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.
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.
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 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 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.
Objective To review researches of treatment of peripheral nerve injury with neuromuscular electrical stimulation (NMES) regarding mechanism, parameters, and cl inical appl ication at home and abroad. Methods The latest original l iterature concerning treatment of peri pheral nerve injury with NMES was extensively reviewed. Results NMES should be used under individual parameters and proper mode of stimulation at early stage of injury. It could promote nerve regeneration and prevent muscle atrophy. Conclusion NMES plays an important role in cl inical appl ication of treating peripheral nerve injury, and implantable stimulation will be the future.
Objective To review the research progress on the three-dimensional (3D) reconstruction and visual ization of peri pheral nerve. Methods Literature about the research on the 3D reconstruction and visual ization of peripheral nerve both at home and abroad were extensively reviewed and thoroughly analyzed. Results The appl ication of 3D reconstruction and visual ization technology was capable of not only reappearing the 3D outer contour and spatial adjacent relationship of peripheral nerve veritably but also displaying, rotating, zooming, dividing and real-time measuring their 3D internal structure and the del icate pathways in any direction either separately or totally. Prel iminary achievements were achievedin terms of brachial plexus, lumbosacral plexus, the functional cluster of nerve trunk, intramuscular nerve distribution pattern, peripheral nerve regeneration and the 3D reconstruction and visual ization research of complex tissue including peripheral nerve. However, the research on the visual ization of peripheral nerve was still in the initial stage since such problems as recognition, segmentation, registration and fusion of the peripheral nerve information were not resolved yet. Conclusion Researching 3D reconstruction and visual ization of the peripheral nerve is of great value for updating the diagnosis and treatment principle of peripheral nerve injury, improving its diagnosis and treatment method and launching a new way for the studying and teaching, which may be a new growing point for the peripheral nerve surgery.
Objective To observe the revascularization process of chemically extracted acellular allogeneous nerve graft in repairing rat sciatic nerve defect. Methods Eighty adult male SD rats were selected. The sciatic nerve trunks from ischial tuberosity to the ramus of tibiofibular nerve of 16 SD rats were obtained and were prepared into acellular nerve stents by chemical reagent. Sixty-four SD rats were used to prepare the models of sciatic nerve defect (1.0 cm) and thereafter were randomized into two groups (n=32): experimental group in which acellular allogeneous nerve grafts were adopted and control group in which orthotopic transplantation of autologous nerve grafts were adopted. Postoperatively, the general conditions of all rats were observed, and the gross and ALP staining observation were conducted at 5, 7, 10, 14, 21, 28 days and 2, 3 months, respectively. Results All the incisions were healed by first intention. Trail ing status and toe’s dysfunction in extension happened to the right hindl imb of rats in two groups and were improved 6 weeks after operation. General observation showed that the grafts of two groups connected well to the nerves, with appearances similar to that of normal nerve. ALP staining demonstrated that the experimental group had no ingrowth of microvessel but the control group had ingrowth of microvessel 5 days after operation; the experimental group had ingrowth of microvessel but both groups had no microvessel 7 days after operation; few longitudinal microvessel throughout the grafts were observed in both groups 10, 14 and 21 days after operation; no obvious difference in capillary network of grafts was observed between two groups 28 days after operation; and the microvascular architecture of grafts in both groups were similar to that of normal nerve 2 and 3 months after operation. Conclusion When the chemically extracted allogeneous nerve graft is adopted to repair the peripheral nerve defect, new blood microvessels can grow into grafts timely and effectively.
