Dar-Yu Yang

Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague-Dawley rats weighing 250-300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Enhanced regeneration in injured sciatic nerve by human amniotic mesenchymal stem cell

OBJECTIVE Amniotic fluid mesenchymal stem cells (MSCs) have the potential to differentiate into neuronal stem cells in vitro. We evaluated using amniotic fluid MSCs to support or enhance the ability of the injured sciatic nerve to cross a nerve gap. MATERIALS AND METHODS We created a 5 mm nerve defect in Sprague Dawley rats. One group received therapy with MSCs embedded into woven oxidised regenerated cellulose gauze (Surgical; Ethicon, Somerville, NJ) and fibrin glue, while a control group received woven Surgicel and fibrin glue only. Evaluation methods included behavioural, electrophysiological and immunohistochemical studies. RESULTS In gait analysis, the angle of the ankles in the treatment and control group were 46.4 degrees (standard deviation [SD]=15 degrees) and 36 degrees (SD=8.2 degrees), respectively, which was statistically significant (p=0.045). Five of 10 treated rats (50%) demonstrated partial foot movement, while none of the control group had any movement. The percentage amplitude of muscle compound action potential in the experimental group was 43% (SD=12.5%) compared to 29% (SD=8.8%) in the control group (p=0.038). The conduction latencies in the control and experimental groups was 2.5 ms (SD=0.45) and 1.7 ms (SD=0.47), respectively (p=0.005). Histological examination demonstrated that 70% of the treatment group achieved a maximum axon diameter percentage across the nerve gap of greater than 50%, compared with 0% in the control group. There were no differences in direction of fibre growth and fibrotic reaction between the two groups. CONCLUSION Amniotic fluid MSC can augment growth of injured nerve across a nerve gap. This effect may be due to neurotrophic or induction effects of the MSC interacting with Schwann cells. Further study is required to determine the underlying mechanism of this effect.

Dual regeneration of muscle and nerve by intravenous administration of human amniotic fluid–derived mesenchymal stem cells regulated by stromal cell–derived factor-1α in a sciatic nerve injury model

OBJECT Human amniotic fluid-derived mesenchymal stem cells (AFMSCs) have been shown to promote peripheral nerve regeneration. The expression of stromal cell-derived factor-1α (SDF-1α) in the injured nerve exerts a trophic effect by recruiting progenitor cells that promote nerve regeneration. In this study, the authors investigated the feasibility of intravenous administration of AFMSCs according to SDF-1α expression time profiles to facilitate neural regeneration in a sciatic nerve crush injury model. METHODS Peripheral nerve injury was induced in 63 Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into 1 of 3 groups: Group I, crush injury as the control; Group II, crush injury and intravenous administration of AFMSCs (5 × 10(6) cells for 3 days) immediately after injury (early administration); and Group III, crush injury and intravenous administration of AFMSCs (5 × 10(6) cells for 3 days) 7 days after injury (late administration). Evaluation of neurobehavior, electrophysiological study, and assessment of regeneration markers were conducted every week after injury. The expression of SDF-1α and neurotrophic factors and the distribution of AFMSCs in various time profiles were also assessed. RESULTS Stromal cell-derived factor-1α increased the migration and wound healing of AFMSCs in vitro, and the migration ability was dose dependent. Crush injury induced the expression of SDF-1α at a peak of 10-14 days either in nerve or muscle, and this increased expression paralleled the expression of its receptor, chemokine receptor type-4 (CXCR-4). Most AFMSCs were distributed to the lung during early or late administration. Significant deposition of AFMSCs in nerve and muscle only occurred in the late administration group. Significantly enhanced neurobehavior, electrophysiological function, nerve myelination, and expression of neurotrophic factors and acetylcholine receptor were demonstrated in the late administration group. CONCLUSIONS Amniotic fluid-derived mesenchymal stem cells can be recruited by expression of SDF-1α in muscle and nerve after nerve crush injury. The increased deposition of AFMSCs paralleled the expression profiles of SDF-1α and its receptor CXCR-4 in either muscle or nerve. Administration of AFMSCs led to improvements in neurobehavior and expression of regeneration markers. Intravenous administration of AFMSCs may be a promising alternative treatment strategy in peripheral nerve disorder.

