Objective To systematically review the effectiveness and safety of autologous implantation of stem cells for diabetic peripheral neuropathy (DPN). Methods Randomized controlled trials on relevant studies were retrieved in databases including CBM (1978-2011.6), CNKI (1979-2011.6), MEDLINE (1950-2011.6), PubMed (1950-2011.6), EMbase (1970-2011.6) and The Cochrane Library (Issue 3, 2011). References of the included studies were also retrieved. Two reviewers independently screened literature according to the inclusion and exclusion criteria, extracted data, and assess the methodological quality of the included studies. Then, meta-analysis was performed using RevMan 5.0 software.Results Four RCTs involving 68 patients (136 limbs) were included, most of which were low in methodological quality. The results of meta-analysis indicated that, autologous stem cell therapy improved or even eliminated DPN symptoms including pain, numbness, and cold sensation in the limbs, intermittent limping, and rest pain. Compared with the routine therapy, autologous stem cell therapy improved tibial sensory nerve conduction velocity (MD=5.75, 95%CI 3.86 to 7.64, Plt;0.000 01), tibial motor nerve conduction velocity (MD=4.04, 95%CI 0.90 to 7.18, P=0.001), sural sensory nerve conduction velocity (MD=7.47, 95%CI 4.00 to 10.94, Plt;0.000 1), and sural motor nerve conduction velocity (MD=3.38, 95%CI 0.07 to 7.58, P=0.05), with no adverse reaction reported. Conclusion Current evidence shows that, autologous stem cell therapy is effective in treating DPN. Due to the lack of high quality studies, more high quality RCTs are needed to verify the above conclusion.
Objective To observe the effect of comprehensive rehabilitation in patients with peripheral nerve injuries after the Wenchuan earthquake. Methods A total of 24 cases of peripheral nerve injuries who were admitted to the Rehabilitation Center for Earthquake Victims of West China Hospital of Sichuan University were treated with comprehensive rehabilitation, including exercise therapy, acupuncture therapy, functional electrical stimulation, and occupational therapy (mainly sensory training and wearing orthosis). After 30 treatment sessions, patient motor and sensory function, upper limb functional activity, and electrodiagnostic parameters were evaluated. Meanwhile, concomitant injuries were also recorded. Results As for the recovery of motor and sensory functions, the effective rate was 41.66%. The difference in the scores of upper limb functional activities was statistically significant before and after treatment (Plt;0.01). As assessed by electromyogram and nerve conduction velocity, the response rate was 87.50%. Patients with more concomitant injuries were likely to have slower recovery. Conclusion Comprehensive rehabilitation is appropriate and effective for patients with peripheral nerve injuries after the Wenchuan earthquake.
目的 探讨原发性干燥综合征周围神经病变的发生与干燥综合征A型/B型抗体（抗SSA/SSB抗体）的关系。 方法 纳入2009年1月－2011年12月期间门诊及住院收治的原发性干燥综合征患者88例。所有患者均接受神经系统检查，采用蛋白质印迹法检测抗SSA抗体和抗SSB抗体，利用全自动化学发光仪检测血清维生素B12水平。 结果 88例原发性干燥综合征患者中有27例（30.7%）存在周围神经病变。有或无周围神经病变的患者在年龄、性别、病程等一般情况方面无明显不同。有周围神经病变和无周围神经病变的原发性干燥综合征患者抗SSA抗体阳性率分别为70.4%（19/27）、70.5%（43/61），差异无统计学意义（χ2=0.000，P=0.991）；抗SSA/SSB抗体双阳性率分别为63.0%（17/27）、14.8%（9/61），差异有统计学意义（χ2=17.416，P=0.000）；血清维生素B12水平分别为（390 ± 55）、（410 ± 86）pg/dL，差异无统计学意义（t=0.908，P=0.370）。 结论 周围神经病变在原发性干燥综合征患者中较常见，且周围神经病变的发生多伴随血清抗SSA/SSB抗体阳性。
Objective To investigate the feasibility of establishing the visualization models of intraneural microvessels of sciatic nerves in Sprague Dawley (SD) rats by systemic infusion of Evan’s blue (EB) or lead oxide and to compare the advantages and disadvantages. Methods Fifteen healthy adult SD rats of either gender, weighing 200-250 g, were randomly divided into traditional group (group A, n=5), fluorescence group (group B, n=5), and radiography group (group C, n=5). Ink, EB, and lead oxide, all mixed with gelatin solution, were injected in groups A, B, and C, respectively. After 2 hours of cryopreservation under 4°C, all sciatic nerves were harvested and observed through stereomicroscope to make sure the filling condition. The two-dimentional (2D) images were then collected via reflexion fluorescent microscope in group B and via micro-CT scan in group C. All images were imported into computer to establish three-dimentional (3D) reconstruction models by Mimics 15.0. Results All groups could show the outline of intraneural microvessels of sciatic nerves under stereomicroscope. Diameters of them were measured under fluorescent microscope, ranging from 10 µm to 30 µm. Both groups B and C could establish 3D reconstruction models from 2D images. These models could clearly reproduce the structure of microvessels. Conclusion Both EB and lead oxide can be used to establish 3D reconstruction models to observe structure of the intraneural vessels. However, EB has some disadvantages, such as predisposition to infiltration, grainy 2D images and time-consuming procedure; it is not suitable for researches of large specimen. Though 2D pictures from lead oxide have lower resolution than EB, it is easier to be manipulated and appropriate for experiments of large specimen.
