• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제31권3호
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• pp.199-218
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• 2005

Purpose of Study: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. Materials and Methods: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with ${\beta}$-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells$(1{\times}10^6)$ or BDNF-Ad infected Schwann cells$(1{\times}10^6)$ were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. Results: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were $1.54{\pm}4.0{\times}10^6$ and $9.66{\pm}9.6{\times}10^6$. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell $0.69\;{\mu}g/{\mu}l$ of DNA was detected and in BDNF-Adenovirus transfected Schwann cell $0.795\;{\mu}g/{\mu}l$ of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. Conclusion: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제17권4호
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• pp.317-330
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• 1995

The purpose of this study is to observe the stimulant effect of the isolated nerve segment on the peripheral nerve regeneration using silicone tube in rats. Sprague-Dawley female albino rats were used as experimental animals and nerve defects were made by resection of 14 mm-long segment of right sciatic nerves. In control group, the defects were bridged with the silicone tubes. In experimental group, the defects were bridged with the same manner and additionally, 2mm-long nerve segments were inserted at the center of the silicone tubes. 3 months later, the regenerated nerve tissue within the silicone tubes were examined by histologic study. Axonal diameters and numbers of axons were measured and all data were analyzed by using SAS package program. The results obtained were as follows : 1. The experimental group was increased than the control group in general diameters of the regenerated nerve fiber.(P<0.05) 2. The diameters of axons were increased in the experimental group compare to the control group.(P<0.05) 3. The numbers of axons were increased in the experimental group compare to the control group.(P<0.05)

• 한방재활의학과학회지
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• 제19권3호
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• pp.15-32
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• 2009

Objectives : The purpose of this Study was to identify the effect of Dokhwalgisaeng-tang(Duhuoqisheng-tang) and Jungsongouhyul pharmacopuncture on pain control and nerve regeneration after crush injury in rat sciatic nerve. Methods : Neuropathic pain was induced by crush-induced model of right sciatic nerve. Animal groups were divided as follows; Group I: no treatment control group, Group II : experimental group treated with Dokhwalgisaeng-tang(Duhuoqisheng-tang), Group III : experimental group treated with Jungsongouhyul pharmacopuncture, and Group IV : experimental group treated with Dokhwalgisaeng-tang(Duhuoqisheng-tang) and Jungsongouhyul pharmacopuncture. To evaluate pain intensity, each group was observed paw withdrawal threshold and immunoreactivity on the c-fos before and after respective treatments in five hours, first, third, and fifth day. To evaluate nerve regeneration, those were observed SFI(Sciatic Functional Index) and GAP-43(Growth Associated Protein 43) after each treatment in seventh and thirteenth day. Results : 1. Paw withdrawal threshold to the mechanical stimuli made the significant difference between group IV and the control group after five days of the experiment. 2. Paw withdrawal threshold to the thermal stimuli made the significant difference between group I and III, I and IV, II and IV. 3. In immunohistochemical response of c-fos, as time passes, the immunoreactivity of all groups was decreased gradually. Especially, group IV was observed the lowest after three days. 4. The differences of sciatic function indexes in each group were significantly between group I and III, I and IV, II and IV after 14 days, and between group I and III, I and IV, II and IV, III and IV after 21 days. 5. In immunohistochemical response of GAP-43, all groups had higher GAP-43 immunoreactivity at the 14 days from post-injury and group IV showed highest immunoreactivity. Conclusions : Based on above the results, it is proposed that Dokhwalgisaeng-tang(Duhuoqisheng-tang) and Jungsongouhyul pharmacopuncture may be helpful as a treatment in neuropathic pain and nerve regeneration in rat model.