Enhanced regeneration in spinal cord injury by concomitant treatment with granulocyte colony-stimulating factor and neuronal stem cells.

Granulocyte colony-stimulating factor (G-CSF) inhibits programmed cell death and stimulates neuronal progenitor differentiation. Neuronal stem cells transplanted into injured spinal cord can survive, differentiating into astroglia and oligodendroglia, and supporting axon growth and myelination. Herein, we evaluate the combined effects of G-CSF and neuronal stem cells on spinal cord injury. For 40 Sprague-Dawley rats (n=10 in each group) transverse spinal cord resections at the T8-9 level were carried out, leaving an approximately 2-mm gap between the distal and proximal ends of the cord. Neuronal stem cells embedded in fibrin glue treated with or without G-CSF (50 microg/kg x 5 days) (groups III and IV) or fibrin glue with or without G-CSF (50 microg/kg x 5 days) (groups I and II) were transplanted into the gap in the injured spinal cord. Spinal cord regeneration was assessed using a clinical locomotor rating scale scores and electrophysiological, histological and immunohistochemical analysis 3 months after injury. Regeneration was more advanced in group IV than in groups III or II according to the clinical motor score, motor evoked potential, and conduction latency. Most advanced cord regeneration across the gap was observed in group IV rats. Higher densities of bromodeoxyuridine in the injured area and higher expression levels of Neu-N and MAP-2 over the distal end of the injured spinal cord were observed in group IV compared with groups II or III, but there was no significant difference in expression of glial fibrillary acid protein. This synergy between G-CSF and neuronal stem cells may be due to increased proliferation of progenitor cells in the injured area and increased expression of neuronal stem cell markers extrinsically or intrinsically in the distal end of injured cord.

Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto

Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.

Neuroprotective effect of atorvastatin in an experimental model of nerve crush injury.

BACKGROUNDStatins have therapeutic benefits for the management of several disorders. A short-term course of a high-dose statin pretreatment has demonstrated neuroprotective effects against neurological diseases. However, the molecular basis underlying the neuroprotective action of statins remains unclear. OBJECTIVEWe investigated whether a short-term course of high-dose atorvastatin pretreatment has beneficial effects in protecting sciatic nerve from crush injury. METHODSAtorvastatin (5 mg/kg) or saline was given orally to Sprague-Dawley rats for 7 days before injury. The rats were subjected to crush injury in the left sciatic nerve with a vessel clamp. Biochemical, functional, electrophysiological, and morphological alterations occurring during injury-induced degeneration/regeneration were examined. RESULTSAtorvastatin improved injury-induced neurobehavioral/electrophysiological changes and axonal loss. Damage-associated alterations, including structural disruption, oxidative stress, inflammation, and apoptosis, were attenuated by atorvastatin. After injury, regeneration-associated genes, including growth-associated protein-43, myelin basic protein, ciliary neurotrophic factor, and collagen, were upregulated by atorvastatin. The suppression of extracellular signal-regulated kinase, AKT, signal transducer and activators of transcription-1, and necrosis factor-κB and the elevated activation of c-Jun N-terminal kinase, Smad2/3, and activating protein-1 were associated with the neuroprotective action of atorvastatin. CONCLUSIONThese findings suggest that a short-term course of high-dose atorvastatin pretreatment can protect against sciatic nerve crush injury through modifying intracellular or extracellular environments, making it favorable for regeneration.

Intracapsular decompression or radical resection followed by Gamma Knife surgery for patients harboring a large vestibular schwannoma