Objective To explore the histochemical staining for distinguishing and local izing nerve fibers and fascicles at histological level in three-dimensional reconstruction of peri pheral nerves. Methods The right median nerve was harvested from one fresh cadaver and embedded in OCT compound. The sample was serially horizontally sl iced with 6 μm thickness. All sections were stained with Karnovsky-Roots method (group A, n=30) firstly and then stained with toluidine blue (group B, =28) and Ponceau 2R (group C, n=21) in proper sequence. The results of each step were taken photos (× 100). After successfully stitching, the two-dimensional panorama images were compared, including texture feature, the number and aver gray level of area showing acetylchol inesterase (AchE) activity, and result of auto microscopic medical image segmentation. Results In groups A, B, and C, the number of AchE-positive area was (21.63 ± 4.06)× 102, (20.64 ± 3.51)× 102, and (20.54 ± 5.71)× 102, respectively, showing no significant difference among 3 groups (F=0.64, P=0.54); the mean gray level was (1.41 ± 0.06)× 102, (1.10 ± 0.05)× 102, and (1.14 ± 0.07)× 102, respectively, showing significant differences between group A and groups B and C (P lt; 0.001). In the image of group A, only AchE-positive area was stained; in the image of group B, myelin sheath was obscure; and in the image of group C, axons and myelin sheath could be indentified, the character of nerve fibers could be distinguished clearly and accurately, and the image segmentation of fascicles could be achieved easier than other 2 images. Conclusion The image of Karnovsky-Roots-toluidine blue-Ponceau 2R staining has no effect on the AchE-positive area in the image of Karnovsky-Roots staining and shows better texture feature. This improved histochemical process may provide ideal image for the three-dimensional reconstruction of peri pheral nerves.
ObjectiveTo establish an efficient method of isolating and culturing high activity and high purity of Schwann cells, and to identify the cells at the levels of transcription and translation. MethodsThe sciatic nerves harvested from a 4-week-old Sprague Dawley rat were digested in the collagenase I for 15 minutes after dissecting, and then the explants were planted in culture flask directly. The cells were cultured and passaged in vitro, the growth state and morphological changes of the cells were observed under inverted phase contrast microscope. MTT assay was used to test the proliferation of cells and the cells growth curve was drawn. RT-PCR and immunohistochemistry staining were used to detect S100 and glial fibrillary acidic protein (GFAP) at the levels of transcription and translation, respectively. The purity of cells was caculated under microscope. ResultsAfter the digestion of collagenase I, fibroblast-like cells appeared around explants within 24 hours, with slender cell body and weak refraction. After tissues were transferred to another culture flask, a large number of dipolar or tripolar cells were seen after 48 hours, with slender ecphyma, plump cell body, and b refraction, and the cells formed colonies within 72 hours. The cells were covered with the bottom of culture flask within 48-72 hours after passaging at a ratio of 1∶2, and spiral colonies appeared. Cells showed vigorous growth and full cytoplasm after many passages. MTT assay results showed that the cells at passage 3 entered the logarithmic growth phase on the 3rd day, reached the plateau phase on the 7th day with cell proliferation, and the growth curve was “S” shape. RT-PCR results showed that the cells expressed S100 gene and GFAP gene, and immunohistochemistry staining showed that most of the cells were positively stained, indicating that the majority of cells expressing S100 protein and GFAP protein. The purity of Schwann cells was 98.37% ± 0.30%. ConclusionHigh activity and high purity of Schwann cells can be acquired rapidly by single-enzyme digestion and explant-culture method.