• Archives of Plastic Surgery
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• 제49권2호
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• pp.200-206
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• 2022

Background Carpal tunnel syndrome (CTS) is the most common entrapment neuropathy. Studies have shown that results of CTS surgery are poorer in patients with diabetes. In this study, the effect of platelet-rich plasma (PRP) on nerve regeneration was investigated through clinical and electromyographic findings in patients with diabetes who underwent CTS surgery. Methods A retrospective analysis of 20 patients with diabetes who had surgically decompressed CTS was conducted. Patients were divided into two groups. The study group received PRP treatment following surgery. The control group did not receive any treatment. Patients were assessed using electromyography and the Boston Carpal Tunnel Syndrome Questionnaire preoperatively as well as postoperatively at 3-month, 6-month, and 1-year follow-ups visits. Results There was a decrease in complaints and an improvement in sensory and motor examinations in both groups. The Boston Carpal Tunnel Syndrome Questionnaire scores did not show any statistically significant differences between the two groups. However, electromyographic findings showed that there were statistical differences between preoperative and postoperative (3 months, 6 months, and 1 year) results in both groups. When the two groups were compared using preoperative and postoperative (3 months, 6 months, and 1 year) electromyographic values, no statistically significant differences were seen. Conclusion Single injections of PRP did not have a significant impact on median nerve regeneration following CTS surgery in patients with diabetes. The effectiveness of multiple PRP injections can be investigated in patients with diabetes in future studies.

• 한방재활의학과학회지
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• 제19권1호
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• pp.39-55
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• 2009

Objectives : This study was designed to evaluate effects of electroacupuncture and Cervi Pantortichum Cornu pharmacopuncture on pain and nerve regeneration after crush injury of sciatic nerve in rats. Methods : We divided the subjects into 5 groups : control group, acupuncture treated group, electroacupuncture treated group, Cervi Pantortichum Cornu pharmacopuncture treated group, electroacupuncture and Cervi Pantortichum Cornu pharmacopuncture group. Hot plate test and degree of Substance P antibody in each group were observed at the 1st, 2nd, 3rd and 5th day. Sciatic function index and change of BDNF immunoreactivity had been estimated at the 7th, 14th, 21st day respectively. Atrophy of the gastrocnemius at the 21st day was observed. Results : All the experimental groups showed significant changes in hot plate test, degree of Substance P antibody, sciatic function index and change of BDNF immunoreactivity compared with control group. Especially, when electroacupuncture and Cervi Pantortichum Cornu pharmacopuncture used together, results were more effective than other groups. Recovery of the gastrocnemius was also the same. Conclusions : It is suggested that electroacupuncture and Cervi Pantortichum Cornu pharmacopuncture may play a significant role on pain decrease and nerve regeneration after crush injury of sciatic nerve in rats and these two treatments could be more effective when used together.

• 한방재활의학과학회지
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• 제21권2호
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• pp.125-142
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• 2011

Objectives : Peripheral nerve injuries are commonly encountered clinical problems and often result in severe functional deficits. The purpose of this study was to evaluate the effects of Haein-tang(Hairen-tang) extract on functional recovery and pain release in the sciatic nerve after crushed sciatic nerve injury in rats. Methods : 1. Sciatic functional index(SFI) were performed on functional recovery. 2. c-Fos immunohistochemistry were performed on c-Fos expressions in the paraventricular nucleus(PVN) and ventrolateral periaqueductal gray(vIPAG). 3. Neurofilament immunohistochemistry were performed on neurofilament regeneration. 4. Western blot were performed on brain-derived neurotrophic factor(BDNF) and nerve growth factor(NGF) expression. Results : 1. Haein-tang(Hairen-tang) extract significantly enhanced the SFI value in the sciatic nerve injury and 100 mg/kg, 200 mg/kg Haein-tang(Hairen-tang)-treated group. 2. Haein-tang(Hairen-tang) extract significantly suppressed the sciatic nerve injury-induced increment of c-Fos expressions in the PVN and vIPAG in the sciatic nerve injury and 100 mg/kg, 200 mg/kg Haein-tang(Hairen-tang)-treated group. 3. Haein-tang(Hairen-tang) extract significantly increased neurofilament expression in the sciatic nerve injury and 50 mg/kg, 100 mg/kg, 200 mg/kg Haein-tang(Hairen-tang)-treated group. 4. Haein-tang(Hairen-tang) extract significantly controled the sciatic nerve injury-induced increment of BDNF and NGF expressions in the sciatic nerve injury and 100 mg/kg, 200 mg/kg Haein-tang(Hairen-tang)-treated group. Conclusions : These results suggest that Haein-tang(Hairen-tang) treatment after sciatic nerve injury is effective for the functional recovery by enhancing of axonal regeneration and suppressing of pain.

