 7th PSD; The facial nerve function in 600 of the rats was almost normal at the 21st PSD. In group 2, all left facial nerve paralyzed at the 1st PSD; The blink reflex partly recovered in 40% of the rats and the vibrissae movement was weak in 80% of the rats at the 7th PSD; 8000 of the rats'BF were almost normal and 40% of the rats' VM completely recovered at the 21st PSD. In group 3, The recovery couldn't happen at anytime. Changes in morphology: In group 1, the size of nerve fiber differed in facial canal segment and some of myelin sheath and axons degenerated at the 7th PSD; The fibres' degeneration turned into regeneration at the 21st PSD; In group 2, the morphologic changes in this group were familiar with the group 1 while the degenerated fibers were more and dispersed in transection at the 7th PSD; Regeneration of nerve fibers happened at the 21st PSD. In group 3, most of the fibers crumbled at the 7th PSD and no regeneration was seen at the 21st PSD. Changes in NT-3: Positive staining of NT-3 was largely observed in axons at the 7th PSD, although little NT-3 was seen in the normal fibers. CONCLUSION Facial palsy of the rats in group 2 was more extensive than that in group 1 and their function partly recovers at the 21st PSD. The fibres' degeneration occurs not only dispersed throughout the injury site but also occurred throught the length of the nerve. NT-3 immunoreactivity increased in activated fibers after partial transection.
Animals ; Facial Nerve ; metabolism ; pathology ; physiopathology ; Facial Nerve Injuries ; classification ; metabolism ; pathology ; physiopathology ; Neurotrophin 3 ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo study the protective effect of local gene therapy with adeno-associated virus (AAV)-mediated neurotrophin-3 (NT-3) on the function and morphology of the cochlea of guinea pigs with gentamicin-induced hearing loss.

METHODSHearing loss was induced with gentamicin (80 mg.kg(-1).day(-1) injected intramuscularly) in 18 pigmented guinea pigs 4 days prior to gene transfer. The guinea pigs were then divided into groups A, B, and C for AAV-mediated NT-3 gene transfer (n=7), AAV infection (n=7) or no particular intervention (n=4), respectively. Mini-Osmotic pump were implanted in either side of the ears in groups A and B, and the guinea pigs were injected with gentamicin (80 mg.kg(-1).day(-1)) intramuscularly since the operation day for 10 consecutive days. In group C, only gentamicin was administrated. Before and 14 days after gentamicin administration, auditory brainstem response audiometry (ABR) and distort-product otoacoustic emissions (DPOAE) were recorded, and the animals sacrificed to observe the morphological changes of the cochlear microscopically.

RESULTSCompared with groups B and C, the animals in group A showed better auditory ability (ABR and DPOAE) and significantly higher surviving rate of the outer hair cells (P<0.05).

CONCLUSIONAAV-mediated NT-3 gene transfer may protect and repair the cochlear hair cells and auditory function damaged by aminoglycoside ototoxicity in guinea pigs, and aseptic procedure is of vital importance in cochlear local gene therapy.

Animals ; Cochlea ; physiopathology ; Dependovirus ; genetics ; metabolism ; Genetic Therapy ; Gentamicins ; adverse effects ; Guinea Pigs ; Hearing Loss ; chemically induced ; therapy ; Neurotrophin 3 ; therapeutic use

OBJECTIVETo explore the method for obtaining olfactory ensheathing cells from human fetal olfactory mucosa by cell culture for selective adhesion in the presence of neurotrophin-3 (NT3) and low-concentration serum.

METHODSThe olfactory ensheathing cells were cultured alternatively in DMEM/F12 culture medium containing 10% fetal bovine serum (FBS) and the medium containing NT3 and 2.5% FBS every 72 h. The cells were observed for morphological changes and identified using immunocytochemistry with P75NTR and GFAP, and the cell purity was estimated.

RESULTSThe olfactory ensheathing cells from human fetal olfactory mucosa were positive for P75(NTR) and GFAP, and in in vitro culture, the cells exhibited dipolar or tripolar appearance with long thin neurites. On the 9th day of cell culture, the purity of the olfactory ensheathing cells reached about 83%.

CONCLUSIONThe olfactory ensheathing cells can be obtained by in vitro culture for selective adhesion in the presence of NT3 and low-concentration serum.