OBJECT Microsurgery is the primary treatment used for patients harboring a large vestibular schwannoma (VS). However, its outcome may lead to hearing impairment and facial nerve dysfunction particularly when resection is extended outside the tumor capsule. When surgery for a large VS consists of intracapsular resection and decompression, better preservation of facial and hearing function are obtained. In this study, the authors compared outcomes of intracapsular decompression followed by Gamma Knife surgery (GKS) with outcomes of standard microsurgery followed by radiosurgery. METHODS Between August 2003 and October 2008, 35 patients harboring large VSs (> 3 cm in diameter) were enrolled in this study. Eighteen patients underwent intracapsular decompression followed by GKS (Group I), and 17 patients underwent radical extracapsular resection followed by GKS (Group II). In all cases GKS was performed with a margin dose of 12 Gy. All patients were followed up for at least 3 years. All patients also underwent periodic audiography, electroneuronography (ENoG), MR imaging, and testing with the SF-36 form. The Student t-test and repeated ANOVA were used for statistical analysis. RESULTS The mean ages of the patients (± SEM) in Groups I and II were 50 ± 3.0 and 49 ± 2.3 years, respectively. The female/male ratios were 8:10 in Group I and 7:10 in Group II. All patients had excellent facial function as measured according to the House-Brackmann Facial Grading System (Grade I or II) preoperatively. After the operation, 16 patients (89%) in Group I retained excellent facial function, whereas only 6 patients (35%) in Group II had excellent facial function (p < 0.01). In Group I, 11 patients had serviceable hearing, and all 11 (100%) retained hearing function after the operation. In Group II, 11 patients had serviceable hearing, but none retained hearing function postoperatively (p < 0.001). In Group I, the mean tumor volume (± SEM) was 17.5 ± 1.1 cm(3), and the postoperative volume was 9.35 ± 1.02 cm(3). In Group II, the mean tumor volume was 16.4 ± 0.95 cm(3), whereas the postoperative volume was 1.1 ± 0.14 cm(3) (p < 0.001). After GKS, the tumor volume was reduced to 5.12 ± 1.1 cm(3) and 0.9 ± 0.1 cm(3) in Groups I and II, respectively. No patients experienced adverse effects after GKS. The mean return-to-work times were 2.4 ± 0.16 and 33.4 ± 4.3 weeks in Groups I and II, respectively (p < 0.001). According to the results obtained using the 36-Item Short Form Health Survey (SF-36), patients in Group I enjoyed more significant improvements in quality of life than patients in Group II (p < 0.001). CONCLUSIONS Intracapsular decompression followed by GKS afforded a better neurological outcome and quality of life than radical extracapsular resection followed by GKS. Further application of this approach in patients harboring large VSs seems warranted.

DuraSeal as a ligature in the anastomosis of rat sciatic nerve gap injury.

BACKGROUND Anastomosis of the nerve especially at narrow surgical field and presence of surgical tension is not easily accessible. DuraSeal demonstrates strong adhesive power without producing neurotoxicity. Herein, we evaluate the possibility of DuraSeal as a substitute in the repair of sciatic nerve gap injury. MATERIALS AND METHODS The nerve gap model was constructed by excising the sciatic nerve (5mm in length) in Sprague Dawley rats leaving a 5mm nerve defect between nerve stumps. Animals were categorized into four groups: Group I: no treatment; Group II: 4 stitches suture; Group III: nerve approximation fixed by tissue glue; Group IV: nerve approximation fixed by DuraSeal. The motor function assessment included the CatWalk and SFI as well as electrophysiological studies. Nerve continuity and regeneration was examined at 1 and 8 wk after injury. The inflammatory cells, Schwann cell apoptosis, and Schwann cell proliferation were also investigated 1 wk after injury. RESULTS The achievement of nerve continuity and myelination by DuraSeal approached that of suture demonstrated by crystal violet and Luxol Fast Blue staining at 1 and 8 wk, respectively. Motor function and electrophysiological parameters were restored in DuraSeal and suture group. Early expression of neurofilament and bromodeoxyuridine (BrdU) was also observed in these two groups. There was no statistically significant difference in deposits of macrophages and neutrophil cells or cell apoptosis among these four groups. CONCLUSIONS DuraSeal achieved the same nerve regeneration compared with that of suture and produced better regeneration than that of the tissue glue or without any treatment. The accomplishment of nerve regeneration and continuity without causing neurotoxicity justifies using DuraSeal as a ligature in the anastomosis of nerve gap injury.

