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

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

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

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

Numerous studies have shown that the health of spiral ganglion neurons is highly important for hearing. As a trophic factor of spiral ganglion neurons, neurotrophin 3 (NT3) is a potential candidate for prevention of spiral ganglion neuron degeneration in human. In our experiments, efficient transduction and long term expression of foreign gene of cochlea cells has been found with adenovirus carried lacZ gene (Ad-lacZ). A model of guinea pig deafness was made by intense noise exposure, which destroyed the entire organ of Corti in the middle part of the cochlea. Seven days after noise exposure, the animals were anesthetized and 1 10(8) recombinant adenoviral particles were injected into the scala tympani through the round window membrane. Animals inoculated with neurotrophin 3 adenovirus(Ad-NT3) were designated as the experimental group, animals inoculated with Ad-lacZ vector served as the control group. Four weeks after the inoculation of the virus, NT3 immunoreactivity was observed in the Ad-NT3 inoculated group. HE histochemical staining results showed that in the Ad-lacZ injected group, the neuronal degeneration was severer and the density of spiral ganglion neurons was significantly lower than those in the Ad-NT3 injected group. Our results demonstrate that with adenovirus-mediated overexpression NT3 may be developed into a new treatment to prevent secondary spiral ganglion degeneration following the damage to Corti organ.
Adenoviridae ; genetics ; Animals ; Cochlea ; pathology ; Gene Transfer Techniques ; Genetic Therapy ; Guinea Pigs ; Hearing Loss, Noise-Induced ; pathology ; In Vitro Techniques ; Neurotrophin 3 ; genetics ; Recombination, Genetic

OBJECTIVES: Estrogen is an important hormone for cell growth, development, and differentiation by transcriptional regulation and modulation of intracellular signaling via second messengers. The reduction in the estrogen level after ovariectomy may lead to cognitive impairments associated with morphological changes in areas of the brain mediate memory. The aim of the present study was to find out the effect of tasks on the cognitive function after ovariectomy in rats. METHODS: The animals used in the experiment were 50 Sprague-Dawley female rats. This study applied a hippocampus-independent task (wheel running) and a hippocampus-dependent task (Morris water maze) after ovariectomy in rats and measured the cognitive performance (object-recognition and object-location test) and growth-associated protein 43 (GAP-43) and neurotrophin 3 (NT-3) expression in the hippocampus, which is an important center for memory and learning. RESULTS: There were meaningful differences between the hippocampus-independent and hippocampus-dependent task groups for the object-location test and GAP-43 and NT-3 expression in the hippocampus, but not the object-recognition test. However, the hippocampus-independent task group showed a significant improvement in the object-recognition test, compared to the control group. CONCLUSION: These results suggest that hippocampus-dependent task training after ovariectomy enhances the hippocampus-related memory and cognitive function that are associated with morphological and functional changes in the cells of the hippocampus.
Animals ; Brain ; Cognition Disorders ; Cognition* ; Estrogens ; Female ; GAP-43 Protein ; Hippocampus ; Humans ; Learning ; Memory ; Neurotrophin 3 ; Ovariectomy* ; Rats* ; Rats, Sprague-Dawley ; Second Messenger Systems ; Water

OBJECTIVE: This study aims to investigate the effect of neurotrophin 3 (NT-3) on the osteogenic differentiation of human dental follicle cells (hDFCs). METHODS: hDFCs were isolated and cultured in vitro. Immunocytochemical staining was used to identify the origin of hDFCs. The effects of different NT-3 concentrations on hDFCs proliferation were detected by using CCK-8 assay. The alkaline phosphatase (ALP) activities and mRNA expression levels of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) were determined to investigate the effects of NT-3 on hDFCs osteogenesis. The difference in the number of mineralized nodules was detected using alizarin red staining. RESULTS: Vimentin and cytokeratin staining results showed that hDFCs originated from the mesenchymal cells. NT-3 exerted no evident effect on hDFCs proliferation. The ALP activity and the BMP-2 and OCN mRNA expression levels of hDFCs were significantly improved under treatment with different NT-3 concentrations (25, 50, and 100 ng·mL ⁻¹) compared with those in the control group. BMP-2 and OCN mRNA relative expression levels of hDFCs reached the highest when the NT-3 concentration was 100 ng·mL ⁻¹. The number of mineralized nodules reached the maximum when the hDFCs were treated with 50 and 100 ng·mL ⁻¹ NT-3. CONCLUSIONS Appropriate mass concentration of NT-3 can promote the osteogenic differentiation of hDFCs.
Alkaline Phosphatase ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; Humans ; Mesenchymal Stem Cells ; Neurotrophin 3 ; pharmacology ; Osteocalcin ; metabolism ; Osteogenesis

OBJECTIVE: To construct an adenoviral vector that codes for both human NT3 and EGFP, to confirm the transduction efficiency in rat cochlear cultures and to assess the protection of NT3 on SGNs survival. METHOD: PAdeasy-1 and pAdTrack CMV were used to constructed Ad/NT3 adenovirus and then to transfer postnatal day 3 rat cochlear cultures. The transduction efficiency was determined by microscope observation. The amounts of SGNs were counted to evaluated protection of Ad/NT3 on SGNs survival. RESULT: EGFP positive cells were observed in all cochlear turns. There was approximately 49% in outer sulcus cells and 27% in the interdental cells; less than 2% of the hair cells and SGN. The amounts of SGN of treated Ad/NT3 adenovirus are more than cochlea SGN only Ad/EGFP adenovirus after cultured for 15 days. CONCLUSION Ad/NT3 adenovirus could transduce EGFP and NT3 in large number of supporting cells, but few hair cells or SGNs. The putative release of NT3 from these supporting cells could enhance cell survival and promote neurite outgrowth from SGNs.
Adenoviridae ; genetics ; Animals ; Basilar Membrane ; cytology ; Cell Survival ; genetics ; Cells, Cultured ; Cochlea ; cytology ; Genetic Vectors ; Hair Cells, Auditory ; cytology ; Humans ; Neurotrophin 3 ; genetics ; Rats ; Rats, Inbred F344 ; Transfection