Cell Culture Techniques ; methods ; Cell Separation ; methods ; Cells, Cultured ; Culture Media ; Fetus ; Humans ; Neurotrophin 3 ; pharmacology ; Olfactory Bulb ; cytology ; Olfactory Mucosa ; cytology

OBJECTIVETo study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.

METHODSSeries of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.

RESULTSEach fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.

CONCLUSIONDok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Neurites ; drug effects ; physiology ; Neurotrophin 3 ; pharmacology ; PC12 Cells ; Rats ; Receptor, trkC ; metabolism ; Transfection

OBJECTIVETo investigate the expression of neurotrophin-3 (NT-3) gene in Schwann cells of rat sciatic nerve introduced by an adenovirus vector in vivo.

METHODSA recombinant adenovirus vector for NT-3 (Ad-NT-3) was propagated in 293 packaging cells and titered with tissue culture infectious dose(50) (TCID(50)). Ad-NT-3 was injected directly into the rat sciatic nerve after transection and immediate repair. Immunohistochemical staining was employed to determine the expression of NT-3 in Schwann cells in rat sciatic nerve and the expressive intensity of the tissue slices of the sciatic nerve was measured with LEICA M550 image analysis system.

RESULTSOn the 2nd day after injection of Ad-NT-3, positive stain in the Schwann cells was apparent in the vicinity of anastomosis. NT-3 expression increased significantly on the 7th day (P<0.01) and then decreased 14-28 days after injection (P<0.01). There was no significant difference of NT-3 expression between the 14th and 28th day groups (P<0.05). Compared with the 2nd day group, the 14th and 28th day groups still maintained a relatively high level of NT-3 (P<0.01). Intact and repaired nerves, which were injected with adenovirus encoding LacZ genes (Ad-LacZ) or physiological saline served as controls, showed no NT-3-positive Schwann cells.

CONCLUSIONSAn adenovirus vector can be used to induce efficiently the expression of NT-3 gene in Schwann cells of rat peripheral nerves following nerve injury and repair, which suggests that neurotrophic factors can be introduced into Schwann cells with an adenovirus vector to promote peripheral nerve regeneration.

Adenoviridae ; genetics ; Animals ; Gene Expression ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Immunohistochemistry ; Lac Operon ; genetics ; Neurotrophin 3 ; genetics ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; metabolism ; Sciatic Nerve ; injuries

The present study aimed to study the changes of neurotrophin-3 (NT-3) expression of intrafusal muscle fibers in rat soleus muscles under simulated weightlessness. The tail-suspension (SUS) rat model was used to simulate weightlessness. Forty mature female Sprague-Dawley rats were randomly assigned to ambulatory control (CON), 3-day SUS, 7-day SUS, 14-day SUS and 21-day SUS groups. Immunohistochemistry ABC staining method and enzyme linked immunosorbent assay (ELISA) were used to detect the NT-3 expression of intrafusal muscle fibers in rat soleus muscles. The results from the immunohistochemistry staining technique showed that the extrafusal muscle fibers did not exhibit the NT-3-like immunoreactivity, and NT-3-like immunoreactivity was mainly expressed in nuclear bag 1 and nuclear bag 2 fibers of the muscle spindles. The ELISA results showed that the expression quantity of NT-3 in rat soleus muscles in control, 3-day SUS, 7-day SUS, 14-day SUS and 21-day SUS groups were (14.23±1.65), (14.11±1.53), (13.09±1.47), (12.45±1.51) and (9.85±1.52) pg/mg of tissue respectively. Compared to the control group, the expression quantity of NT-3 was significantly decreased after 14 days of SUS (P<0.05). After 21 days of SUS, the NT-3 expression was further reduced (P<0.01). These results suggest that simulated weightlessness induces an obvious decrease in the NT-3 expression level of intrafusal fibers in rat soleus muscles. Accompanying the simulated weightlessness extension, NT-3 expression in rat soleus muscle spindles is progressively decreased. These changes may contribute to the proprioceptive adaptations to microgravity.
Animals ; Down-Regulation ; Female ; Hindlimb Suspension ; Muscle Spindles ; metabolism ; Muscle, Skeletal ; metabolism ; Neurotrophin 3 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Weightlessness Simulation