Objective To investigate the effects of chitosan/polyvinyl alcohol (PVA) nerve conduits for repairing radial nerve defect in Macaques. Methods Twelve adult Macaques weighing 3.26-5.35 kg were made the models of radial nerve defect (2 cm in length) and were randomly divided into 3 groups according to nerve grafting, with 4 Macaques in each group. Chitosan/PVA nerve conduit, non-graft, and autografts were implanted in the defects in groups A, B, and C, respectively. And the right radial nerves were used as normal control. At 8 months postoperatively, the general observation,electrophysiological methods, and histological examination were performed. Results At 8 months postoperatively, theregenerated nerve bridged the radial nerve defect in group A, but no obvious adhesion was observed between the tube and the peripheral tissue. The regenerated nerve had not bridged the sciatic nerve defect in group B. The adhesions between the implanted nerve and the peri pheral tissue were significant in group C. Compound muscle action potentials (CMAP) were detected in group A and group C, and no CMAP in group B. Peak ampl itude showed a significantly higher value in normal control than in groups A and C (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). Nerve conduction velocity and latency were better in normal control than in groups A and C, and in group C than in group A, all showing significant differences (Plt; 0.05). The density of myl inated fibers in groups A and C was significantly lower than that in normal control (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). The diameter and the myel in sheath thickness of the myl inated fibers in normal control were significantly higher than those in groups A and C, and in group C than in group A, all showing significant differences (P lt; 0.05). Conclusion The chitosan/PVA nerve conduits can promote the peripheral nerve regeneration, and may promise alternative to nerve autograft for repairing peripheral nerve defects.
Objective Peri pheral nerve injury is a common cl inical disease, to study the effects of the physical therapy on the regeneration of the injured sciatic nerve, and provide a reference for cl inical treatment. Methods Sixty-four female adult Wistar rats (weighing 252-365 g) were chosen and randomly divided into 4 groups (n=16): group A, group B, groupC, and group D. The experimental model of sciatic nerve defect was establ ished by crushing the right sciatic nerve in groups B, C, and D; group A served as the control group without crushing. At 2 days after injury, no treatment was given in group B, electrical stimulation in group C, and combined physical therapies (decimeter and infrared ray) in group D. At 0, 7, 14, and 30 days after treatment, the sciatic nerve function index (SFI) and the motor nerve conduction velocity (MNCV) were measured, and morphological and transmission electron microscopy (TEM) examinations were done; at 30 days after treatment, the morphological evaluation analysis of axons was performed. Results At 0 and 7 days after treatment, the SFI values of groups B, C, and D were significantly higher than that of group A (P lt; 0.05); at 14 and 30 days after treatment, the SFI value of group D decreased significantly, no significant difference was observed between group D and group A (P gt; 0.05) at 30 days; whereas the SFI values of groups B and C decreased, showing significant difference when compared with the value of group A (P lt; 0.05). At 0, 7, and 14 days after treatment, the MNCV values of groups B, C, and D were significantly lower than that of group A (P lt; 0.05), and there were significantly differences between group B and groups C, D (P lt; 0.05); at 14 days, the MNCV value of group D was significantly higher than that of group C (P lt; 0.05); and at 30 days, the MNCV values of groups B and C were significantly lower than that of group A (P lt; 0.05), but there was no significant difference between group D and group A (P gt; 0.05). At 0 and 7 days, only collagen and l i pid were observed by TEM; at 14 and 30 days, many Schwann cells and perineurial cells in regeneration axon were observed in groups B, C, and D, especially in group D. Automated image analysis of axons showed that there was no significant difference in the number of myelinated nerve fibers, axon diameter, and myelin sheath thickness between group D and group A (P gt; 0.05), and the number of myelinated nerve fibers and axon diameter of group D were significantly higher than those of groups B and C (P lt; 0.05). Conclusion Physical therapy can improve the regeneration of the injured sciatic nerve of rats.