Objective To observe the delaying effect of neural stem cell (NSC) transplantation on denervated muscle atrophy after peri pheral nerve injury, and to investigate its mechanism. Methods NSCs were separated from the spinal cords of green fluorescent protein (GFP) transgenic rats aged 12-14 days mechanically and were cultured and induced to differentiate in vitro. Thirty-two F344 rats, aged 2 months and weighed (180 ± 20) g, were randomized into two groups (n=16 per group). The animal models of denervated musculus triceps surae were establ ished by transecting right tibial nerve and commom peroneal nerve 1.5 cm above the knee joints. In the experimental and the control group, 5 μL of GFP-NSCsuspension and 5 μL of culture supernatant were injected into the distal stump of the tibial nerve, respectivel. The generalcondition of rats after operation was observed. At 4 and 12 weeks postoperatively, the wet weight of right musculus tricepssurae was measured, the HE staining, the Mallory trichrome staining and the postsynaptic membrane staining were adopted for the histological observation. Meanwhile, the section area of gastrocnemius fiber and the area of postsynaptic membrane were detected by image analysis software and statistical analysis. Results The wounds in both groups of animals healed by first intension, no ulcer occurred in the right hind l imbs. At 4 and 12 weeks postoperatively, the wet weight of right musculus triceps surae was (0.849 ± 0.064) g and (0.596 ± 0.047) g in the experimental group, respectively, and was (0.651 ± 0.040) g and (0.298 ± 0.016) g in the control group, respectively, showing a significant difference (P lt; 0.05). The fiber section area of the gastrocnemius was 72.55% ± 8.12% and 58.96% ± 6.07% in the experimental group, respectively, and was 50.23% ± 4.76% and 33.63% ± 4.41% in the control group, respectively. There were significant differences between them (P lt; 0.05). Mallory trichrome staining of muscle notified that there was more collagen fiber hyperplasia of denervated gastrocnemius in the control group than that in the experimental group at 4 and 12 weeks postoperatively. After 12 weeks of operation, the area of postsynaptic membrane in the experimental group was (137.29 ± 29.14) μm2, which doubled that in the control group as (61.03 ± 11.38) μm2 and was closer to that in normal postsynaptic membrane as (198.63 ± 23.11) μm2, showing significant differences (P lt; 0.05). Conclusion The transplantation in vivo of allogenic embryonic spinal cord NSCs is capable of delaying denervated muscle atrophy and maintaining the normal appearance of postsynaptic membrane, providing a new approach to prevent and treat the denervated muscle atrophy cl inically.
Objective To investigate the effects of icariin and mixed prescri ption of icariin, radix hedysari polysaccharide, and l iquid extracted from earthworm on peri pheral nerve regeneration. Methods Twenty male SD rats weighing (200 ± 10) g were selected and randomized into four groups (n=5 per group): sham operated group (group A), model group (group B), icariin group (group C), and mixed l iquid group (group D). In group A, the left sciatic nerves of the rats were only exposed, and treated at fixed time from the following day with the NS (2 mL/d). In groups B, C, D, the models were made by clamping sciatic nerve and treated with NS, icariin and mixed l iquid, respectively (2 mL/d). The general state ofanimals was observed after the treatment daily. The nerve function index, motor nerve conductive velocity and the morphous and number of myel inated sciatic nerve fibers were measured at 21 days. Results Animals in various groups were all in good state. After 21 days, the weights of rats in groups A, B, C and D were (366.9 ± 14.0), (370.1 ± 16.3), (373.3 ± 19.6) and (374.0 ± 11.4) g, respectively, and there was no significant difference among these groups (P gt; 0.05). For sciatic function index, there was no significant difference between group A and group D (P gt; 0.05), between group B and group C (P gt; 0.05), while there was significant difference between group B and group D (P lt; 0.05). For tibial function index, there was significant difference between group A and groups B, C, D (Plt; 0.05), there was no significant difference between group B and groups C, D (Pgt; 0.05). For peroneal function index, there was no significant difference between group A and groups C, D (P gt; 0.05), between group B and groups C, D (P gt; 0.05). The sciatic motor nerve conductive velocities of group A, B, C and D were (45.0 ± 2.9), (8.0 ± 2.6), (13.4 ± 6.8), and (19.6 ± 9.3) m/s, respectively, there was no significant difference between group B and group C (P gt; 0.05), and there was significant difference between group A and groups B, C, D and between group B and group D (P lt; 0.05). The size of individual myel inated sciatic nerve fibers of regenerated nerves in groups B, C, and D was significantly smaller than that in group A. Comparing with group A, the number of myel inated sciatic nerve fibers in groups B, C, and D was 93.3% ± 35.6%, 90.6% ± 37.1%, and 115.4% ± 40.6%, respectively, but there was no significant difference among four groups (P gt; 0.05). Conclusion Icariin and mixed prescription are safe. The improving peripheral nerve regeneration effect of mixed prescription is more obvious than that of icariin, indicating the comprehensive study of modified formula radixhedysari is necessary to find the effective part or mixture of effective compounds with fixed percentage.