Upper thoracic sympathectomy for axillary osmidrosis or bromidrosis

The difference between axillary osmidrosis (AO) and axillary bromidrosis (AB) is the degree of odor and quantity of sweat, which is associated with selection of therapeutic modality theoretically. Upper thoracic sympathectomy has been used for both diseases but its effect needs to be further evaluated with more clinical data. We collected 108 patients with AO or AB treated by upper thoracic sympathectomy from July 1995 to July 2002. Of these patients, 42 suffered AO alone, 17 had AB (AO with axillary hyperhidrosis [AH]), and 49 had AO with palmar hyperhidrosis (PH). Ninety-two patients (183 sides) received anterior subaxillary transthoracic endoscopic sympathectomy (TES) and 17 patients (33 sides) received posterior percutaneous thoracic phenol sympathicolysis (PTPS). The levels of sympathectomy or sympathicolysis were T3-4 for AO and AB, and T2-4 for AO with PH. Mean follow-up period was 45.2 months (13-97 months). The satisfaction rates of patients were 52.4%, 70.6% and 61.2% for AO, AB and AO with PH, respectively. The rates of patients with improvement and satisfaction were 78.6%, 88.2% and 85.7% for AO, AB, and AO with PH, respectively. These results suggest that upper thoracic sympathectomy may be an acceptable treatment for AB or AO with PH rather than AO only.

Analysis of factors associated with volumetric data errors in gamma knife radiosurgery.

Object: Gamma knife (GK) surgery is an important part of the treatment armamentarium for benign and malignant brain tumors. In general, quantitative volumetrical analysis of the tumor on neuroimaging studies is the most reliable method of assessment of the tumor’s response and is critical for accurate dose planning. This study evaluated various factors contributing to volumetric data error of tumors treated with GK radiosurgery. Method: Three differently shaped phantoms (spherical, rectangular, and irregular morphology) were created by immersing like shaped objects into 2% agarose gel. The volumes of phantoms were measured by laser scanning with errors <1%. MRI sequence and parameters including time of flight (TOF), T1, T2, different slice thickness, size of field of view (FOV), phase FOV as well as different position and axis of phantoms were retrieved and transferred to a Perfexion Gamma Knife Workstation (PGK-WS) and Picture Archiving and Communication System (PACS) for data analysis. The volumetric data errors were presented as the volume difference between those computed on the PGK-WS and actual volume measured by laser scanning divided by the actual laser scanning volume. The systemic error was defined as volume discrepancy between Perfexion and PACS over that in Perfexion. One-way ANOVA was used for evaluation of data errors between different methods as well as for factor analysis. Results: The MRI-computed volume of the various phantoms approached the laser-scanned volume within 2% when the slice number was ≥30. The volumetrical data errors (10/5 slices) associated with various MRIs for phantoms were 6.94 ± 0.04%/9.45 ± 0.35% (spherical phantom), 12.3 ± 0.2%/ 20.06 ± 0.7% (rectangular phantom), and 9.29 ± 0.078%/ 15.67 ± 0.6% (irregular phantom) (p < 0.001 and p < 0.001), respectively. The system errors (10/5 slices) associated with various MRIs for the phantoms were 3.17 ± 0.11%/3.9 ± 0.13% (spherical phantom), 3.61 ± 0.12%/4.01 ± 0.12% (rectangular phantom), and 4.39 ± 0.07%/4.75 ± 0.13% (irregular phantom) (p < 0.001 and p = 0.01), respectively. The volumetric data errors were related to the number of slices and the shape of phantom, but the systemic errors were only related to the irregularity of phantom morphology. The volumetrical data errors were not related to size of the FOV, phase FOV, sequence of T1, T2, TOF, and position of phantom. For the rectangular phantom, the data error was related to slice orientation of imaging acquisition (p < 0.001). Conclusion: Volume discrepancies existed between those volumes computed by the PGK-WS and volumes determined by laser scanning. The volumetric data errors were reduced through the acquisition of more slices through the phantom and a more spherical morphology of the phantom. Relatively few system volume errors were observed between those by the PGK-WS and PACS except for a significant discrepancy for the irregular surface phantom. For the rectangular-shaped phantom, the volumetric data errors were significantly related to slice orientation of measurement. When measuring the tumor response in GK radiosurgery or follow-up, an error of as large as 20% is possible for irregularly shaped object and with MRIs using ≤5 slices through the region of interest.