• 생명과학회지
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• 제16권3호
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• pp.464-471
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• 2006

본 연구에서는 흰쥐의 좌골신경을 손상시킨 후 트레드밀 운동을 적용하여 신경돌기 성장과 좌골신경의 축삭 재생 및 신경성장 인자 발현 그리고 신경기능지수의 변화를 연구했다. 본 연구결과 좌골손상 후 트레드밀 운동을 실시한 그룹이 비운동군에 비해 축삭재생이 촉진되었고, 원위부의 좌골신경에서도 NGF, BDNF단백질 발현이 상당히 증가된 것으로 나타났다. 또한 좌골신경지수를 검사한 결과에서도 운동을 실시한 흰쥐가 비운동 흰쥐에 비해 기능적 회복이 상당히 빠른 것으로 나타났다. 이러한 결과는 좌골손상 후 운동의 실시가 좌골신경의 축삭재생 촉진과 신경영양인자의 발현증가를 통해 기능적 회복에 도움이 될 수 있음을 보여주는 것이다.

• Archives of Reconstructive Microsurgery
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• 제6권1호
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• pp.9-28
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• 1997

Adequate vascularization is pivotally essential for a successful nerve graft. Theoretically, the immediate vascularization will inhibit fibroblast infiltration and stimulate nerve cell regeneration. In this study, histomorphological and electrophysiological studies were performed to determine if vascularized grafts are functionally superior. In rat model, a 4cm segment of the sciatic nerve was obtained and placed as a non vascularized graft on one side, and as a vascularized graft connected to the inferior gluteal vessels on the opposite side. To determine the compound action potential of the gastrocnemius muscle, electromyography was done after 2, 3 and 4 months. Histomorphologically, the distribution of myelinated nerve fibers and Schwann cell were evaluated after toluidine blue staining, The following resutls were obtained: 1. The electrophysiological studies showed no difference between the nonvascularized and vascularized grafts. 2. Two and three months after grafting, myelinated nerve fibers were more abundant in the vascularized proximal, middle and distal areas in all nerve fibers of varying diameters. 3. In the post-nonvascularized graft 2-month group, a few myelinated nerve fibers were present in the proximal and middle areas, but none distally. In the post-vascularized graft 2 month group, myelinated nerve fibers ranging $2-8{\mu}m$ were present in all three areas. 4. In the post-nonvascularized graft 3 month group, a few myelinated nerve fibers ranging in $2-6{\mu}m$ were present in all three areas, but in the post-vascularized graft 3 month group, many myelinated nerve fibers ranging in $2-10{\mu}m$ were present in all three areas. 5. In the post-graft 4-month group, more myelinated nerve fibers were present in all three areas of the vascularized grafts. However, nerve fibers of less than $2{\mu}m$ in diameter were more abundant in the non vascularized grafts. 6. Schwann cells were more abundant in the proximal, middle and distal areas of the post-vascularized 2, 3 and 4-month grafts. Based on these findings, the immediate restoration of circulation in vascularized nerve grafts allows for the increased number of surviving Schwann cells, rapid healing of the axon and myelin sheath changes which occur during Wallerian degeneration, and thus is able to stimulate a morphologically optimal regeneration.

• 한국고분자학회:학술대회논문집
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• 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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• pp.181-181
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• 2006

We developed a novel method to fabricate a nerve guide conduit (NGC) with the porosity of submicron pore sizes (to prevent fibrous tissue infiltration) and hydrophilicity (for effective oxygen and nutrient permeation) using poly(lactic-co-glycolic acid) (PLGA) and Pluronic F127 by a modified immersion precipitation method designed by our laboratory. It was recognized that the hydrophilized PLGA/F127 (3 wt%) tube can be a good candidate as a NGC from the analyses of its morphology, mechanical strength, hydrophilicity, model nutrient permeability and in vivo nerve regeneration behavior using a rat model.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권5호
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.