Objective To observe the histomorphology and the biocompatibil ity of acellular nerve prepared by different methods, to provide the experimental evidence for the selection of preparation of acellular nerve scaffold. Methods Forty-eight adult Sprague Dawley rats, male or female, weighing 180-220 g, were selected. The sciatic nerves were obtained from 30 rats and were divided into groups A, B, and C (each group had 20 nerves). The acellular sciatic nerves were prepared by the chemical methods of Dumont (group A), Sondell (group B), and Haase (group C). The effect to remove cells was estimated by the degree of decellularization, degree of demyel ination, and intergrity of nerve fiber tube. The histocompatibil ity was observed by subcutaneous implant test in another 18 rats. Three points were selected along both sides of centre l ine on the back of rats, and the points were randomly divided into groups A1, B1, and C1; the acellular nerve of groups A, B, and C were implanted in the corresponding groups A1, B1, and C1. At 1, 2, and 4 weeks after operation, the rats were sacrificed to perform the general observation and histological observation. Results The histomorphology: apart of cells and the dissolved scraps of axon could be seen in acellular never in the group A, and part of Schwann cell basilar membrane was broken. In group B, the cells in the acellular never were not removed completely, the Schwann cell basilar membrane formed bigger irregular hollows, part of the Schwann cell basilar membrane was broken obviously. But in the group C, the cells were completely removed, the Schwann cell basilar membrane remained intactly. Group C was better than group A and group B in the degree of decellularization, degree of demyel ination, integrity of nerve fiber tube and total score, showing significant differences (P lt; 0.05). The subcutaneous implant test: there were neutrophils and lymphocytes around the acellular nerve in 3 groups at 1 week after implant. A few of lymphocytes were observed around the acellular nerve in 3 groups at 2 weeks after implant. The inflammation was less in groups A1, B1, and C1 at 4 weeks after implant, part of the cells grew into the acellular nerve and arranged along the Schwann cell basilar membrane. The reaction indexes of the inflammational cells in group A1 and group B1 were higher than that in group C1 at 1, 2, and 4 weeks after implant, showing significant differences (P lt; 0.01), but there was no significant difference between group A1 and group B1 (P gt; 0.05). Conclusion The acellular sciatic nerves prepared by Haase method has better acellular effect and the histocompatibil ity than those by the methods of Dumont and Sondell.
Objective To explore and solve the key technologies of the three dimensional (3D) visual ization reconstruction of functional fascicular groups inside long segmented peri pheral nerve. Methods A 20 cm ulnar nerve from upper arm of fresh adult dead body was embedded by OCT with four pieces of woman’s hair which was used as locating material, then the samples were serially horizontally sl iced into 400 sl ices with 15 μm thickness and 0.5 mm interval. All sl iceswere stained with acetylcholinesterase (AchE) histochemical staining. After that, the 2D panorama images of the same sl ice were obtained with Olympus stereomicroscope and MSHOT MD90 micro figure image device before and after AchE staining. Using the layer processing technique of Photoshop image processing software, the recomposition images including complete 4 location pots were obtained, based on which the algorithm of optimized least square support vector machine (Optimized LS-SVM) and space transformation method was used to fulfill automatic registration. Finally, with artificial assistant outline obtaining, the 3D visual ization reconstruction model of functional fascicular groups of 20 cm ulnar nerve was made using Amira 4.1, and the effects of reverse reduction and the suitabil ity of 3D reconstruction software were evaluated. Results The two-time imaging technique based on the layer process of Photoshop image processing software had the advantages: the image outline had high goodness of fit; the locating pots of merging image was accurate; and the whole procedure was simple and fast. The algorithm of Optimized LS-SVM had high degree of accuracy, and the error rate was only 8.250%. The 3D reconstruction could display the changes of the chiastopic fusion of different nerve functional fascicular groups directly. It could extract alone, merge and combine arbitrarily, and revolve at any angles. Furthermore, the reverse reduction on arbitrarily level dissection of the 3D model was very accurately. Conclusion Based on the two-time imaging technique and computer image layer processing technology, the compute algorithm of auto-registration can be developed and appl ied to 3D visual ization reconstruction of long segmented peripheral nerve. The technological processes is fast, and the reconstruction effect is good.